Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=imbc20

Download by: [Smithsonian Astrophysics Observatory] Date: 10 January 2018, At: 13:05

Molecular Membrane Biology

ISSN: 0968-7688 (Print) 1464-5203 (Online) Journal homepage: http://www.tandfonline.com/loi/imbc20

The Sec translocon mediated protein transport inprokaryotes and eukaryotes

Kärt Denks, Andreas Vogt, Ilie Sachelaru, Narcis-Adrian Petriman, RenukaKudva & Hans-Georg Koch

To cite this article: Kärt Denks, Andreas Vogt, Ilie Sachelaru, Narcis-Adrian Petriman,Renuka Kudva & Hans-Georg Koch (2014) The Sec translocon mediated protein transportin prokaryotes and eukaryotes, Molecular Membrane Biology, 31:2-3, 58-84, DOI:10.3109/09687688.2014.907455

To link to this article: https://doi.org/10.3109/09687688.2014.907455

Published online: 24 Apr 2014.

Submit your article to this journal

Article views: 1506

View related articles

View Crossmark data

Citing articles: 49 View citing articles

2014

http://informahealthcare.com/mbcISSN: 0968-7688 (print), 1464-5203 (electronic)

Mol Membr Biol, 2014; 31(2–3): 58–84! 2014 Informa UK Ltd. DOI: 10.3109/09687688.2014.907455

REVIEW ARTICLE

The Sec translocon mediated protein transport in prokaryotes andeukaryotes

Kart Denks1,2, Andreas Vogt1,2,3, Ilie Sachelaru1,2, Narcis-Adrian Petriman1,2, Renuka Kudva1,2,3, andHans-Georg Koch1,3

1Institute of Biochemistry and Molecular Biology, University of Freiburg, Freiburg, Germany, 2Faculty of Biology, University Freiburg, Freiburg,Germany, and 3Spemann Graduate School of Biology and Medicine (SGBM), Freiburg, Germany

Abstract

Protein transport via the Sec translocon represents an evolutionary conserved mechanismfor delivering cytosolically-synthesized proteins to extra-cytosolic compartments. The Sectranslocon has a three-subunit core, termed Sec61 in Eukaryotes and SecYEG in Bacteria.It is located in the endoplasmic reticulum of Eukaryotes and in the cytoplasmic membraneof Bacteria where it constitutes a channel that can be activated by multiple partner proteins.These partner proteins determine the mechanism of polypeptide movement across thechannel. During SRP-dependent co-translational targeting, the ribosome threads the nascentprotein directly into the Sec channel. This pathway is in Bacteria mainly dedicated formembrane proteins but in Eukaryotes also employed by secretory proteins. The alternativepathway, leading to post-translational translocation across the Sec translocon engages anATP-dependent pushing mechanism by the motor protein SecA in Bacteria and a ratchetingmechanism by the lumenal chaperone BiP in Eukaryotes. Protein transport and biogenesisis also assisted by additional proteins at the lateral gate of SecY/Sec61a and in the lumen of theendoplasmic reticulum or in the periplasm of bacterial cells. The modular assembly enablesthe Sec complex to transport a vast array of substrates. In this review we summarize recentbiochemical and structural information on the prokaryotic and eukaryotic Sec translocons andwe describe the remarkably complex interaction network of the Sec complexes.

Keywords

BiP, protein targeting, Sec61/SecYEG,SecA, SRP

History

Received 10 December 2013Revised 27 February 2014Accepted 10 March 2014Published online 23 April 2014

Introduction

The sorting of proteins between different cellular compart-ments is mediated by a large diversity of protein transportsystems (Lithgow & Waksman, 2013; Papanikou et al., 2007).In prokaryotes, the cytoplasmic membrane is responsible forasymmetric protein distribution between the cytosol andperiplasmic space or the outer membrane in Gram-negativeBacteria. Targeting, sorting and transport systems inEukaryotes are more complex, owing to the presence oforganelles. The protein transport gateway to most organelles(secretory pathway) is the endoplasmic reticulum (ER) andthe Sec translocon constitutes the major protein transport sitein the ER membrane. The Sec translocon is also universallyconserved in the cytoplasmic membrane of Bacteria andArchaea (Kudva et al., 2013; Zimmermann et al., 2011). Inaddition, the Sec translocon is present in the thylakoidmembrane in chloroplasts, but absent in most mitochondria

with the exception of certain protozoans (Albiniak et al.,2012; Tong et al., 2011).

Although the Sec translocon primarily functions as anaqueous conduit for proteins, it differs from most other poresas its channel forming subunit (SecY/Sec61a) is able to openboth transversally into the periplasm/ER lumen and alsolaterally to the lipid membrane (Figure 1). Transverse openingfacilitates protein translocation across the membrane, whereaslateral opening of the translocon allows lipid insertion ofmembrane proteins. The lateral gate of the translocon iscomposed of four flexible a-helices of the SecY/Sec61asubunit (Van den Berg et al., 2004). The translocation ofsecretory proteins and large hydrophilic domains of mem-brane proteins through the SecY/Sec61 pore requires ATPhydrolysis by the ATPase SecA on the cis side of the bacterialmembrane or by chaperones like BiP on the trans side of theER membrane (Kudva et al., 2013; Zimmermann et al., 2011).The proton motive force (pmf) also contributes to theenergetics of protein translocation across the bacterial mem-brane (van der Laan et al., 2004) although it is not essentialfor protein transport in vitro (Koch & Muller, 2000).

A major challenge during protein transport is prevent-ing premature and unproductive folding of proteinsbefore they reach the Sec translocon. This is especiallythe case for hydrophobic membrane proteins which are

Correspondence: Hans-Georg Koch, Institut fur Biochemie undMolekularbiologie, ZBMZ, Albert-Ludwigs-Universitat Freiburg,Germany. Tel: +49 761/203 5250. E-mail: hans-georg.koch@biochemie-uni-freiburg.deKart Denks, Institut fur Biochemie und Molekularbiologie, ZBMZ,Albert-Ludwigs-Universitat Freiburg, Germany. Tel: +49 761/203 5222.E-mail: kaert.denks@biochemie.uni-freiburg.de

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Figure 1. Protein targeting pathways in bacterial and mammalian cells (A) Bacteria engage two targeting pathways for delivering proteins to theSecYEG translocon. The SecA-dependent pathway is used by periplasmic and outer membrane proteins, which contain a cleavable signal sequence.Cytosolic chaperones, including trigger factor and tetrameric SecB keep the nascent polypeptide in a translocation-competent state during its journey tothe membrane. The pre-protein is then transferred to SecA, which drives translocation through the SecYEG channel by ATP hydrolysis. Although it isgenerally assumed that SecA acts post-translationally, some data indicate that SecA can bind to ribosome-nascent chains (RNCs), i.e. that it can also actco-translationally. The SecYEG translocon interacts at least transiently with the SecDFYajC complex, which might support the proton-motif force(pmf)-dependent steps during protein transport. The co-translational SRP-pathway is mainly used for inner membrane proteins and initiated by theribosome-bound SRP. SRP-RNCs bind to the SecYEG-bound SRP receptor FtsY, RNCs dock onto the SecYEG translocon and the SRP-FtsY complexdissociates in a GTP-dependent manner. During the lateral exit from the SecYEG channel, the nascent membrane protein contactsYidC. YidC is shownto support SecYEG during membrane protein insertion, and it also acts as a SecYEG independent insertase for small or closely spaced membraneproteins. Targeting of membrane proteins to YidC also appears to be SRP-dependent. The translocon associates transiently with several additionalproteins, that are required for cleaving the signal sequences of secretory proteins (signal peptidase, SPase), for protein folding (the periplasmicchaperones Skp and PpiD) or for quality control (the membrane-bound protease FtsH). (B) In eukaryotes, the Sec61-mediated insertion and thetranslocation occurs both co-translationally and post-translationally. During post-translational targeting, fully synthesized pre-proteins are kept in atransport-competent state by members of the Hsp90, Hsp70 and Hsp40 chaperone families. Translocation is mediated by the Sec61 complex inassociation with Sec62/Sec63 and the chaperone BiP at the lumenal side of the ER membrane. BiP binds to the translocating substrates in the ER lumenand prevents their back-sliding by an ATP-dependent ratcheting mechanism. The eukaryotic SRP pathway delivers both membrane proteins andsecretory proteins co-translationally to the Sec61 complex. The eukaryotic SRP receptor consists of two unrelated GTPases, SRa (homologous to FtsY)and SRb. The Sec61 translocon associates in a substrate-dependent manner with additional proteins that either bind to RNCs (Ramp4) or are suggestedto assist membrane protein folding (TRAM, Sec63). BiP is also required for co-translational transport. Like in bacteria, additional proteins are involvedin processing and quality control (SPase; TRAP [translocon associated protein], oligosacharyl transferase [OST], the Hsp40-homologue Erj1 or AAA-proteases). This Figure is reproduced in color in the online version of Molecular Membrane Biology.

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 59

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

aggregation-prone. Targeting of many proteins thereforebegins at the ribosome during translation. Co-translationaltargeting is initiated by the signal recognition particle (SRP),which binds to the tunnel exit of the ribosome to recognizeits substrate early during synthesis (Berndt et al., 2009;Bornemann et al., 2008). SRP-ribosome-associated nascentchains (SRP-RNCs) are delivered to the membrane-boundSRP receptor (SR), and the ribosome thereafter aligns withthe channel of the Sec translocon to facilitate co-translationaltransport of the nascent polypeptide (Gilmore et al., 1982b;Koch et al., 1999; Valent et al., 1998). In bacteria, theSRP pathway is mainly dedicated to the targeting of innermembrane proteins, while the eukaryotic SRP delivers bothER membrane proteins and secretory proteins to the Sec61complex.

The Sec translocon also transports proteins post-transla-tionally, i.e. upon termination of protein synthesis. In bacteria,the post-translational mode is preferentially employed byperiplasmic and outer membrane proteins and involves thecytosolic ATPase SecA (Koch et al., 1999; Koch & Muller,2000). SecA also cooperates with the SRP pathway for theinsertion of membrane proteins with periplasmic domainslonger than approx. 30 amino acids (Deitermann et al., 2005;Neumann-Haefelin et al., 2000; Saaf et al., 2009). Ineukaryotes, post-translational transport requires the associ-ation of Sec61 with the Sec62/Sec63 complex (Meyer et al.,2000; Panzner et al., 1995).

The folding and processing of transported proteins isfacilitated by periplasmic and lumenal chaperones (Gordon &Kindt, 1976; Missiakas et al., 1996), signal peptidases (Changet al., 1978; Zwizinski & Wickner, 1980) and oligosaccharyltransferases (Lau et al., 1983). The final topology ofmembrane proteins is further affected by the lipid composi-tion of the membrane (Dowhan & Bogdanov, 2009). Thedynamic interplay of the core translocon with many additionalfactors is common to both prokaryotes and eukaryotes, andthis probably ensures efficient transport and biogenesis of avast array of substrates.

This review provides an insight to the current know-ledge on the Sec translocon. Most of the data on theprokaryotic Sec translocon is based on the Gram-negativemodel organism E. coli. For reviews covering proteintranslocation in Archaea and Gram positive bacteria pleasesee Pohlschroder et al. (2005), Calo & Eichler (2011) andYuan et al. (2010).

Composition and architecture of the coreSec complex

Most components of the Sec pathway were identified fromgenetic screens conducted in E. coli and Saccharomycescerevisiae (Deshaies & Schekman, 1987; Emr et al., 1981;Oliver & Beckwith, 1981). Mutations that caused proteinsecretion defects were referred to as sec alleles and mapped tosecA, secD, secE, secF and secY in E.coli; prl mutations(suppressors of signal sequence mutations) allowed thesecretion of proteins with defective signal sequences andwere mapped to secA (prlD), secE (prlG), secG (prlH)and secY (prlA) (Bieker & Silhavy, 1990; Emr et al.,1981, Ito et al., 1983; Oliver & Beckwith, 1981; Schatz &

Beckwith, 1990). The three-dimensional crystal structure ofthe Sec translocon from the archaeon Methanocaldococcusjanaschii confirmed many structural and functional predic-tions that were based on these early genetic screens (Smithet al., 2005; Van den Berg et al., 2004). Subsequentstudies found that the general architecture observed for theMethanocaldococcus jananschii Sec complex appears to beuniversally conserved in both prokaryotes and eukaryotes(Becker et al., 2009; Clemons et al., 2004; Egea & Stroud,2010; Frauenfeld et al., 2011; Gogala et al., 2014; Gumbartet al., 2009; Li et al., 2007; Menetret et al., 2005; 2007; 2008;Mitra et al., 2005; Park et al., 2014; Tsukazaki et al., 2008;Zimmer et al., 2008).

The core Sec translocon consists of three protein subunits– SecY, SecE and SecG in bacteria, and Sec61a, Sec61g andSec61b in eukaryotes (Zimmermann et al., 2011). SecYEand Sec61ag exhibit significant sequence conservation andare essential (Kudva et al., 2013; Park & Rapoport, 2012).The Sec61b subunit found in Eukaryotes and Archaea isnot homologous to the eubacterial SecG subunit, andneither Sec61b nor SecG are essential for protein transport(Finke et al., 1996; Nishiyama et al., 1994).

SecY and Sec61a are comprised of 10 transmembranea-helical domains (TMs) each and have similar molecularmasses, i.e. 48 kDa for E. coli SecY and 52 kDa for Homosapiens Sec61a (Rensing & Maier, 1994). When visualizedfrom the top, the 10 TMs are divided into two halves thatresemble a clamshell surrounding a central pore (Figure 2D;Van den Berg et al., 2004). The two halves (TMs 1–5 and6–10) are connected by a periplasmic loop, which is referredto as the hinge region or the back of the translocon. A sidesection through the SecYEb complex reveals an hourglass-shaped structure with two funnels, one opening to thecytoplasmic face and the other one to the periplasmic faceof the membrane (Figure 2A). The two funnels are separatedby a central constriction, which is called the pore ring. It iscomprised of amino acid residues with bulky hydrophobicside chains, e.g. by six isoleucine residues in E. coli SecY(Van den Berg et al., 2004). The translocon assumes a closedconformation in the resting state: the cytoplasmic funnelis empty and its periplasmic counterpart is plugged by ashort a-helix (Helix 2a or plug) (Tsukazaki et al., 2008;Van den Berg et al., 2004). Mutagenesis studies and structuraldata suggest that the central constriction and the plug mayplay a role in the controlled opening and closing of the Secpore (Egea & Stroud, 2010; Harris & Silhavy, 1999; Van denBerg et al., 2004). Molecular dynamics simulations supportthe view that the plug participates in sealing the pore andmaintaining substrate selectivity of the translocon (Gumbart& Schulten, 2008). However, deleting the plug domain ofthe Sec channel does not affect growth of either E. coli orS. cerevisiae (Junne et al., 2006; Li et al., 2007; Maillardet al., 2007) although it decreases the selectivity of thetranslocon for its substrates (Li et al., 2007; Maillard et al.,2007). The X-ray structure of the plug-less SecYE channelshows that neighbouring residues can replace the functionof the plug (Li et al., 2007).

The exit of TMs into the lipid phase is facilitated bystructural rearrangements in the lateral gate of the SecY/Sec61a channel and involves TMs 2b and 3 on one side

60 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

and helices 7 and 8 on the other side of SecY (Hizlan et al.(2012), Figure 2C). Signal sequences might be maintained atthe lateral gate since cross-linking data have shown that signalsequences contact lipids during insertion (Higy et al., 2005;Martoglio et al., 1995) and that they are intercalatedbetween transmembrane helices 2 and 7 of SecY/Sec61a(Plath et al., 1998).

Most bacteria have a SecE molecule with only one TM.In contrast, E. coli SecE consists of three TMs and hasa molecular mass of 14 kDa. However, only the third TM ofSecE is essential for protein transport and cell viability(Schatz et al., 1991). In eukaryotes, including H. sapiens,Sec61g is a single-spanning membrane protein of approx.8 kDa (Hartmann et al., 1994). SecE is located at the backof SecY (Figure 2D) stabilizing the two halves of SecY(Van den Berg et al., 2004). Indeed, SecY molecules that havebeen proteolytically cleaved at the hinge region remainactive if SecE is present (Lycklama a Nijeholt et al., 2013).SecY in E. coli is rapidly degraded by the membrane proteaseFtsH in the absence of SecE (Kihara et al., 1995).

SecG in E. coli is a 12 kDa protein consisting of two TMsconnected by a cytoplasmic loop. Cross-linking studies havelocated SecG next to the cytosolic loops C2 and C3 of SecY(Satoh et al., 2003; van der Sluis et al., 2002), although SecG/Sec61b are thought to have only limited contacts to SecY andSecE (Van den Berg et al., 2004; Zimmer et al., 2008). SecGis not essential for protein transport in vitro (Brundage et al.,1990) but SecG deletion strains exhibit protein transportdefects in vivo (Flower, 2001; Flower et al., 2000). In E. coli,the function of SecG has been linked to the SecA-dependentpost-translational transport across the Sec channel (Duong &Wickner, 1997b; Morita et al., 2012; Nishiyama et al., 1996).As SecA is absent in Eukaryotes with the exception of

chloroplasts and since it is also not found in Archaea, afunctional connection between SecA and SecG would explainits presence in Bacteria only. SecG has been proposed toundergo reversible topology inversions for facilitating SecA-SecY interaction (Morita et al., 2012; Nishiyama et al., 2012;Sugai et al., 2007). Although several dual topology proteinshave been identified in E. coli (Daley et al., 2005; Rapp et al.,2006), a topologically fixed SecG derivative does not preventSecA-dependent protein translocation (van der Sluis et al.,2006). Thus, the physiological significance of the topologyswitch needs to be further analysed. Recent data suggest thatthe orientation of SecG depends on a non-proteinaceousglycolipozyme that was shown to influence membrane proteininsertion and translocation (Moser et al., 2013).

The non-homologous Sec61b in Eukaryotes and Archaeais slightly smaller than SecG and contains only one TM(Hartmann et al., 1994; Kalies et al., 1998). Sec61b wasshown to interact with the SRP receptor (Helmers et al., 2003)and with the signal peptidase in yeast (Kalies et al., 1998).Furthermore, the role of Sec61b might not be limited totranslocation since it interacts with Rtn1p, a protein involvedin ER tubule formation (Zhao & Jantti, 2009), and it appearsto be required for plasma membrane targeting of Gurken, theligand of epidermal growth factor receptor in Drosophila(Kelkar & Dobberstein, 2009).

Additional subunits, partner proteins and themembrane environment of the bacterial andeukaryotic Sec complex

The Sec complex has a modular nature. Some of theinteractions of the core Sec translocon have been observedin all domains of life, e.g. with ribosomes, the SRP receptor

Figure 2. The Sec translocon. Schematicrepresentation of the Sec translocon in theclosed (A) and the open (B) conformationviewed from the front in the membrane plane(left), as a transverse section through themiddle of the pore in the membrane plane(middle) and from the cytoplasmic side (top,right). The open and the closed conformationrefer to the lateral gate being closed or openas shown in the front and top representation,respectively. The transverse section and thetop view show the pore ring and the plugbeing displaced for the accommodation of asubstrate. (C) Surface representation of theArchaeal SecYEb translocon in the plane ofthe membrane (adapted from Van den Berget al. [2004]; pdb: 1RHZ). The lateral gatehelices (TM2b, TM3, TM7 and TM8) ofSecY and a short helix (helix 2a), called theplug, are highlighted. The plug is suggestedto be involved in sealing the channel. Thecytoplasmic loops C4, C5 and C6 of SecY arethe major cytoplasmic contact sites for FtsY,SecA and the ribosome. (D) The top view ofSec61YEb from the cytoplasmic site showsthe plug (dark green) sealing the channel andSecE embracing SecY at the back. ThisFigure is reproduced in color in the onlineversion of Molecular Membrane Biology.

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 61

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

and signal peptidases; yet many interactions are characteristicto either Bacteria or Eukaryotes. A number of proteins havebeen shown to interact at least transiently with the Sectranslocon (Table 1). Attempts to determine the structure of aholo-translocon (Duong & Wickner, 1997b) comprisingseveral modules attached to the Sec core complex havebeen successful for SecA-SecYE complexes (Zimmer et al.,2008) and for ribosome-SecYEG/ribosome-Sec61 complexes(Frauenfeld et al., 2011; Gogala et al., 2014; Menetret et al.,2005, 2007, 2008; Mitra et al., 2005; Park & Rapoport, 2012;Park et al., 2014). The Sec translocon interactions withadditional partners depend on the nature of the substrate andtherefore the exact composition of the holo-translocon isprobably rather flexible in vivo.

The interaction of ribosomes with Sec complex

The ability of the Sec complex to bind to ribosomes is anessential feature and ribosome binding sites on the transloconare evolutionarily conserved (Becker et al., 2009; Frauenfeldet al., 2011; Houben et al., 2005; Prinz et al., 2000).The cytosolic loops of SecY/Sec61a between TM 6 and 7(C4 loop) and TM 8 and 9 (C5 loop) mediate ribosomebinding (Cheng et al., 2005; Frauenfeld et al., 2011; Kuhnet al., 2011; 2014; Park & Rapoport, 2012; Park et al., 2014;

Raden et al., 2000). The universal ribosome adaptor siteconsisting of the proteins L23, L24 and L29 (E. colinomenclature, Figure 3), and conserved rRNA helices,establish contacts to the Sec translocon both in prokaryotesand eukaryotes (Becker et al., 2009; Frauenfeld et al., 2011).Recent comparative cryo-EM reconstructions show that bothtranslating and non-translating ribosomes provide the samebinding sites for the translocon although rather largeconformational changes take place within the transloconupon substrate binding (Gogala et al., 2014; Park et al., 2014).

The tunnel exit area of the ribosome contacts not onlythe Sec translocon, but acts as the binding platform for SRP,SecA, trigger factor, and nascent chain modifying enzymes(Figure 3), (Baram et al., 2005; Ferbitz et al., 2004;Frauenfeld et al., 2011; Gu et al., 2003; Huber et al., 2011;Kramer et al., 2002; Kramer et al., 2009; Kuhn et al., 2011;Schlunzen et al., 2005; Ullers et al., 2003). Similarly, thecorresponding area of the eukaryotic ribosome also controlsthe binding of SRP, methionine aminopeptidase 1 and thechaperones NAC (nascent chain associated complex) andSsb1/2 in a substrate-specific manner (Raue et al., 2007). It isnot clear how binding of that many ribosomal tunnel exitligands is coordinated in space and time.

The interaction of other ribosomal regions with the mem-brane might also facilitate the contact to the Sec translocon.

Table 1. Sec-translocon associated proteins and their conservation. Dark grey represents the proteins present in all or most species; light greyrepresents the proteins found in some species; blank – no known homologue. The paralogues are not indicated.

Yeast Mammals Bacteria Archaea Mitochondria Chloroplasts References

TransloconSecY/Sec61a * 1,2,3,4

SecE/Sec61g * 1,2,3,4

SecG 1,2,3,4

Sec61b 1,2,3,5

Translocon-associated proteinsSecD/SecF 6,7,8

YajC 2,6

YidC/Oxa1/Alb3 9

Sec62 3,10

Sec63 3,10

Sec71/72 10

ERj1 11

TRAM 3

TRAP 3

Ramp4/Ysy6p 12

SecA 3,13

BiP/Kar2 3

Calmodulin 14

Targeting factorsSRP 2,3

SR 2,3

Processing enzymesSpase1 15

OST 16,17,18

FtsH** 19

ChaperonesSecB 20

Skp 21

PpiD 22

1Hartmann et al. (1994); 2Cao & Saier (2003); 3Pohlschroder et al. (2005); 4Cline & Dabney-Smith (2008); 5Kinch et al. (2002); 6Eichler (2003);7Hand et al. (2006); 8Tseng et al. (1999); 9Zhang et al. (2009b); 10Tyedmers et al. (2000); 11Dudek et al. (2002); 12Yamaguchi et al. (1999); 13Noharaet al. (1995); 14Nakashima et al. (2012); 15Dalbey et al. (1997); 16Calo & Eichler (2011); 17Nothaft & Szymanski (2010); 18Aebi et al. (2013);19Janska et al. (2013); 20van der Sluis & Driessen (2006); 21Gatsos et al. (2008).

*SecYE is only found in mitochondria of some protozoans (Albiniak et al., 2012; Tong et al., 2011); **Eukaryotes have other members ofAAA-metalloproteases.

62 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

One such region is the eukaryotic ribosome expansionsegment 27 (ES27L) which has been shown to contact theER membrane by in situ cryo-EM tomography in caninepancreas microsomes (Pfeffer et al., 2012). Conformationalrearrangements of ES27L might play a role in ribosomerelease from the ER membrane (Pfeffer et al., 2012). WhetherES27L interacts with the membrane lipids or proteins is notyet clear but its flexibility suggests that it might respond toevents on the ribosomal tunnel exit.

The interaction of the SRP receptor with Sec complex

The transfer of RNCs from the SRP to the Sec complex is thefinal and most crucial step during co-translational targeting.Biochemical and genetic evidence suggest that membrane-bound SR interacts directly with the Sec61 complex (Jianget al., 2008; Song et al., 2000). The bacterial SR is termedFtsY and it is homologous to the eukaryotic SRa subunit(Luirink et al., 1994). FtsY and SRa belong to the SIMIBIfamily of GTPases harboring a characteristic NG-domain(Figure 4C). However, both proteins use different strategiesfor membrane binding. The X-domain of SRa dimerizes withSRb, an integral 30 kDa ER membrane protein present only inEukaryotes (Figure 4E) (Schwartz & Blobel, 2003; Schlenkeret al., 2006). SRb belongs to the Ras superfamily of smallGTPases and it requires GTP-activation to allow stableSRa binding (Fulga et al., 2001; Schwartz & Blobel, 2003).The observation that Sec61 regulates the nucleotide occu-pancy of SRb has led to the idea that Sec61b functions as

nucleotide exchange factor for SRb (Helmers et al., 2003).The interaction with Sec61b could keep SRb in its GTP-bound state, which would prime it for the subsequentinteraction with SRa.

There is no SRb-homologue present in bacterial mem-branes and as FtsY does not have an X-domain, it uses bothits NG domain and an enterobacteria-specific A-domain formembrane attachment. FtsY binds to negatively chargedphospholipids and to the cytosolic loops of SecY (Angeliniet al., 2005; 2006; Braig et al., 2009; Kuhn et al., 2011; Parlitzet al., 2007). In E. coli, FtsY is present in large excess overSecYEG and it is likely that a large portion of the SecYEGtranslocons are in contact with FtsY (Drew et al., 2003;Kudva et al., 2013). Importantly, the same conserved residuesof SecY that are in contact with FtsY also bind to theribosome (Kuhn et al., 2011). Therefore, it appears likely thatFtsY occupies the ribosome binding site of SecY until it isdisplaced by SRP-RNCs. FtsY also competes with SecA forSecYEG binding (Kuhn P, Koch HG, unpublished work),but it is unknown how access of FtsY or SecA to SecYEG isregulated in vivo. Although the A-domain of FtsY has beenshown to interact with SecY, deleting the A-domain reducesthe efficiency of co-translational targeting only moderately(Eitan & Bibi, 2004; Weiche et al., 2008). In contrast, deletingthe two lipid-binding helices in the N-terminus and at theinterface of A and N domains of FtsY completely inhibitsco-translational targeting (Parlitz et al., 2007; Weiche et al.,2008). This could indicate that membrane attachment of FtsYand not its ability to bind to SecY is crucial for its function(Mircheva et al., 2009). However, a second SecY binding siteis proposed to exist within the NG-domain of FtsY, whichcould facilitate its binding to SecY independently of theA-domain (Kuhn et al., 2011). In addition, the membranearound the bacterial Sec complex is likely enriched withphosphatidylglycerol and cardiolipin, which are required forSecYEG activity (Gold et al., 2010). As FtsY also bindspreferentially to negative phospholipids (Braig et al., 2009),sufficient amounts of FtsY are probably located in closeproximity to the SecYEG complex even in the absence of theA-domain.

The interaction of signal peptidase with Sec complex

The majority of non-cytosolic proteins originally bear a signalsequence which is recognized by targeting factors. After thepre-protein is targeted to the translocase, the signal sequenceis eventually removed by membrane-embedded signal pep-tidases (SPases). The signal peptide is subsequently degradedby the membrane bound signal peptide peptidases (SPPase)(Nam & Paetzel, 2013; Voss et al., 2013; Wang et al., 2008)and the amino acids are recycled.

Many different SPases are found in all domains oflife. Prokaryotic SPases are classified as Spase I, II andIV (Auclair et al., 2012). The Spase I (LepB in E. coli)is an essential and conserved serine-protease, specific tothe non-lipoprotein substrates of the Sec and Tat (twin-arginine-dependent translocation) translocons (Auclair et al.,2012; Nyathi et al., 2013). Its catalytic domain is locatedin the periplasm and its TMs probably assist in signal peptideprocessing (Paetzel et al., 1998). The Spase I of E. coli has

Figure 3. The ribosomal tunnel exit as a binding platform for targetingfactors, chaperones, nascent chain processing enzymes and thetranslocon. L23, L24 and L29 constitute a universal ribosomal adaptorsite. Data are collected from: TF and peptidyl formylase (PDF): (Krameret al., 2002; Bingel-Erlenmeyer et al., 2008); SecY: (Frauenfeld et al.,2011); SecA: (Huber et al., 2011); SRP: (Gu et al., 2003; Halic et al.,2006; Schaffitzel et al., 2006); methionine amino peptidase (MAP):(Sandikci et al., 2013); YidC: (Kohler et al., 2009; Seitl et al., 2013;Welte et al., 2012). This Figure is reproduced in color in the onlineversion of Molecular Membrane Biology.

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 63

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

two transmembrane domains while Bacillus subtilis hasone (Tjalsma et al., 1998). Eukaryotic signal peptidasesare organized in multi-subunit complexes termedSPC. However, the catalytic activity of SPC is locatedat LepB homologue which is Sec11 in yeast (Figure 5)(VanValkenburgh et al., 1999) and Spc18/Spc21 in mammals(Liang et al., 2003).

Although signal sequences of Sec substrates are cleavedoff during the translocation (Josefsson & Randall, 1981a;1981b), evidence for a direct interaction between SPase andthe translocon is limited. So far, only the yeast Sec61b wasshown to interact with signal peptidase during translocation(Antonin et al., 2000; Kalies et al., 1998).

The effect of the lipid environment on proteintransport

The lipid bilayer constitutes the permeability barrier ofthe cell and influences directly the activity of multiplemembrane protein complexes, including the Sec translocon.Phospholipids also affect the stability and final topology ofnewly synthesized membrane proteins (Dowhan & Bogdanov,

2009). This is achieved by the charged phospholipid headgroups, their asymmetric distribution and the nature of theacyl chains.

The ER membrane and the cytoplasmic membrane ofGram-negative bacteria consist largely of zwitterionicphospholipids while Gram-positive bacteria have moreanionic membrane lipids (Epand & Epand, 2011). Acomparison of the lipid composition of E. coli membraneand the ER membrane is given in Table 2.

The zwitterionic phospholipid phosphotidylethanolamine(PE) is important for membrane elasticity and curvature(Raetz, 1978). PE probably supports conformational flexibil-ity of the Sec complex during protein transport (Rietveldet al., 1995) and its depletion has been shown to reducetranslocation efficiency (Mikhaleva et al., 2001; van der Doeset al., 2000). PE has also been suggested to affect membranebinding of FtsY (Millman et al., 2001).

Phospholipids with negatively charged head groupslike phosphatidylglycerol (PG), phosphatidylserine (PS) andphosphatidylinositol (PI) have the most pronounced effect onmembrane protein biogenesis (Table 3). The activities of bothSecA and FtsY are stimulated by negatively charged

Figure 4. Structure of the signal recognitionparticle (SRP) and its receptor (SR) (A)Cryo-EM reconstitution of the eukaryoticSRP with the conserved SRP54 subunit, theadditional eukaryotic SRP subunits and the7.5 S RNA (adapted from: (Halic et al.,2004); pdb: 1RY1). (B) Crystal structure ofthe prokaryotic SRP (adapted from (Ataideet al., 2011); pdb: 2XXA). The conservedprotein subunit Ffh (fifty-four homologue)and the 4.5 S RNA. (C) Crystal structure ofthe NG-subunit of the bacterial SRP receptorFtsY (adapted from (Ataide et al., 2011); pdb:2XXA) (D) Complex of the prokaryotic SRPand the NG-domain of FtsY (adapted from(Ataide et al., 2011); pdb: 2XXA) (E) Crystalstructure of the eukaryotic X-domain of SRain complex with the cytoplasmic domain ofSRb (adapted from Schwartz & Blobel[2003]; pdb: 1NRJ). This Figure is repro-duced in color in the online version ofMolecular Membrane Biology.

64 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

phospholipids (Bahari et al., 2007; Braig et al., 2009;Lam et al., 2010; Lill et al., 1990; Parlitz et al., 2007) andthe absence of PG severely impairs protein transport in E. coli(de Vrije et al., 1988; van der Does et al., 2000). Cardiolipin(CL) also stabilizes the SecYEG dimer and creates a highaffinity binding surface for the motor protein SecA(Gold et al., 2010). Acidic phospholipids induce the dissoci-ation of dimeric SecA exposing its binding interface toSecYEG (Alami et al., 2007). The amount of CL boundto SecYEG is proportional to the ATPase activity of SecA(Gold et al., 2010).

Sterols seem to inhibit protein translocation initiation,most likely hindering RNC binding to Sec61 (Nilssonet al., 2001; Yamamoto et al., 2012). This is probablythe reason why sterols are scarce in the ER membrane(van Meer et al., 2008).

The interaction network of bacterial Sec complex

Some Sec complex-associated proteins like SecA arepresent only in bacteria while others like periplasmic

chaperones also have functional homologues in eukaryoticcells (Table 1).

SecA

SecA is (Figure 6) probably best studied partner protein of thebacterial Sec complex. It was identified in the genetic screensduring the discovery of the sec and prl alleles (Emr et al.,1981; Oliver & Beckwith, 1981). It has a dual role as it actsas an ATP-fueled motor supporting protein transport acrossthe inner membrane and as a targeting factor for the post-translational pathway.

SecA binds to SecYEG with an affinity of 20–40 nM(Douville et al., 1995) and is considered to function as asoluble subunit of the Sec translocon. SecA and FtsY have anoverlapping binding sites on SecY, however SecA binds toadditional residues on SecY that are distributed acrosscytosolic loops C2–C6 (Mori & Ito, 2006). The crystalstructure of SecA in complex with the bacterial translocon(Zimmer et al., 2008) shows SecA:SecYEG in a 1:1stoichometry but biochemical data also support a 1:2 or 2:2stoichometry (Deville et al., 2011; Osborne & Rapoport,2007). The PBD domain (peptide binding domain);also called PPXD; (pre-protein cross-linking domain) ofSecA, provides the major contact site with SecY (Figure 6C).The PBD domain of free SecA is closely packed against thehelical wing domain (HWD) (Hunt et al., 2002;Vassylyev et al., 2006) but in the SecY-bound SecA structurethe PBD is rotated towards the nucleotide-binding domain 2(NBD-2) (Zimmer et al., 2008). It is thought that the PBDdomain functions as a flexible trap that captures thetranslocating substrate in a groove formed by PBD andHWD (Park & Rapoport, 2012). In the SecA-SecYE structure,the groove is aligned with the SecY channel, allowing thesubstrate to move through the groove into the channel(Zimmer et al., 2008).

Figure 5. The topology of yeast Sec62,Sec63, Sec71, Sec72 and Sec11. The lume-nal J-domain of Sec63 is important for therecruitment of BiP, a chaperone which isessential for Sec61-mediated translocation.The negatively charged C-terminus of Sec63binds to the N-terminus of Sec62 to collect-ively support post-translational translocation.Sec71 and Sec72 form the complex withSec62/Sec63. Sec11 is the catalytic subunit ofthe yeast signal peptidase complex. ThisFigure is reproduced in color in the onlineversion of Molecular Membrane Biology.

Table 2. The phospholipid composition of the E. coli inner membrane and the ER membrane.

Structural lipids of the E. coli inner membrane1 Structural lipids in ER membrane2,3

PE PG CL PC PE PS PI

Charge zwitterionic anionic anionic zwitterionic zwitterionic anionic anionicCoverage 70–75% 20–25% 5–10% 50–60% 25–30% 1–5% 10–15%Bilayer formation* ! + ! + ! + +

PE, phosphatidylethanolamine; PG, Phosphatidylglycerol; CL, Cardiolipin; PC, Phosphatidylcholine; PS, Phosphatidylserine; PI, Phosphatidylinositol.*Bilayer formation designates the ability to spontaneously organize into a lipid bilayer in the correct solvent. 1van der Does et al. (2000); 2Raetz(1978); 3van Meer et al. (2008).

Table 3. Influence of structural lipids of the E. coli inner membrane andthe ER membrane on targeting and function of Sec translocon.

Structural lipids ofE. coli inner membrane

Structural lipidsof ER membrane

PE PG CL PC PE PS

Targeting ++ 6 ++5 ++4,5 n.a. +2 +7

Insertion +10 +3,5 ++3,5 n.a. ++2 n.a.Translocation ++3 ++3,6 ++4 + ++2 +7

Folding ++1,2,8,9 ++1 ++1 ++ ++2,5 n.a.

++ important; + important but incompletely investigated; n.a., noinformation available. 1Bogdanov et al. (1996); 2Vitrac et al. (2011);3van Dalen & de Kruijff (2004); 4Gold et al. (2010); 5Braig et al.(2011); 6Kusters et al. (1991); 7Yamamoto et al. (2013); 8Bogdanov &Dowhan (2012); 9Bogdanov & Dowhan (1998); 10Dowhan &Bogdanov (2009).

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 65

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

SecDFYajC

The trimeric SecDFYajC complex is a low-abundant integralmembrane protein complex that was shown to interact withthe SecYEG (Duong & Wickner, 1997b). The deletion ofSecD/SecF negatively affects bacterial growth and theirpresence stimulates protein export (Pogliano & Beckwith,1994). SecDFYajC might support the pmf-and SecA-depend-ent steps of protein transport (Duong & Wickner, 1997a;Tsukazaki et al., 2011). However, Archaea lack SecA but haveSecDF, thus their SecA-associated role is not clear (Handet al., 2006). As SecDFYajC binds to the YidC insertase,it was proposed to tether YidC to the SecYEG channel(Nouwen & Driessen, 2002). However, a recent study showedthat SecY and YidC interact even in the absence of SecDF(Sachelaru et al., 2013).

YidC

YidC is an essential membrane protein, present in Bacteria,some Archaea, mitochondria (Oxa1) and chloroplasts (Alb3,Alb4) (for review see Dalbey et al., 2011; Kudva et al., 2013).It acts as a co-insertase/chaperone supporting the integrationof membrane proteins via the Sec complex (Beck et al., 2001;Nagamori et al., 2004). YidC was recently shown to establishextensive contacts to all four TMs of the lateral gate of SecY(Sachelaru et al., 2013). Apart from that, YidC can also serveas a Sec-independent insertase for a broad range of innermembrane proteins (Chen et al., 2002; Samuelson et al., 2001;Welte et al., 2012). YidC substrates are mainly hydrophobicwithout long periplasmic stretches (Welte et al., 2012). Whiletargeting of substrates to YidC has been shown to requirethe SRP pathway (Facey et al., 2007; Welte et al., 2012),it remains to be investigated whether this is a general rule fortargeting. It also remains to be studied how YidC-mediatedinsertion of membrane proteins occurs in vivo.

PpiD and Skp

The periplasm of Gram-negative bacteria hosts a myriadof chaperones engaged in protein folding and quality control

(for review see Merdanovic et al., 2011). Two of theseperiplasmic chaperones, PpiD and Skp (seventeen-kilodalton-protein) are known to act in the immediate vicinity ofthe SecYEG translocon. PpiD and Skp are periplasmicchaperones that influence the assembly of numerous outermembrane and periplasmic proteins (Chen & Henning, 1996;Dartigalongue & Raina, 1998; Jarchow et al., 2008). Skp wasshown to interact with its substrate in the vicinity of theplasma membrane (Schafer et al., 1999) and before thepreprotein is fully translocated by the Sec complex (Harmset al., 2001). Although this suggests that Skp is in closeproximity to the Sec complex, direct evidence for aninteraction between the two is lacking. This is different forPpiD, another non-essential and membrane-anchored peri-plasmic chaperone. Cross-linking data show that PpiDestablishes extensive contacts with the lateral gate of SecY(Sachelaru et al., 2013). PpiD is thought to mediate therelease of the nascent chain from the translocon and it couldplay a role in the early folding of translocated proteins(Antonoaea et al., 2008; Matern et al., 2010).

FtsH and Syd

FtsH is an essential zinc-metalloprotease which plays a role inmembrane protein quality control in bacteria, mitochondriaand chloroplasts. It is proposed to degrade misfoldedsubstrates in an ATP-dependent fashion (Dalbey et al.,2012; Ito & Akiyama, 2005). FtsH has been shown todegrade the SecY subunit of the translocon when SecE is notpresent in stoichiometric amounts (Kihara et al., 1995).This could be mediated by the small SecY-binding cytosolicprotein Syd which might recognize the compromised status ofthe translocon (Dalal et al., 2009). FtsH has also been found inthe complex with YidC indicating that the latter mightparticipate in the quality control of transport processes(van Bloois et al., 2008).

MPiase

MPiase is a glycolipid composed of diacylglycerol and aglycan chain of three acetylated aminosugars linked through

Figure 6. Structure of SecA, the motorprotein of the post-translational transport inbacteria. (A) Schematic domain organisationof SecA (NBD, Nucleotide binding domains;PBD, peptide-cross-linking domain; HSD,helical scaffold domain; HWD, helical wingdomain; CTD, C-terminal domain). (B)Crystal structure of SecA from Thermotogamaritima (adapted from Zimmer et al.(2008); pdb: 3DIN). The colour code is thesame as in (A). (C) Crystal structure of SecAin complex with the SecYEG translocon(adapted from Zimmer et al. (2008); pdb:3DIN). The helices of the lateral gate of SecYare highlighted. This Figure is reproduced incolor in the online version of MolecularMembrane Biology.

66 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

pyrophosphate. MPiase was shown to exhibit chaperone-likeactivity driving subsequent membrane integration of sub-strates (Nishiyama et al., 2012). A role during proteintranslocation and a direct interaction with the Sec complexhas also been suggested based on the observation that thetopology inversion of SecG occurs only when MPiaseassociates with SecYEG (Moser et al., 2013).

The interaction network of eukaryotic Sec complex

The core Sec complexes in eukaryotes have additionalsubunits like Sec62 and Sec63, which are involved in post-translational protein transport. The intricate interaction net-work of the Sec61 translocon also includes proteins requiredfor energizing protein transport (BiP) and substrate foldingand modification (TRAP, TRAM, OST). A recent studyidentified also O-mannosyltransferase in complex with theSec translocon and found that mannosylation can take placeduring translocation of the substrate protein (Loibl et al.,2014). The eukaryotic translocon also establishes transientcontacts to protein kinases and protein acetylases since Sec61is a subject of co-translational and post-translational regula-tion. Sec61b, ERj1 and Sec63 have been shown to bephosphorylated by protein kinase C and casein kinase 2(Ampofo et al., 2013; Gotz et al., 2009; Gruss et al., 1999).Yeast Sec62 and Sec61b (Sbh1) appear to be co-translation-ally acetylated by NatA (Soromani et al., 2012).

Sec62/Sec63

Sec62 and Sec63 are integral ER membrane proteinsfacilitating Sec61-dependent translocation. Sec62 has twomembrane-spanning helices and a positively chargedN-terminal cytoplasmic domain (Wittke et al., 2000). Sec63belongs to the Hsp40 family of heat shock proteins andcontains a characteristic J-domain between its second andthird TM (Figure 5) (Skowronek et al., 1999). The J-domain islocated in the ER lumen where it interacts with the Hsp70-family protein BiP (Brodsky et al., 1995). The negativelycharged C-terminus of Sec63 contacts the N-terminus ofSec62 (Lang et al., 2012).

Sec62 is an essential protein involved in posttranslationaltranslocation (Deshaies & Schekman, 1989; Lang et al., 2012;Ng et al., 1996). Sec63, on the other hand, influences both,post-translational and co-translational transport (Brodskyet al., 1995; Young, 2001). For the latter, Sec63 actsindependently of Sec62 (Jermy et al., 2006; Mades et al.,2012). However, Sec63 is not essential in mammalian cells(Lang et al., 2012; Meyer et al., 2000; Tyedmers et al., 2000),since it could be functionally replaced by a similar proteinErj1 (Kroczynska et al., 2004). The mammalian homologueof Sec62 has gained a ribosome-binding site alluding to apossible contribution to co-translational transport (Mulleret al., 2010).

Sec62/Sec63 complex assembles in a 1:1 ratio with thecore translocon (Meyer et al., 2000). Yeast has two additionalsubunits, Sec71 and Sec72 (Figure 5) in complex with Sec62/Sec63 (Deshaies et al., 1991; Feldheim et al., 1993; Plathet al., 2004). Although mutations in sec71 or sec72 impairprotein transport, they are not essential and their role is notunderstood (Fang & Green, 1994).

BiP (binding immunoglobulin protein)

BiP, also known as 78 kDa glucose-regulated protein (GRP-78), heat shock 70 kDa protein 5 (HSPA5) or Kar2p in yeast,is an essential lumenal Hsp70-family chaperone. BiP hasmultiple functions during ER transport; it assists the insertionof pre-proteins into the Sec complex (Dierks et al., 1996),helps in gating the Sec complex (Alder et al., 2005; Hammanet al., 1998) and serves as a molecular ratchet duringtranslocation (Nicchitta & Blobel, 1993; Tyedmers et al.,2003). Binding of BiP to the Sec complex occurs via itsco-chaperone Sec63 (Lyman & Schekman, 1995, 1997) andErj1 (Dudek et al., 2002). In addition, BiP has multiple lipidbinding sites (Keller, 2011). Recently, the lumenal loop 7 ofSec61a was shown to contact BiP (Schauble et al., 2012).

Calmodulin

The Sec61 pore is responsible for passive Ca2+ efflux fromthe ER into the cytoplasm (Erdmann et al., 2011; Flourakiset al., 2006) and preventing Ca2+ leakage requires channelgating. The chaperone BiP has been shown to seal thelumenal opening of the Sec61a during early translocationevents (Alder et al., 2005; Hamman et al., 1998). In highereukaryotes, Ca2+ leakage is further reduced by the cytoplas-mic protein calmodulin. Calmodulin is a universal mediatorof Ca2+-controlled activity of numerous enzymes, ion chan-nels, aquaporins and other proteins (Zhou et al., 2013).Recently, a high affinity binding site for calmodulin (IQmotif) was identified on the cytosolic N-terminus of Sec61a(Erdmann et al., 2011). Ca2+-bearing calmodulin binds to thetranslocon and limits ion flux (Erdmann et al., 2011) but theefficiency of calmodulin in restricting Ca2+ leakage largelydepends on the presence of BiP (Schauble et al., 2012).Calmodulin might also have an additional role in the post-translational targeting pathway (Shao & Hegde, 2011).

TRAM (translocating chain associated membrane protein)

TRAM is a 37-kDa glycoprotein that spans the ER membraneeight times with both N- and C-termini facing the cytoplasm(Tamborero et al., 2011). TRAM was identified as a majorcross-linking partner of several secretory proteins in mam-malian cells (Gorlich et al., 1992; Krieg et al., 1989) and wasshown to be required for protein transport in reconstitutedproteoliposomes (Gorlich & Rapoport, 1993). Crosslinkingdata demonstrate that TRAM remains in contact with nascentchains even after their release from Sec61a (Liao et al., 1997;Sadlish et al., 2005). TRAM is suggested to act as a chaperoneduring the integration of less hydrophobic TM segmentsinto the bilayer (Cross & High, 2009; Heinrich et al., 2000;Shao & Hegde, 2011). It is likely that TRAM cooperates withthe Sec complex to assemble multiple TMs, a function similarto the proposed role of YidC during bacterial membraneinsertion.

TRAP (translocon associated protein complex)

TRAP is a hetero-tetrameric protein complex that bindsstoichiometrically to Sec61 (Hartmann et al., 1993; Menetretet al., 2008). TRAP associates with Sec61 and oligosaccharyltransferase (OST, see below) to form the most abundant

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 67

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

protein complexes associated with membrane-bound ribo-somes (Potter & Nicchitta, 2002). In situ cryo electrontomography studies have mapped TRAP in complex withmonomeric Sec61 (Pfeffer et al., 2012). TRAP acceleratestransport of various substrates, but its precise function isunknown (Fons et al., 2003). Recent studies have suggestedthat TRAP is involved in the topogenesis of membraneproteins, affecting the translocation of charged residues(Sommer et al., 2013).

OST (oligosaccharyl transferase)

Approximately 70% of the eukaryotic secretome is potentiallyglycosylated (Zafar et al., 2011). Errors in glycosylation lead tomisfolded proteins, giving rise to many congenital diseases(Schachter & Freeze, 2009). Asparagine-linked (N-linked)glycosylation takes place in the lumen of the ER and iscatalyzed by OST, which transfers the dolichol-linked sugarunit to the corresponding sequon in the substrate protein (Tai &Imperiali, 2001). OST is a membrane-embedded hetero-oligomeric complex with at least eight different subunits inyeast (Karaoglu et al., 1997). The catalytic subunit STT3 ishighly conserved across the species (Burda & Aebi, 1999). N-glycosylation can occur co-translationally in a supramolecularcomplex of OST, the translating ribosome and Sec61 (Haradaet al., 2009). OST binds to the ribosome near the tunnel exit(Harada et al., 2009) and to the Sec61 complex (Chavan et al.,2005; Pfeffer et al., 2014; Wang & Dobberstein, 1999).Shibatani et al. (2005) showed that mammalian OSTcomplexes also have a high affinity to TRAM.

N-linked glycosylation is one of the most common post-translational modifications in eukaryotes, but is also con-served in several prokaryotes (Aebi et al., 2013; Baker et al.,2013). The catalytic subunit of the bacterial OST is calledPglB and exhibits significant sequence similarity to theeukaryotic STT3 (Schwarz & Aebi, 2011), however a directinteraction of PglB with the bacterial SecYEG complexremains to be demonstrated.

Calnexin

Calnexin is a type I ER membrane protein that serves as aconstituent of the ER chaperone machinery for glycoproteins(Aebi et al., 2010). Calnexin can bind to its substratesboth post-translationally and co-translationally, suggesting itsproximity to the Sec61 translocon (Chen et al., 1995). A directinteraction between calnexin and the Sec61 complex wasconfirmed by two-hybrid analyses and co-immuneprecipita-tion (Boisrame et al., 2002). Recently, it was shown thatpalmitoylated calnexin is part of the ribosome-translocon

complex and makes contact to the Sec61 associated TRAPasubunit (Lakkaraju et al., 2012a). Interestingly, the calnexin-ribosome-translocon complex appears to require the actincytoskeleton for stabilization, which adds to the emergingconcept that the cytoskeleton serves as an organizer andregulator of multiple cellular processes (Jaqaman &Grinstein, 2012; Kim & Coulombe, 2010).

RAMP4 (ribosome-associated membrane protein)

RAMP4 is a 7-kDa single-spanning membrane proteinassociating with the active ribosome-Sec61 complex(Gorlich et al., 1992). In cells with high secretory activitylike hepatocytes, the unfolded protein response is induced inthe absence of RAMP4 (Hori et al., 2006). Overexpression ofRAMP4 in ER-stressed HEK293 cells supresses aggregationand degradation of newly synthesized membrane proteins(Yamaguchi et al., 1999). This indicates that RAMP4 isinvolved in membrane protein folding. RAMP4 has also beenshown to regulate N-linked glycosylation of nascent secretoryproteins (Lee et al., 2003; Schroder et al., 1999). Moreover,RAMP4 could be involved in the early sensing of a nascentchain since the eukaryotic ribosomal protein Rpl17 (E. coliL22 homologue) crosslinks to RAMP4 only if the nascent TMsegment is buried inside the ribosomal tunnel (Pool, 2009).

Erj1

ERj1 is a Sec63-related mammalian ER-membrane residentprotein belonging to the Hsp40 family (Dudek et al., 2002).Erj1 contacts the ribosomal tunnel exit and BiP in theperiplasm (Blau et al., 2005; Dudek et al., 2005), regulatingprotein translation in a BiP-dependent manner (Benedix et al.,2010; Dudek et al., 2005).

Protein targeting to the Sec complex

Signal sequences

Signal peptides determine the cellular localization of proteins(Hegde & Bernstein, 2006). Proteins that are translocatedvia the Sec complex usually possess an N-terminal signalsequence, which has three parts: a positively chargedN-terminal region, followed by a central hydrophobicH-region and a polar C-terminal region (Figure 7, (vonHeijne, 1985). In secretory proteins, the C-region containsa cleavage site for signal peptidases (von Heijne, 1984).The general architecture of signal sequences is conserved,but they are highly variable in their primary sequence andlength (von Heijne, 1986). Eukaryotic and prokaryoticsignal sequences are interchangeable (von Heijne, 1985).

Figure 7. The signal sequence. The signalpeptides of the Sec translocon susbtrates ineukaryotes and prokaryotes share a commonarchitecture, with a short, positively chargedN-terminal region (N-region), a central,hydrophobic region (H-region) and a polarC-terminal region (C-region). The best con-served part of the signal peptide is theC-region, which can also contain a signalpeptidase (SPase) cleavage site. This Figureis reproduced in color in the online version ofMolecular Membrane Biology.

68 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Several studies, however, have shown that the signal peptideis not the sole determinant for efficient targeting to the Sectranslocon. Mature alkaline phosphatase C-terminal portioncontributes to the SecA and translocon binding independentlyof the signal peptide (Gouridis et al., 2009). Furthermore,the superoxide dismutase in proteobacteria is transported tothe periplasm despite of the lack of a canonical signal peptide(Krehenbrink et al., 2011).

Most integral membrane proteins lack cleavable signalsequences and their highly hydrophobic N-terminal TMserves as a signal for recognition instead (von Heijne,1990). This TM is a stretch of about 20 mostly non-polaramino acids and is referred to as a signal anchor sequence.

Co-translational targeting via the SRP pathway

In 1970, Blobel and Sabatini proposed that the cellularlocalization of a protein is dictated by a region encoded in itsN-terminus. SRP was discovered nine years later as acytosolic agent restoring the translocation activity of high-salt washed canine pancreatic ER microsomes (Walter &Blobel, 1980; Walter et al., 1981). SRP was also shown toselectively arrest the translation of a secretory pre-proteinin vitro (Walter & Blobel, 1981). This translation elongationarrest was rescued by an ER membrane-embedded factor(Walter & Blobel, 1981), which was identified to be theSRP receptor (SR) (Gilmore et al., 1982a, 1982b). Soon after,SRP was found to bind to the N-terminus of its substrate(Kurzchalia et al., 1986). The SRP pathway is highlyconserved and essential in all organisms (Bernstein et al.,1993; Larsen & Zwieb, 1993; Poritz et al., 1988a, 1988b;Romisch et al., 1990), with the known exception of the yeastS. cerevisiae (Hann & Walter, 1991) and some Streptococcusspecies (Gutierrez et al., 1999; Hasona et al., 2005).

Initial attempts to link prokaryotic SRP to protein targetingevents were unsuccessful and it was assumed that SRP inbacteria might function as a chaperone. This assumptionwas supported by the fact that the SRP components were notidentified in the initial genetic screens for mutants impaired inprotein secretion. It became clear only later, that the E. coliSRP pathway is predominantly engaged in targeting ofmembrane proteins (de Gier et al., 1996; Luirink et al.,1994; Macfarlane & Muller, 1995) which explains why theSRP pathway was not identified in the initial genetic screensusing secretory proteins as substrates. Subsequent biochem-ical studies revealed that the E. coli SecA/SecB pathway andthe SRP pathway constitute two largely non-overlappingtargeting pathways for secretory and membrane proteins,respectively (Koch et al., 1999; Valent et al., 1998).Re-defined genetic screens then also confirmed the require-ment of the SRP pathway for membrane protein targeting(Tian & Beckwith, 2002).

Structure of the SRP

The structure of SRP varies largely across species. EukaryoticSRP is composed of six proteins (SRP9/SRP14/SRP19/SRP54/SRP68/SRP72) and the 7S RNA (Figure 4A; Walter & Blobel,1982). Bacteria have a minimal version of SRP, consistingof the protein Ffh, (Fifty-Four Homologue, due to its homologyto the eukaryotic SRP54 subunit), and 4.5S RNA (Figure 4B;

Poritz et al., 1990) both being essential for targeting. Ffh canfunctionally replace mammalian and yeast SRP54 (Bernsteinet al., 1993; Powers & Walter, 1997). Hence, the bacterial SRPrepresents the minimal functional SRP.

Ffh can be divided into two domains: The methionine-richM-domain is responsible for signal sequence binding(Lutcke et al., 1992; Zheng & Gierasch, 1997; Zopf et al.,1990), while the N-terminal NG-domain contains the GTPasecentre and provides the binding surface for the SRP receptor(Egea et al., 2004; Focia et al., 2004; Zopf et al., 1993). Thehigh methionine content in the M-domain is thought toprovide structural flexibility for accommodating signalsequences of different sizes and compositions (Bernsteinet al., 1993; Keenan et al., 1998). The 4.5S RNA interactswith both domains of Ffh. It constitutes together with theM-domain a binding groove for signal sequences, and it isrequired for stable complex formation between Ffh and SRby modulating their GTP hydrolysis activity during thetargeting cycle (Ataide et al., 2011; Jagath et al., 2001; Milleret al., 1994; Zheng & Gierasch, 1997). The presence of RNAin SRP probably alludes to its evolutionary age (Hartman &Smith, 2010). Interestingly, SRP RNA is lacking in theplastids of spermatophytes, where it has been functionallyreplaced by the protein cpSRP43 (Trager et al., 2012).In other plants, cpSRP43 and RNA subunits are both requiredfor functionality of the SRP complex (Trager et al., 2012).

Our knowledge on prokaryotic SRP is mainly basedon studies using E. coli as a model organism. However,additional SRP subunits or SRP-like GTPases have beenfound in other bacteria and Archaea (Bange & Sinning, 2013;Zwieb & Bhuiyan, 2010).

The most significant difference between the bacterialand the eukaryotic SRP is the presence of the Alu domain inthe latter. The Alu domain consists of the SRP subunits 9 and14 and domain I of 7.5S RNA (Siegel & Walter, 1985; Siegel& Walter, 1986). It has been shown to arrest translationelongation immediately after the signal sequence emergesfrom the ribosome (Ogg & Walter, 1995). SRP9 and SRP14interact with the ribosome at the interface of the small andlarge ribosomal subunits (Terzi et al., 2004). Cryo-EM studiesindicate that the Alu domain reaches into the elongation factorbinding site to compete with EF-Tu binding (Halic et al.,2004). Elongation arrest likely increases the time windowfor efficient targeting to avoid that substrates exceeding acritical length lose their translocation competence (Flanaganet al., 2003). Elongation arrest may depend on the concen-tration of free SR on the ER membrane (Lakkaraju et al.,2008). E. coli SRP does not contain the Alu domain andtherefore lacks the ability to arrest translation (Powers &Walter, 1997).

The SRP19 subunit of eukaryotic SRP mediates 7S RNAbinding to SRP54 (Egea et al., 2008; Hainzl et al., 2002;2005; Wild et al., 2001). The SRP68/72 subunits probablycooperate with SRP19 to support the last step, SRP54binding, of SRP complex assembly (Leung & Brown, 2010).

The SRP cycle

The SRP cycle (Figure 8) starts with substrate recognitionat the tunnel exit of the ribosome. SRP and ribosomes

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 69

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

establish a high-affinity contact before the substantial partof the nascent chain has emerged from the tunnel (Bornemannet al., 2008; Flanagan et al., 2003). E. coli SRP rapidly scansribosomes and its binding to the ribosomal tunnel exit is onlystabilized in the presence of a signal anchor sequence(Holtkamp et al., 2012). After the M-domain of SRP bindsthe signal sequence, the complex is targeted to the membranewhere contacts between SRP and SR are established.The NG-domains of SRa/FtsY and SRP build a pseudo-homodimer (Figure 4D) (Egea et al., 2004; Focia et al., 2004)forming a composite GTPase site. GTP binding stablilizes theinteraction between SRP and SR (Bacher et al., 1996) andinitiates the transfer of signal sequence from SRP to the Seccomplex (Rapiejko & Gilmore, 1997). The Sec transloconregulates conformational changes in the compound SRP-SRcomplex and activates GTP hydrolysis (Akopian et al.,2013a). SRP and FtsY stimulate GTP hydrolysis reciprocally(Powers & Walter, 1997). Released SRP then cycles backto the cytosol to start a new targeting reaction. For recentreviews on the SRP cycle see Leung & Brown (2010),Akopian et al. (2013b), Kudva et al. (2013), Nyathi et al.(2013), and Kuhn et al. (2014).

Eukaryotic SRP recognizes both secretory pre-proteinsand membrane proteins, while E. coli SRP has a higheraffinity for hydrophobic signal anchor sequences (Beck et al.,2000; Lee & Bernstein, 2001; Valent et al., 1995). Yet E. coliSRP is also required for the targeting of a few periplasmicproteins, with cleavable but unusually hydrophobic signalsequences (Huber et al., 2005). This probably explains whyE. coli SRP was also found to bind to the signal sequence ofthe eukaryotic secretory protein preprolactin (Luirink et al.,1992) and why E. coli Ffh can replace SRP54 in eukaryotes(Bernstein et al., 1993; Powers & Walter, 1997). How SRPselects its substrates is still not entirely clear, but it is thoughtto be mainly influenced by the hydrophobicity of the substrateprotein (Beck et al., 2000; Neumann-Haefelin et al., 2000;

Ng et al., 1996; Valent et al., 1998) and by the absence ofhelix breaking amino acids (Beha et al., 2003). Additionally,translational speed and specific proofreading steps whichcontrol the GTPase cycle of the SRP-SR targeting complexprobably contribute to substrate selection (Zhang et al.,2009a, 2010).

SRP-independent targeting to the Sec complex

In vitro studies performed with E. coli inverted membranevesicles have determined that SecA, SecB and the pmfare necessary and sufficient for the translocation of Sec-dependent secretory proteins, i.e. proteins of the periplasmicspace or the outer membrane (Brundage et al., 1990;Cunningham et al., 1989; Koch et al., 1999). SecA is onlyfound in Bacteria and SecB in Proteobacteria (Muller et al.,2001). How Archaea have compensated for the loss of SecAis not known, but in some species the Sec-independentpost-translational Tat (twin-arginine translocation) pathwaymight be responsible for the export of many secretory proteins(Palmer & Berks, 2012).

Also eukaryotic cells employ SRP-independent pathwaysfor secretory and membrane proteins. These alternativepathways initially seemed to be required in S. cerevisiaewhere the SRP-pathway is inessential (Hann & Walter, 1991).However, it is becoming increasingly apparent that post-translational transport across the ER membrane is utilizedby a significant number of proteins in all eukaryotes.One SRP-independent pathway in eukaryotes is mediated bythe ER-embedded Sec62/Sec63 proteins, which cooperatewith Sec61. Another known pathway is the Get3/TRC40pathway, which transports membrane proteins withC-terminal transmembrane domains (tail-anchored [TA] pro-teins). Although, SRP was found to interact with modelTA proteins (Abell et al., 2004; Leznicki et al., 2010), it isgenerally assumed that the GET3/TRC40 pathway does not

Figure 8. Schematic view of the SRP-SRcycle. The model was adapted from (Akopianet al., 2013b). (1) SRP rapidly scans ribo-somes and binds stably only to thosetranslating a SRP substrate (2) The presenceof the correct substrate increases the affinityof SRP to its receptor (SR), resulting intargeting of the ribosome nascent chain(RNC) to the membrane. This conformationof the SRP-SR complex is termed the earlycomplex. In bacteria, the SRP-SR affinity isfurther increased if SR is in contact withnegatively charged phospholipids. (3) In thepresence of the correct substrate, the GTPasedomains of SRP and SR align to form acomposite GTPase centre (closed complex).To prevent premature dissociation of theSRP-SR complex, GTP hydrolysis is delayeduntil contacts to the Sec translocon areestablished. (4) GTP hydrolysis induces thedissociation of SRP-SR complex. SRP-SRGTPases do not require GTP-activating pro-teins or nucleotide-exchange factors. ForSRP, the contact with the ribosome might besufficient to exchange GDP against GTP.This Figure is reproduced in color in theonline version of Molecular MembraneBiology.

70 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

involve the Sec complex (Denic et al., 2013; Hegde &Keenan, 2011).

Bacterial SecA/SecB pathway

Secretory pre-proteins in E. coli are targeted to the transloconby the 100 kDa-ATPase SecA, which in some cases is assistedby the cytosolic chaperone SecB (Figure 1A) (Driessen &Nouwen, 2008; Kudva et al., 2013; Park & Rapoport, 2012).SecA binds to signal sequences (Akita et al., 1990;Karamyshev & Johnson, 2005), and is therefore required forthe targeting of pre-proteins to the membrane (Gelis et al.,2007; Lill et al., 1990; Randall & Hardy, 1977). However,it is not clear where the signal sequence binding occurs.The classical model suggests that it takes place after thepre-protein is targeted to the membrane by SecB and otherchaperones (Fekkes et al., 1998; Randall et al., 1998). Recentevidence for SecA-ribosome binding indicates however thatSecA may also recognize its substrates co-translationally(Huber et al., 2011).

SecB is a 17 kDa cytosolic protein that forms ahomotetramer (Xu et al., 2000) and is thought to hold thesecretory pre-protein in an unfolded state (Randall et al.,1998; Watanabe & Blobel, 1989). Its role in pre-proteintargeting is disputed since SecB does not bind the signalsequence specifically, but rather has a general affinity tohydrophobic stretches (Knoblauch et al., 1999). Furthermore,SecB is not essential suggesting that protein transport is notstrictly dependent on it (de Cook & Tommassen, 1991; vander Sluis & Driessen, 2006). The number of E. coli proteinsfor which a clear SecB dependency during transport has beenshown is rather low (520), suggesting that it is dispensableduring transport of most E. coli proteins.

In vitro studies have identified another chaperone, TriggerFactor (TF), as one of earliest and major contact partners of anascent secretory protein (Beck et al., 2000; Deuerling et al.,2003; Valent et al., 1995). However, recent in vivo evidencefrom ribosome profiling studies suggests that TF mainly bindsto substrates after they have reached a length of more than100 residues (Oh et al., 2011). TF preferentially associateswith ribosomes engaged in translation of secretory proteins(Oh et al., 2011). Ribosome-binding is required for theactivity of TF which is to keep the nascent polypeptide ina translocation-competent loosely folded shape (Hoffmannet al., 2012). We suggest a recent review by Castanie-Cornetet al. (2013) for more details on the chaperone networkfacilitating protein targeting in E. coli.

Eukaryotic Sec62/Sec63 pathway

Yeast cells lacking SRP suffer from severe growth defects(Hann & Walter, 1991), but their translocation abilityrecovers over time (Ogg et al., 1992). This indicates theexistence of alternative SRP-independent protein targetingpathways. Attempts to identify these salvage pathways soonled to the discovery of SEC62/SEC63 alleles since theirmutations caused secretion defects of SRP-independentpre-proteins (Deshaies & Schekman, 1989; Ng et al., 1996).In fact, a large-scale analysis of yeast S. cerevisiae signalsequences predicts that approx. 43% of its secretome wouldutilize an SRP-independent pathway (Ast et al., 2013). It is

important to emphasize that post-translational transport stillrequires the Sec61 channel and BiP (Matlack et al., 1999;Panzner et al., 1995).

Although Sec62 has been mainly associated with post-translational translocation, Sec63 might also contribute toco-translational transport (Deshaies & Schekman, 1989; Nget al., 1996). Nevertheless, the precise function of bothproteins remains elusive. Sec63 is thought to affect the gatingof the Sec61 translocon in a precursor dependent fashion(Lang et al., 2012). Furthermore, overexpression of Sec63leads to a considerable decrease in the steady-state levelsof multi-spanning membrane proteins (Mades et al., 2012),indicating that Sec63 might also have a regulatory function.

The C-terminus of yeast Sec62 was found in closeproximity to the signal sequence of prepro-alpha-factorin vivo (Dunnwald et al., 1999). This was confirmed bycross-linking experiments, which indicate that the signalsequence establishes contacts with both Sec61 and Sec62(Plath et al., 2004). Further systematic studies of substrateswith different signal sequences indicate that Sec62 is indeedinvolved in recognition of moderately hydrophobic signalsequences (Reithinger et al., 2013).

The transport of two discrete sets of proteins has beenshown to be dependent on Sec62: the translocation of smallproteins (100–160 residues) (Lakkaraju et al., 2012b) and thetransport of GPI-anchored cell surface proteins (Ast et al.,2013). Small proteins, including hormones, chemokines, andantimicrobial peptides are abundant in metazoa (Ingolia et al.,2009). Why small proteins escape SRP pathway is not clearbut they may simply be too short for efficient co-translationalrecognition. GPI-anchored proteins carry an N-terminalsignal sequence and a characteristic C-terminal GPI signaturesequence, which is potentially recognized by the Get3/TRC40pathway (Ast et al., 2013). Both motifs are required forefficient translocation, revealing the interplay between twodifferent targeting systems (Ast et al., 2013).

It is not entirely clear how substrates are routed to thepost-translational targeting pathway in eukaryotes. Some, likeprepro-alpha factor appear to engage both SRP-dependentand -independent targeting pathways in yeast, but areexclusively co-translationally targeted by SRP when analyzedwith mammalian ER membranes (Garcia & Walter, 1988; Nget al., 1996). Pathway selection is not only determined by thesignal sequence or the length of a preprotein, but additionalfeatures within the mature domain are also important(Johnson et al., 2013; Shao & Hegde, 2011). A recent studyalso suggests that SRP and Sec62-mediated targeting mightnot be mutually exclusive (Reithinger et al., 2013).

What are the cytosolic targeting factors for Sec62/Sec63pathway remains to be elucidated. The ATP-dependentHsp70/Hsp90 chaperone network has been shown to bindpost-translationally transported proteins (Ast et al., 2013;Chirico et al., 1988; Deshaies et al., 1988). Surprisingly, theCa2+-binding protein calmodulin binds selectively to thehydrophobic signal sequences of short precursors in a Ca2+-dependent fashion (Shao & Hegde, 2011). Like SRP,calmodulin contains a methionine-rich domain responsiblefor recognizing a diverse set of hydrophobic targets (O’Neil &DeGrado, 1990). Calmodulin maintains the translocationcompetence of the small proteins protecting them from

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 71

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

ubiquitination (Shao & Hegde, 2011). How calmodulinreleases its substrate is not known, but it was shown tointeract with Sec61 (Erdmann et al., 2011). The frequencyand significance of calmodulin-mediated protein targetingneeds to be further studied.

Translation-independent targeting

Asymmetric mRNA localization is a tool to determine thelocation of protein synthesis in eukaryotes. Developmentalabnormalities and neuronal disorders are caused by mRNAtrafficking defects (Xing & Bassell, 2013). mRNA transport isa common paradigm in polarized cells but recent evidenceindicates that ER localization of proteins might also bedetermined by ribosome-free mRNA targeting (Hermesh &Jansen, 2013). The ER acts as a major site for proteinsynthesis in eukaryotes (Reid & Nicchitta, 2012; Stephenset al., 2005). The translation of cytosolic and nuclear mRNAscan be initiated by ER-bound ribosomes but the lack of asignal sequence induces ribosome detachment from the ERmembrane (Potter & Nicchitta, 2000). It has also been shownthat the large ribosomal subunit remains at the membraneafter synthesis of a secretory or a membrane protein isterminated (Potter & Nicchitta, 2000). This probably reducesthe need for SRP-dependent targeting of RNCs. Targeting ofmRNAs to the ER membrane in S. cerevisiae occursindependently of translation and SRP (Kraut-Cohen et al.,2013). Recent studies have revealed that prokaryotic mRNAsalso tend to localize at the place where their correspondingprotein products execute their function (Broude, 2011;Montero Llopis et al., 2010; Nevo-Dinur et al., 2011;Valencia-Burton et al., 2007).

What promotes ribosome-independent mRNA localizationto the ER or the bacterial inner membrane is not known.Both the involvement of the cytoskeleton (Czaplinski &Singer, 2006; Palacios, 2007) and mRNA-binding proteins(to facilitate diffusion) have been suggested (Hermesh &Jansen, 2013). It is also not clear on what are the targetingdecisions based. It is proposed that cis-acting sequenceswithin mRNAs may serve as targeting signals. Membrane-spanning domains are encoded by the codons showing astrong uracil bias (Prilusky & Bibi, 2009). Indeed, theU-enriched stretches of the transcript are required to localizemRNA properly (Bibi, 2012; Kraut-Cohen & Gerst, 2010).

The Sec61 complex is probably the main ribosomedocking site in eukaryotes, because after Sec61 depletion60% less ER-bound polysomes have been observed (Langet al., 2012). The relative amount of membrane-boundribosomes is thought to be much lower in bacteria(Herskovits & Bibi, 2000; Randall & Hardy, 1977) and theclassical model suggests that ribosomes detach from themembrane after synthesis and insertion of membrane proteinsis completed. However, even non-translating ribosomesexhibit a significant affinity for the Sec translocon in E.coli (Prinz et al., 2000; Schaletzky & Rapoport, 2006).Membrane-bound ribosomes could re-initiate the translationof SRP substrates and thus would not depend on cytosolicSRP for targeting. In support of this, a recent study has shownthat membrane-anchored SRP maintains its targeting activity(Braig et al., 2011), indicating that recognition of signal

sequences does not necessarily need to occur in the cytosol.Furthermore, it was suggested that FtsY, the bacterial SRPreceptor, is co-translationally targeted to the bacterial mem-brane by an SRP-independent mechanism (Herskovits et al.,2002). This would constantly replenish the membrane-boundribosome pool (Bibi, 2011). However, this model requiresfurther experimental verification.

Mechanisms of SecY/Sec61 mediatedco-translational and post-translationalprotein transport

The Sec complex constitutes a passive pore that needs to beactivated by an interaction with its partner proteins (Table 1).Although the function of some of these proteins is known,in most cases it is still unclear how the biological functionof these proteins is coordinated with the transport activity ofthe Sec complex.

A single copy of SecY/Sec61 is sufficient for proteintransport

The oligomeric state of the Sec complex in its resting andactive state has been a matter of intense debate. Depending onexperimental procedures and the expression level of the Seccomplex, monomeric, dimeric, tetrameric and higher oligo-mers of SecY and Sec61 complexes have been found (see Park& Rapoport, 2012; Kudva et al., 2013 for recent reviews).Although there appears to be now a general agreement that asingle copy of the Sec complex is sufficient for proteintranslocation and insertion, pore sizes not compatible with asingle SecY copy were detected by fluorescence quenchingexperiments (Hamman et al., 1997) and electrophysiologymeasurements (Wirth et al., 2003). Thus, issues about theoligomeric state of the Sec complex and in particular theinfluence of substrates or Sec accessory proteins need to beresolved.

The active Sec complex during post-translationalprotein translocation

Translocation of a secretory protein through the Sec complexcommences with loop-like insertion of the substrate into thechannel. The signal sequence remains in contact with thechannel wall, while the downstream segment resides inthe pore (Plath et al., 1998; Shaw et al., 1988). Substrateinsertion requires an open channel, and in the bacterial Seccomplex, this channel opening might be induced upon bindingof SecA (Zimmer et al., 2008). The crystal structure of theSecA-bound Sec complex shows a partially opened lateralgate and a displaced plug domain, although the latter appearsto still seal the channel (Zimmer et al., 2008). Signalsequences have been cross-linked to both TMs 2b and 7 ofSec61 channel (Plath et al., 1998) and to phospholipids(Martoglio et al., 1995). This probably indicates that afterinitial SecA-dependent weakening of the lateral gate itfurther opens by the intercalation of the signal sequence.This is supported by the data with 2D crystals of the SecYEGcomplex in contact with a preprotein mimic (Hizlan et al.,2012). These data show that TM2b is tilted away fromthe lateral gate and that TM7 is relocated closer to TM5 andTM10 at the back of the channel. This movement displaces

72 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

the plug even further and brings it closer to TM3 of SecE.The movement of TM7 as observed in the pre-proteinactivated 2D (Hizlan et al., 2012) structure has not beenobserved in the SecA-activated SecYE structure (Zimmeret al., 2008), and therefore both structures probably representconsecutive steps of the activation of the Sec complex prior totranslocation. Recent cryo-EM reconstitution indicates thatthe plug does not shift prominently in the translocatingSec61-ribosome complex; rather the TM10 moves outward,disrupting the central constriction pore (Gogala et al., 2014).The observed conformational changes explain the phenotypeof several prl mutations, which allow the translocation ofsubstrates with defective or missing signal sequences. Manyof the prl mutations were mapped to TM2a (plug), TM7 andTM10 (Osborne & Silhavy, 1993; Smith et al., 2005). In thesemutants, lateral gate opening and plug movement is probablylargely signal sequence independent.

Substrates are translocated through the channel in discretesteps of approx. 30–40 amino acids (Schiebel et al., 1991;Tomkiewicz et al., 2006) and each step is linked to ATPhydrolysis by SecA. How ATP binding and hydrolysis drivetranslocation of the substrate across the channel, is stillunknown. One model predicts that two short a-helices ofSecA, the two-helix finger, inserts into the channel upon ATPbinding. After ATP hydrolysis, the two helix finger wouldreset and insert again upon binding the next ATP, therebyproviding the mechanical force for the step-wise translocation(Erlandson et al., 2008). Although highly attractive, thismodel is questioned by the observation that translocationproceeds even when the two-helix finger is trapped in placeby chemical cross-linking (Whitehouse et al., 2012).Alternative models have been proposed (Tomkiewicz et al.,2007), but none of them is currently fully supported by theexperimental data.

Post-translational transport across the bacterial SecYEGcomplex is also stimulated by the proton motive force (pmf)as first described by (Driessen & Wickner, 1991). Their studyalso showed that both components of the pmf, i.e. theelectrochemical gradient (D ) and proton gradient (DpH) arerequired for stimulation. Pmf reduces the amount of ATPrequired for the in vitro translocation of secretory proteins(Shiozuka et al., 1990) and is thought to act at a late stage oftranslocation (Schiebel et al., 1991). On the other hand, pmfwas also proposed to help orienting the signal sequencewithin the Sec channel, which occurs at an early phase oftranslocation (Nouwen et al., 1996). Conformational changestriggered by the proton transfer could allow SecDFYajC topull out substrates from the periplasmic side of the Secchannel (Tsukazaki et al., 2011). A similar functionwas also proposed for periplasmic chaperones like Skp(Schafer et al., 1999) or PpiD (Antonoaea et al., 2008).However, it is unknown whether this function of Skp or PpiDis linked to the pmf.

In eukaryotes, the pre-proteins are transported to thetranslocon with the help of cytosolic chaperones of the Hsp90,Hsp70 and Hsp40 families (Chirico et al., 1988; Zimmermannet al., 1988). These chaperones keep pre-proteins competentfor interaction with the Sec complex. During translocation,the Sec61 core associates with the Sec62/Sec63 complex,which in yeast involves also Sec71 and Sec72 (reviewed in

Zimmermann et al., 2011). The cytosolic domains of thesemembrane proteins might interact with the chaperones, butthis needs to be experimentally verified. Post-translationaltranslocation in eukaryotes also requires energy, but unlikebacterial SecA, the Hsp70-type chaperone BiP (called Kar2pin yeast) binds to the incoming substrates in the ER lumen andprevents their back-sliding by ATP hydrolysis. This mechan-ism is referred to as Brownian ratchet because it confersdirectionality to the otherwise undirected Brownian motion.The Brownian ratchet model is also proposed as a possiblemode of action for for SecA (Tomkiewicz et al., 2007).

The binding of the J-domain of Sec63 or ERj1 stimulatesthe ATP hydrolysis by BiP and induces the closure of itssubstrate binding pocket thereby trapping the translocatedpre-protein (for review, see Park & Rapoport, 2012). Once thenext segment of the translocated polypeptide emerges fromthe Sec channel, the next BiP molecule binds. Release of BiPfrom its substrate requires nucleotide exchange factors likeSil1p or Lhs1p (Tyson & Stirling, 2000).

The active Sec complex during co-translationalprotein transport

After SRP-mediated membrane targeting the ribosome docksonto the cytosolic loops C4 and C5 of SecY/Sec61a and thecytoplasmic helix of SecE/Sec61g (Becker et al., 2009; Chenget al., 2005; Frauenfeld et al., 2011). This involves conservedbinding sites within the rRNA and universal ribosome adaptorsite (Behrens et al., 2013; Frauenfeld et al., 2011; Prinz et al.,2000). Cryo-EM structures do not reveal any significantconformational changes within Sec complex upon ribosomebinding (Becker et al., 2009; Gogala et al., 2014; Park et al.,2014), although electrophysiology experiments show thatribosomes induce channel opening of the SecYEG translocon(Knyazev et al., 2013). The Cryo-EM structures of an RNC-bound Sec complex on the other hand, display a partiallyopened lateral gate (Frauenfeld et al., 2011; Gogala et al.,2014; Park et al., 2014), similarly to the partially openedSecA-SecY complex (Zimmer et al., 2008). The structureof a SecYE complex form Pyrococcus furiosus was solvedwith a part of a juxtaposed SecYE molecule inserted intothe channel (Egea & Stroud, 2010). This second copy issuggested to mimic a polypeptide in transit and in agreementwith this assumption; the structure shows a wider openingof the lateral gate. However, the plug was not displacedand was still occupying the central restriction (Egea &Stroud, 2010).

A recent study showed that upon binding of RNCs bearinga DsbA signal sequence to the Sec translocon (Park et al.,2014), the lateral gate opens and the conversion from theclosed to the open structure appears to involve mainly rigidbody movements of the N-terminal half of SecY. Additionalchanges are observed for TM7 and the cytoplasmic part ofTM8, which is displaced towards the membrane surface.These movements of SecY are accompanied by movements ofSecE and SecG (Park et al., 2014). The plug only slightlychanges its position, but instead movements of the lateralgate helices generate a cavity adjacent to the plug domainthat is confined by the signal sequence intercalated in theopen lateral gate.

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 73

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

The portioning of TMs from the Sec channel into the lipidphase is mainly determined by the hydrophobicity of theTM (Hessa et al., 2005) and follows the ‘‘positive-insiderule’’ (von Heijne & Gavel, 1988). The Sec translocon canactively discriminate between TMs and less hydrophobicstretches (Hessa et al., 2005). The hydrophobicity thresholdfor integration is partly controlled by the central constrictionof Sec61 pore (Demirci et al., 2013) and this threshold seemsto be lower in prokaryotes (Ojemalm et al., 2013).

It has been recently shown that the Sec complex exerts abiphasic pulling force on a nascent chain (Ismail et al., 2012).It is not entirely clear where the TMs are exactly positionedduring each phase, but the data are consistent with asequential and ordered integration pathway as suggestedalso by (Houben et al., 2005; Sadlish et al., 2005). However,this linear membrane integration might not apply for everymulti-spanning membrane protein. The translocon offers aflexible environment for membrane protein biogenesis as itcan specifically delay the integration of selected TMs(Cross & High, 2009; Ismail et al., 2008) and reorient theothers (Goder et al., 1999). The insertion of less hydrophobicTMs can be assisted by the neighboring more hydrophobicTM (Heinrich & Rapoport, 2003; Ojemalm et al., 2012).En bloc insertion has been shown by FRET analyses tooccur pairwise (Hou et al., 2012) and several TMs can bemaintained by the translocon simultaneously (Enquist et al.,2009; Sadlish et al., 2005).

It is not yet clear to which extent translocon-associatedproteins affect the final topology of membrane proteins.Although SecYEG proteoliposomes together with SRP andFtsY are sufficient for membrane protein insertion in vitro(Braig et al., 2009; Welte et al., 2012), several studies haveshown that TMs exiting the Sec complex contact YidC (Becket al., 2001; Urbanus et al., 2001). The lateral gate of SecYitself also establishes contacts to the YidC (Sachelaru et al.,2013). Intriguingly, in vitro experiments demonstrated that theSecY-YidC interaction is influenced by the presence of RNCs(Sachelaru et al., 2013). It appears likely that that thesubstrate-induced movements of the lateral gate (Park et al.,2014) directly affect the SecY-YidC interface. In eukaryotes,TRAM and TRAP are shown to facilitate membrane proteininsertion (Hegde et al., 1998; Saurı et al., 2007).

To support the biogenesis of membrane proteins with largeperiplasmic portions, the Sec-translocon must operate in acomposite insertion-translocation mode. SecA is required tosupport the translocation of periplasmic domains longer than30 amino acids (Deitermann et al., 2005). As the ribosomeand SecA have overlapping binding sites on SecYEG (Kuhnet al, 2011) and as they compete with each other for SecYEGbinding (Wu et al., 2012), it remains open how thesimultaneous activity of ribosome and SecA is coordinated.The corresponding process in eukaryotes could be mediatedby BiP, although this needs to be verified experimentally.

Conclusion and perspectives

The biochemical, biophysical and structural characterizationof the Sec-dependent transport over the last decades hasprovided a detailed picture of this highly conserved andessential cellular process. A key conclusion is that protein

transport machineries are modular and vastly dynamic innature. This is exemplified by the observation that theSec translocon cooperates with the seemingly independenttwin-arginine translocation machinery during the insertionof a membrane protein (Keller et al., 2012). Revealing thecoordinated interaction of the Sec translocon with its multiplepartner proteins in response to substrate, will be an importantnext step in understanding the molecular mechanism ofprotein transport.

There are probably yet undiscovered cytosolic and mem-brane-bound partner proteins that support efficient transportvia the Sec complexes in living cells. For instance, qualitycontrol is an important aspect in biological processes, butlittle is known on how the Sec translocon cooperates withproteases and chaperones for facilitating proper foldingand eventually the removal of misfolded or mistargetedproteins.

SecA and SRP have been the subjects of extensiveresearch. However, it needs to be elucidated how SecAlinks ATP-hydrolysis to polypeptide movement and how theproton motif force and the SecDFYajC complex contributeto translocation. Furthermore, the mechanism of SecA-dependent signal recognition and targeting is not entirelyclear. The SecYEG independent insertion of membraneproteins via YidC raises the question of how SRP routescertain substrates into the YidC pathway instead of theSecYEG pathway.

Our knowledge on protein targeting is based on thedetailed analyses of only a few substrates. A more compre-hensive catalogue of validated substrates would reveal howthey are specifically recognized and what determinestheir post-translational or co-translational targeting mode.In particular, the role of the translation speed in pathwayselection requires further analysis. Likewise, the contributionof translation-independent targeting to the Sec complexes is alargely unexplored and exciting area of research.

Although a couple of high-resolution structures haveilluminated intricate details of protein transport, additionalstructures are needed, i.e. structures of the Sec complextranslocating a polypeptide, structures of the eukaryotic Seccomplex and structures of the Sec translocon associatedwith FtsY, YidC, TRAM to name only a few. Cryo Electrontomography and other advanced microscopy techniquespromise to provide in-depth analyses of protein transport atthe cellular level.

The exciting developments during the last years haveallowed a deeper insight into the molecular mechanisms thatsustain protein transport via the Sec translocon. Nevertheless,the enormous complexity of this system requires furtherexploration.

Declaration of interest

The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of the paper.

This works was supported by grants of the DeutscheForschungsgemeinschaft (DFG) (FOR929, FOR967 andGRK1478); and the Excellence Initiative grant of theGerman Federal and State Governments – GSC-4 SpemannGraduate School of Biology and Medicine to HGK. NAP was

74 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

supported by a PhD fellowship of the German AcademicExchange Service (DAAD).

References

Abell BM, Pool MR, Schlenker O, Sinning I, High S. 2004. Signalrecognition particle mediates post-translational targeting in eukary-otes. EMBO J 23:2755–2764.

Aebi M, Bernasconi R, Clerc S, Molinari M. 2010. N-glycan structures:Recognition and processing in the ER. Trends Biochem Sci 35:74–82.

Aebi M, Schuldiner M, Schwappach B. 2013. N-linked proteinglycosylation in the ER. Biochim Biophys Acta – Mol Cell Res1833:2430–2437.

Akita M, Sasaki S, Matsuyama S, Mizushima S. 1990. SecA interactswith secretory proteins by recognizing the positive charge at theamino terminus of the signal peptide in Escherichia coli. J Biol Chem265:8164–8169.

Akopian D, Dalal K, Shen K, Duong F, Shan S-o. 2013a. SecYEGactivates GTPases to drive the completion of cotranslational proteintargeting. J Cell Biol 200:397–405.

Akopian D, Shen K, Zhang X, Shan S-o. 2013b. Signal recognitionparticle: An essential protein-targeting machine. Annu Rev Biochem82:693–721.

Alami M, Dalal K, Lelj-Garolla B, Sligar SG, Duong F. 2007. Nanodiscsunravel the interaction between the SecYEG channel and its cytosolicpartner SecA. EMBO J 26:1995–2004.

Albiniak AM, Baglieri J, Robinson C. 2012. Targeting of lumenalproteins across the thylakoid membrane. J Experim Bot 63:1689–1698.

Alder NN, Shen Y, Brodsky JL, Hendershot LM, Johnson AE. 2005. Themolecular mechanisms underlying BiP-mediated gating of the Sec61translocon of the endoplasmic reticulum. J Cell Biol 168:389–399.

Ampofo E, Welker S, Jung M, Muller L, Greiner M, Zimmermann R,Montenarh M. 2013. CK2 phosphorylation of human Sec63 regulatesits interaction with Sec62. Biochim Biophys Acta – Gen Subj 1830:2938–2945.

Angelini S, Boy D, Schiltz E, Koch HG. 2006. Membrane binding of thebacterial signal recognition particle receptor involves two distinctbinding sites. J Cell Biol 174:715–724.

Angelini S, Deitermann S, Koch HG. 2005. FtsY, the bacterial signal-recognition particle receptor, interacts functionally and physicallywith the SecYEG translocon. EMBO Rep 6:476–481.

Antonin W, Meyer HA, Hartmann E. 2000. Interactions between Spc2pand other components of the endoplasmic reticulum translocationsites of the yeast Saccharomyces cerevisiae. J Biol Chem 275:34068–34072.

Antonoaea R, Furst M, Nishiyama K-i, Muller M. 2008. The periplasmicchaperone PpiD interacts with secretory proteins exiting from theSecYEG translocon. Biochemistry 47:5649–5656.

Ast T, Cohen G, Schuldiner M. 2013. A network of cytosolic factorstargets SRP-independent proteins to the endoplasmic reticulum.Cell 152:1134–1145.

Ataide SF, Schmitz N, Shen K, Ke A, Shan SO, Doudna JA, Ban N.2011. The crystal structure of the signal recognition particle incomplex with its receptor. Science 331:881–886.

Auclair SM, Bhanu MK, Kendall DA. 2012. Signal peptidase I: Cleavingthe way to mature proteins. Protein Sci 21:13–25.

Bacher G, Lutcke H, Jungnickel B, Rapoport TA, Dobberstein B. 1996.Regulation by the ribosome of the GTPase of the signal-recognitionparticle during protein targeting. Nature 381:248–251.

Bahari L, Parlitz R, Eitan A, Stjepanovic G, Bochkareva ES, Sinning I,Bibi E. 2007. Membrane Targeting of ribosomes and their releaserequire distinct and separable functions of FtsY. J Biol Chem 282:32168–32175.

Baker JL, Celik E, DeLisa MP. 2013. Expanding the glycoengineeringtoolbox: The rise of bacterial N-linked protein glycosylation. TrendsBiotechnol 31:313–323.

Bange G, Sinning I. 2013. SIMIBI twins in protein targeting andlocalization. Nat Struct Mol Biol 20:776–780.

Baram D, Pyetan E, Sittner A, Auerbach-Nevo T, Bashan A, Yonath A.2005. Structure of trigger factor binding domain in biologicallyhomologous complex with eubacterial ribosome reveals its chaperoneaction. Proc Natl Acad Sci USA 102:12017–12022.

Beck K, Eisner G, Trescher D, Dalbey RE, Brunner J, Muller M,Muller M. 2001. YidC, an assembly site for polytopic Escherichia coli

membrane proteins located in immediate proximity to the SecYEtranslocon and lipids. EMBO Rep 2:709–714.

Beck K, Wu L-F, Brunner J, Muller M. 2000. Discrimination betweenSRP- and SecA/SecB-dependent substrates involves selective recog-nition of nascent chains by SRP and trigger factor. EMBO J 19:134–143.

Becker T, Bhushan S, Jarasch A, Armache J-P, Funes S, Jossinet F, et al.2009. Structure of monomeric yeast and mammalian Sec61complexes interacting with the translating ribosome. Science 326:1369–1373.

Beha D, Deitermann S, Muller M, Koch HG. 2003. Export of beta-lactamase is independent of the signal recognition particle. J BiolChem 278:22161–22167.

Behrens C, Hartmann E, Kalies KU. 2013. Single rRNA helices bindindependently to the protein-conducting channel SecYEG. Traffic 14:274–281.

Benedix J, Lajoie P, Jaiswal H, Burgard C, Greiner M, Zimmermann R,et al. 2010. BiP modulates the affinity of its co-chaperone ERj1 forribosomes. J Biol Chem 285:36427–36433.

Berndt U, Oellerer S, Zhang Y, Johnson AE, Rospert S. 2009. A signal-anchor sequence stimulates signal recognition particle binding toribosomes from inside the exit tunnel. Proc Natl Acad Sci USA 106:1398–1403.

Bernstein HD, Zopf D, Freymann DM, Walter P. 1993. Functionalsubstitution of the signal recognition particle 54-kDa subunit by itsEscherichia coli homolog. Proc Natl Acad Sci USA 90:5229–5233.

Bibi E. 2011. Early targeting events during membrane protein biogenesisin Escherichia coli. Biochim Biophys Acta – Biomembr 1808:841–850.

Bibi E. 2012. Is there a twist in the Escherichia coli signal recognitionparticle pathway? Trends Biochem Sci 37:1–6.

Bieker KL, Silhavy TJ. 1990. The genetics of protein secretion in E. coli.Trends Genet 6:329–334.

Bingel-Erlenmeyer R, Kohler R, Kramer G, Sandikci A, Antolic S,Maier T, et al. 2008. A peptide deformylase-ribosome complex revealsmechanism of nascent chain processing. Nature 452:108–111.

Blau M, Mullapudi S, Becker T, Dudek J, Zimmermann R, Penczek PA,Beckmann R. 2005. ERj1p uses a universal ribosomal adaptor site tocoordinate the 80S ribosome at the membrane. Nat Struct Mol Biol12:1015–1016.

Blobel G, Sabatini DD. 1970. Controlled proteolysis of nascentpolypeptides in rat liver cell fractions. I. Location of the polypeptideswithin ribosomes. J Cell Biol 45:130–145.

Bogdanov M, Dowhan W. 1998. Phospholipid-assisted protein folding:Phosphatidylethanolamine is required at a late step of the conform-ational maturation of the polytopic membrane protein lactosepermease. EMBO J 17:5255–5264.

Bogdanov M, Dowhan W. 2012. Lipid-dependent generation of dualtopology for a membrane protein. J Biol Chem 287:37939–37948.

Bogdanov M, Sun J, Kaback HR, Dowhan W. 1996. A phospholipid actsas a chaperone in assembly of a membrane transport protein. J BiolChem 271:11615–11618.

Boisrame A, Chasles M, Babour A, Beckerich J-M, Gaillardin C. 2002.Sbh1p, a subunit of the Sec61 translocon, interacts with the chaperonecalnexin in the yeast Yarrowia lipolytica. J Cell Sci 115:4947–4956.

Bornemann T, Jockel J, Rodnina MV, Wintermeyer W. 2008. Signalsequence-independent membrane targeting of ribosomes containingshort nascent peptides within the exit tunnel. Nat Struct Mol Biol 15:494–499.

Braig D, Bar C, Thumfart J-O, Koch HG. 2009. Two cooperating helicesconstitute the lipid-binding domain of the bacterial SRP receptor.J Mol Biol 390:401–413.

Braig D, Mircheva M, Sachelaru I, van der Sluis EO, Sturm L,Beckmann R, Koch HG. 2011. Signal sequence-independent SRP-SRcomplex formation at the membrane suggests an alternative targetingpathway within the SRP cycle. Mol Cell Biol 22:2309–2323.

Brodsky JL, Goeckeler J, Schekman R. 1995. BiP and Sec63p arerequired for both co- and posttranslational protein translocation intothe yeast endoplasmic reticulum. Proc Natl Acad Sci USA 92:9643–9646.

Broude NE. 2011. Analysis of RNA localization and metabolismin single live bacterial cells: Achievements and challenges.Mol Microbiol 80:1137–1147.

Brundage L, Hendrick JP, Schiebel E, Driessen AJM, Wickner W. 1990.The purified E. coli integral membrane protein SecYE is sufficient

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 75

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

for reconstitution of SecA-dependent precursor protein translocation.Cell 62:649–657.

Burda P, Aebi M. 1999. The dolichol pathway of N-linked glycosylation.Biochim Biophys Acta – Gen Subj 1426:239–257.

Calo D, Eichler J. 2011. Crossing the membrane in Archaea, thethird domain of life. Biochim Biophys Acta – Biomembr 1808:885–891.

Cao TB, Saier Jr MH. 2003. The general protein secretory pathway:phylogenetic analyses leading to evolutionary conclusions. BiochimBiophys Acta – Biomembr 1609:115–125.

Castanie-Cornet M-P, Bruel N, Genevaux P. 2013. Chaperone network-ing facilitates protein targeting to the bacterial cytoplasmic mem-brane. Biochim Biophys Acta – Mol Cell Res. doi: 10.1016/j.bbamcr.2013.11.007.

Chang CN, Blobel G, Model P. 1978. Detection of prokaryotic signalpeptidase in an Escherichia coli membrane fraction: Endoproteolyticcleavage of nascent f1 pre-coat protein. Proc Natl Acad Sci USA 75:361–365.

Chavan M, Yan A, Lennarz WJ. 2005. Subunits of the translocon interactwith components of the oligosaccharyl transferase complex. J BiolChem 280:22917–22924.

Chen M, Xie K, Jiang F, Yi L, Dalbey REE. 2002. YidC, a newly definedevolutionarily conserved protein, mediates membrane protein assem-bly in bacteria. Biol Chem 383:1565–1572.

Chen R, Henning U. 1996. A periplasmic protein (Skp) of Escherichiacoli selectively binds a class of outer membrane proteins.Mol Microbiol 19:1287–1294.

Chen W, Helenius J, Braakman I, Helenius A. 1995. Cotranslationalfolding and calnexin binding during glycoprotein synthesis. Proc NatlAcad Sci USA 92:6229–6233.

Cheng Z, Jiang Y, Mandon EC, Gilmore R. 2005. Identification ofcytoplasmic residues of Sec61p involved in ribosome binding andcotranslational translocation. J Cell Biol 168:67–77.

Chirico WJ, Waters MG, Blobel G. 1988. 70K heat shock relatedproteins stimulate protein translocation into microsomes. Nature 332:805–810.

Clemons WM, Menetret J-F, Akey CW, Rapoport TA. 2004. Structuralinsight into the protein translocation channel. Curr Opin Struct Biol14:390–396.

Cline K, Dabney-Smith C. 2008. Plastid protein import and sorting:Different paths to the same compartments. Curr Opin Plant Biol 11:585–592.

Cross BC, High S. 2009. Dissecting the physiological role of selectivetransmembrane-segment retention at the ER translocon. J Cell Sci122:1768–1777.

Cunningham K, Lill R, Crooke E, Rice M, Moore K, Wickner W, OliverD. 1989. SecA protein, a peripheral protein of the Escherichia coliplasma membrane, is essential for the functional binding andtranslocation of proOmpA. EMBO J 8:955–959.

Czaplinski K, Singer RH. 2006. Pathways for mRNA localization in thecytoplasm. Trends Biochem Sci 31:687–693.

Dalal K, Nguyen N, Alami M, Tan J, Moraes TF, Lee WC, et al. 2009.Structure, binding, and activity of Syd, a SecY-interacting protein.J Biol Chem 284:7897–7902.

Dalbey RE, Lively MO, Bron S, Dijl JMV. 1997. The chemistryand enzymology of the type I signal peptidases. Protein Sci 6:1129–1138.

Dalbey RE, Wang P, Kuhn A. 2011. Assembly of bacterial innermembrane proteins. Annu Rev Biochem 80:161–187.

Dalbey RE, Wang P, van Dijl JM. 2012. Membrane proteases in thebacterial protein secretion and quality control pathway. Microbiol MolBiol Rev 76:311–330.

Daley DO, Rapp M, Granseth E, Melen K, Drew D, von Heijne G. 2005.Global topology analysis of the Escherichia coli inner membraneproteome. Science 308:1321–1323.

Dartigalongue C, Raina S. 1998. A new heat-shock gene, ppiD, encodesa peptidyl-prolyl isomerase required for folding of outer membraneproteins in Escherichia coli. EMBO J 17:3968–3980.

de Cook H, Tommassen J. 1991. Conservation of components of theEscherichia coli export machinery in prokaryotes. FEMS MicrobiolLett 80:195–199.

de Gier J-WL, Mansournia P, Valent QA, Phillips GJ, Luirink J,von Heijne G. 1996. Assembly of a cytoplasmic membrane proteinin Escherichia coli is dependent on the signal recognition particle.FEBS Lett 399:307–309.

de Vrije T, de Swart RL, Dowhan W, Tommassen J, de Kruijff B. 1988.Phosphatidylglycerol is involved in protein translocation acrossEscherichia coli inner membranes. Nature 334:173–175.

Deitermann S, Sprie GS, Koch HG. 2005. A dual function for SecA inthe assembly of single spanning membrane proteins in Escherichiacoli. J Biol Chem 280:39077–39085.

Demirci E, Junne T, Baday S, Berneche S, Spiess M. 2013. Functionalasymmetry within the Sec61p translocon. Proc Natl Acad Sci USA110:18856–18861.

Denic V, Dotsch V, Sinning I. 2013. Endoplasmic reticulum targetingand insertion of tail-anchored membrane proteins by the GETpathway. Cold Spring Harb Perspect Biol 5:a013334.

Deshaies RJ, Koch BD, Werner-Washburne M, Craig EA, Schekman R.1988. A subfamily of stress proteins facilitates translocation ofsecretory and mitochondrial precursor polypeptides. Nature 332:800–805.

Deshaies RJ, Sanders SL, Feldheim DA, Schekman R. 1991. Assemblyof yeast Sec proteins involved in translocation into the endoplasmicreticulum into a membrane-bound multisubunit complex. Nature 349:806–808.

Deshaies RJ, Schekman R. 1987. A yeast mutant defective at an earlystage in import of secretory protein precursors into the endoplasmicreticulum. J Cell Biol 105:633–645.

Deshaies RJ, Schekman R. 1989. SEC62 encodes a putative membraneprotein required for protein translocation into the yeast endoplasmicreticulum. J Cell Biol 109:2653–2664.

Deuerling E, Patzelt H, Vorderwulbecke S, Rauch T, Kramer G,Schaffitzel E, et al. 2003. Trigger factor and DnaK possessoverlapping substrate pools and binding specificities. Mol Microbiol47:1317–1328.

Deville K, Gold VAM, Robson A, Whitehouse S, Sessions RB,Baldwin SA, et al. 2011. The oligomeric state and arrangement ofthe active bacterial translocon. J Biol Chem 286:4659–4669.

Dierks T, Volkmer J, Schlenstedt G, Jung C, Sandholzer U, Zachmann K,et al. 1996. A microsomal ATP-binding protein involved in efficientprotein transport into the mammalian endoplasmic reticulum. EMBOJ 15:6931–6942.

Douville K, Price A, Eichler J, Economou A, Wickner W. 1995. SecYEGand SecA are the stoichiometric components of preprotein translocase.J Biol Chem 270:20106–20111.

Dowhan W, Bogdanov M. 2009. Lipid-dependent membrane proteintopogenesis. Annu Rev Biochem 78:515–540.

Drew D, Froderberg L, Baars L, de Gier J-WL. 2003. Assembly andoverexpression of membrane proteins in Escherichia coli. BiochimBiophys Acta – Biomembr 1610:3–10.

Driessen AJ, Wickner W. 1991. Proton transfer is rate-limiting fortranslocation of precursor proteins by the Escherichia coli translocase.Proc Natl Acad Sci USA 88:2471–2475.

Driessen AJM, Nouwen N. 2008. Protein translocation across thebacterial cytoplasmic membrane. Annu Rev Biochem 77:643–667.

Dudek J, Greiner M, Muller A, Hendershot LM, Kopsch K,Nastainczyk W, Zimmermann R. 2005. ERj1p has a basic role inprotein biogenesis at the endoplasmic reticulum. Nat Struct Mol Biol12:1008–1014.

Dudek J, Volkmer J, Bies C, Guth S, Muller A, Lerner M, et al. 2002.A novel type of co-chaperone mediates transmembranerecruitment of DnaK-like chaperones to ribosomes. EMBO J 21:2958–2967.

Dunnwald M, Varshavsky A, Johnsson N. 1999. Detection of transientin vivo interactions between substrate and transporter during proteintranslocation into the endoplasmic reticulum. Mol Cell Biol 10:329–344.

Duong F, Wickner W. 1997a. The SecDFyajC domain of preproteintranslocase controls preprotein movement by regulating SecA mem-brane cycling. EMBO J 16:4871–4879.

Duong F, Wickner W. 1997b. Distinct catalytic roles of the SecYE, SecGand SecDFyajC subunits of preprotein translocase holoenzyme.EMBO J 16:2756–2768.

Egea PF, Napetschnig J, Walter P, Stroud RM. 2008. Structures ofSRP54 and SRP19, the two proteins that organize the ribonucleic coreof the signal recognition particle from Pyrococcus furiosus. PLoS One3:e3528.

Egea PF, Shan SO, Napetschnig J, Savage DF, Walter P, Stroud RM.2004. Substrate twinning activates the signal recognition particle andits receptor. Nature 427:215–221.

76 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Egea PF, Stroud RM. 2010. Lateral opening of a translocon upon entryof protein suggests the mechanism of insertion into membranes.Proc Natl Acad Sci USA 107:17182–17187.

Eichler J. 2003. Evolution of the prokaryotic protein translocationcomplex: A comparison of archaeal and bacterial versions of SecDF.Mol Phylogen Evol 27:504–509.

Eitan A, Bibi E. 2004. The core Escherichia coli signal recognitionparticle receptor contains only the N and G domains of FtsY.J Bacteriol 186:2492–2494.

Emr SD, Hanley-Way S, Silhavy TJ. 1981. Suppressor mutations thatrestore export of a protein with a defective signal sequence. Cell 23:79–88.

Enquist K, Fransson M, Boekel C, Bengtsson I, Geiger K, Lang L, et al.2009. Membrane-integration characteristics of two ABC transporters,CFTR and P-glycoprotein. J Mol Biol 387:1153–1164.

Epand RM, Epand RF. 2011. Functional consequences of the lateralorganization of biological membranes. In: Yeagle PL, ed. TheStructure of Biological Membranes, 3rd ed. Boca Raton, FL: CRCPress.

Erdmann F, Schauble N, Lang S, Jung M, Honigmann A,Ahmad M, et al. 2011. Interaction of calmodulin with Sec61alimits Ca2+ leakage from the endoplasmic reticulum. EMBO J 30:17–31.

Erlandson KJ, Miller SBM, Nam Y, Osborne AR, Zimmer J, RapoportTA. 2008. A role for the two-helix finger of the SecA ATPase inprotein translocation. Nature 455:984–987.

Facey SJ, Neugebauer SA, Krauss S, Kuhn A. 2007. The mechan-osensitive channel protein MscL is targeted by the SRP to the novelYidC membrane insertion pathway of Escherichia coli. J Mol Biol365:995–1004.

Fang H, Green N. 1994. Nonlethal sec71-1 and sec72-1 mutationseliminate proteins associated with the Sec63p-BiP complex from S.cerevisiae. Mol Cell Biol 5:933–942.

Fekkes P, De Wit JG, Van Der Wolk JPW, Kimsey HH, Kumamoto CA,Driessen AJM. 1998. Preprotein transfer to the Escherichia colitranslocase requires the co-operative binding of SecB and the signalsequence to SecA. Mol Microbiol 29:1179–1190.

Feldheim D, Yoshimura K, Admon A, Schekman R. 1993. Structural andfunctional characterization of Sec66p, a new subunit of the polypep-tide translocation apparatus in the yeast endoplasmic reticulum.Mol Cell Biol 4:931–939.

Ferbitz L, Maier T, Patzelt H, Bukau B, Deuerling E, Ban N. 2004.Trigger factor in complex with the ribosome forms a molecular cradlefor nascent proteins. Nature 431:590–596.

Finke K, Plath K, Panzner S, Prehn S, Rapoport TA, Hartmann E,Sommer T. 1996. A second trimeric complex containing homologs ofthe Sec61p complex functions in protein transport across the ERmembrane of S. cerevisiae. EMBO J 15:1482–1494.

Flanagan JJ, Chen JC, Miao Y, Shao Y, Lin J, Bock PE, Johnson AE.2003. Signal recognition particle binds to ribosome-bound signalsequences with fluorescence-detected subnanomolar affinity that doesnot diminish as the nascent chain lengthens. J Biol Chem 278:18628–18637.

Flourakis M, Van Coppenolle F, Lehen’kyi Vy, Beck B, Skryma R,Prevarskaya N. 2006. Passive calcium leak via translocon is a first stepfor iPLA2-pathway regulated store operated channels activation.FASEB J 20:1215–1217.

Flower AM. 2001. SecG function and phospholipid metabolism inEscherichia coli. J Bacteriol 183:2006–2012.

Flower AM, Hines LL, Pfennig PL. 2000. SecG is an auxiliarycomponent of the protein export apparatus of Escherichia coli.Mol Gen Genet 263:131–136.

Focia PJ, Shepotinovskaya IV, Seidler JA, Freymann DM. 2004.Heterodimeric GTPase core of the SRP targeting complex. Science303:373–377.

Fons RD, Bogert BA, Hegde RS. 2003. Substrate-specific function of thetranslocon-associated protein complex during translocation across theER membrane. J Cell Biol 160:529–539.

Frauenfeld J, Gumbart J, v d Sluis EO, Funes S, Gartmann M,Beatrix B, et al. 2011. Cryo-EM structure of the ribosome-SecYEcomplex in the membrane environment. Nat Struct Mol Biol 18:614–621.

Fulga TA, Sinning I, Dobberstein B, Pool MR. 2001. SRbeta coordinatessignal sequence release from SRP with ribosome binding to thetranslocon. EMBO J 20:2338–2347.

Garcia PD, Walter P. 1988. Full-length prepro-alpha-factor can betranslocated across the mammalian microsomal membrane only iftranslation has not terminated. J Cell Biol 106:1043–1048.

Gatsos X, Perry AJ, Anwari K, Dolezal P, Wolynec PP, Likic VA, et al.2008. Protein secretion and outer membrane assembly inAlphaproteobacteria. FEMS Microbiol Rev 32:995–1009.

Gelis I, Bonvin AMJJ, Keramisanou D, Koukaki M, Gouridis G,Karamanou S, et al. 2007. Structural basis for signal-sequencerecognition by the translocase motor SecA as determined by NMR.Cell 131:756–769.

Gilmore R, Blobel G, Walter P. 1982a. Protein translocation across theendoplasmic reticulum. I. Detection in the microsomal membraneof a receptor for the signal recognition particle. J Cell Biol 95:463–469.

Gilmore R, Walter P, Blobel G. 1982b. Protein translocation across theendoplasmic reticulum. II. Isolation and characterization of the signalrecognition particle receptor. J Cell Biol 95:470–477.

Goder V, Bieri C, Spiess M. 1999. Glycosylation can influencetopogenesis of membrane proteins and reveals dynamic reorientationof nascent polypeptides within the translocon. J Cell Biol 147:257–266.

Gogala M, Becker T, Beatrix B, Armache J-P, Barrio-Garcia C,Berninghausen O, Beckmann R. 2014. Structures of the Sec61complex engaged in nascent peptide translocation or membraneinsertion. Nature 506:107–110.

Gold VAM, Robson A, Bao H, Romantsov T, Duong F, Collinson I.2010. The action of cardiolipin on the bacterial translocon. Proc NatlAcad Sci USA 107:10044–10049.

Gordon SM, Kindt TJ. 1976. BIP: A low molecular weight proteincoisolated with beta-2 microglublin. Biochem a Biophys ResCommun 72:984–990.

Gorlich D, Rapoport TA. 1993. Protein translocation into proteolipo-somes reconstituted from purified components of the endoplasmicreticulum membrane. Cell 75:615–630.

Gorlich D, Hartmann E, Prehn S, Rapoport TA. 1992. A protein of theendoplasmic reticulum involved early in polypeptide translocation.Nature 357:47–52.

Gotz C, Muller A, Montenarh M, Zimmermann R, Dudek J. 2009. TheER-membrane-resident Hsp40 ERj1 is a novel substrate for proteinkinase CK2. Biochem a Biophys Res Commun 388:637–642.

Gouridis G, Karamanou S, Gelis I, Kalodimos CG, Economou A. 2009.Signal peptides are allosteric activators of the protein translocase.Nature 462:363–367.

Gruss OJ, Feick P, Frank R, Dobberstein B. 1999. Phosphorylation ofcomponents of the ER translocation site. Eur J Biochem 260:785–793.

Gu SQ, Peske F, Wieden HJ, Rodnina MV, Wintermeyer W. 2003. Thesignal recognition particle binds to protein L23 at the peptide exit ofthe Escherichia coli ribosome. RNA 9:566–573.

Gumbart J, Schulten K. 2008. The roles of pore ring and plug in theSecY protein-conducting channel. J Gen Physiol 132:709–719.

Gumbart J, Trabuco LG, Schreiner E, Villa E, Schulten K. 2009.Regulation of the protein-conducting channel by a bound ribosome.Structure 17:1453–1464.

Gutierrez JA, Crowley PJ, Cvitkovitch DG, Brady LJ, Hamilton IR,Hillman JD, Bleiweis AS. 1999. Streptococcus mutans ffh, a geneencoding a homologue of the 54 kDa subunit of the signal recognitionparticle, is involved in resistance to acid stress. Microbiology 145:357–366.

Hainzl T, Huang SH, Sauer-Eriksson AE. 2002. Structure of the SRP19-RNA complex and implications for signal recognition particleassembly. Nature 417:767–771.

Hainzl T, Huang SH, Sauer-Eriksson AE. 2005. Structural insights intoSRP RNA: An induced fit mechanism for SRP assembly. RNA 11:1043–1050.

Halic M, Becker T, Pool MR, Spahn CM, Grassucci RA, Frank J,Beckmann R. 2004. Structure of the signal recognition particleinteracting with the elongation-arrested ribosome. Nature 427:808–814.

Halic M, Blau M, Becker T, Mielke T, Pool MR, Wild K, Sinning I,Beckmann R. 2006. Following the signal sequence from ribosomaltunnel exit to signal recognition particle. Nature 444:507–511.

Hamman BD, Chen J-C, Johnson EE, Johnson AE. 1997. The aqueouspore through the translocon has a diameter of 40–60 A duringcotranslational protein translocation at the ER membrane. Cell 89:535–544.

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 77

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Hamman BD, Hendershot LM, Johnson AE. 1998. BiP maintains thepermeability barrier of the ER membrane by sealing the lumenal endof the translocon pore before and early in translocation. Cell 92:747–758.

Hand NJ, Klein R, Laskewitz A, Pohlschroder M. 2006. Archaeal andbacterial SecD and SecF homologs exhibit striking structural andfunctional conservation. J Bacteriol 188:1251–1259.

Hann BC, Walter P. 1991. The signal recognition particle inS. cerevisiae. Cell 67:131–144.

Harada Y, Li H, Lennarz WJ. 2009. Oligosaccharyltransferase directlybinds to ribosome at a location near the translocon-binding site. ProcNatl Acad Sci USA 106:6945–6949.

Harms N, Koningstein G, Dontje W, Muller M, Oudega B, Luirink J,de Cock H. 2001. The early interaction of the outer membrane proteinphoe with the periplasmic chaperone Skp occurs at the cytoplasmicmembrane. J Biol Chem 276:18804–18811.

Harris CR, Silhavy TJ. 1999. Mapping an interface of SecY (PrlA) andSecE (PrlG) by using synthetic phenotypes and in vivo cross-linking.J Bacteriol 181:3438–3444.

Hartman H, Smith TF. 2010. GTPases and the origin of the ribosome.Biol Direct 5:36.

Hartmann E, Gorlich D, Kostka S, Otto A, Kraft R, Knespel S, et al.1993. A tetrameric complex of membrane proteins in the endoplasmicreticulum. Eur J Biochem 214:375–381.

Hartmann E, Sommer T, Prehn S, Gorlich D, Jentsch S, Rapoport TA.1994. Evolutionary conservation of components of the proteintranslocation complex. Nature 367:654–657.

Hasona A, Crowley PJ, Levesque CM, Mair RW, Cvitkovitch DG,Bleiweis AS, Brady LJ. 2005. Streptococcal viability and diminishedstress tolerance in mutants lacking the signal recognition particlepathway or YidC2. Proc Natl Acad Sci USA 102:17466–17471.

Hegde RS, Bernstein HD. 2006. The surprising complexity of signalsequences. Trends Biochem Sci 31:563–571.

Hegde RS, Keenan RJ. 2011. Tail-anchored membrane protein insertioninto the endoplasmic reticulum. Nat Rev Mol Cell biol 12:787–798.

Hegde RS, Voigt S, Rapoport TA, Lingappa VR. 1998. TRAM regulatesthe exposure of nascent secretory proteins to the cytosol duringtranslocation into the endoplasmic reticulum. Cell 92:621–631.

Heinrich SU, Mothes W, Brunner J, Rapoport TA. 2000. The Sec61pcomplex mediates the integration of a membrane protein by allowinglipid partitioning of the transmembrane domain. Cell 102:233–244.

Heinrich SU, Rapoport TA. 2003. Cooperation of transmembranesegments during the integration of a double-spanning protein intothe ER membrane. EMBO J 22:3654–3663.

Helmers J, Schmidt D, Glavy JS, Blobel G, Schwartz T. 2003. The beta-subunit of the protein-conducting channel of the endoplasmicreticulum functions as the guanine nucleotide exchange factor forthe beta-subunit of the signal recognition particle receptor. J BiolChem 278:23686–23690.

Hermesh O, Jansen R-P. 2013. Take the (RN)A-train: Localization ofmRNA to the endoplasmic reticulum. Biochim Biophys Acta – MolCell Res 1833:2519–2525.

Herskovits AA, Bibi E. 2000. Association of Escherichia coli ribosomeswith the inner membrane requires the signal recognition particlereceptor but is independent of the signal recognition particle. ProcNatl Acad Sci USA 97:4621–4626.

Herskovits AA, Shimoni E, Minsky A, Bibi E. 2002. Accumulation ofendoplasmic membranes and novel membrane-bound ribosome-signalrecognition particle receptor complexes in Escherichia coli. J CellBiol 159:403–410.

Hessa T, Kim H, Bihlmaier K, Lundin C, Boekel J, Andersson H, et al.2005. Recognition of transmembrane helices by the endoplasmicreticulum translocon. Nature 433:377–381.

Higy M, Gander S, Spiess M. 2005. Probing the environment of signal-anchor sequences during topogenesis in the endoplasmic reticulum.Biochemistry 44:2039–2047.

Hizlan D, Robson A, Whitehouse S, Gold Vicki A, Vonck J, Mills D,et al. 2012. Structure of the SecY complex unlocked by a preproteinmimic. Cell Rep 1:21–28.

Hoffmann A, Becker Annemarie H, Zachmann-Brand B, Deuerling E,Bukau B, Kramer G. 2012. Concerted action of the ribosome and theassociated chaperone trigger factor confines nascent polypeptidefolding. Mol Cell 48:63–74.

Holtkamp W, Lee S, Bornemann T, Senyushkina T, Rodnina MV,Wintermeyer W. 2012. Dynamic switch of the signal recognition

particle from scanning to targeting. Nat Struct Mol Biol 19:1332–1337.

Hori O, Miyazaki M, Tamatani T, Ozawa K, Takano K, Okabe M, et al.2006. Deletion of SERP1/RAMP4, a component of the endoplasmicreticulum (ER) translocation sites, leads to ER stress. Mol Cell Biol26:4257–4267.

Hou B, Lin PJ, Johnson AE. 2012. Membrane protein TM segmentsare retained at the translocon during integration until the nascentchain cues FRET-detected release into bulk lipid. Mol Cell 48:398–408.

Houben EN, Zarivach R, Oudega B, Luirink J. 2005. Early encounters ofa nascent membrane protein: specificity and timing of contacts insideand outside the ribosome. J Cell Biol 170:27–35.

Huber D, Boyd D, Xia Y, Olma MH, Gerstein M, Beckwith J. 2005. Useof thioredoxin as a reporter to identify a subset of Escherichia colisignal sequences that promote signal recognition particle-dependenttranslocation. J Bacteriol 187:2983–2991.

Huber D, Rajagopalan N, Preissler S, Rocco MA, Merz F, Kramer G,Bukau B. 2011. SecA interacts with ribosomes in order to facilitateposttranslational translocation in bacteria. Mol Cell 41:343–353.

Hunt JF, Weinkauf S, Henry L, Fak JJ, McNicholas P, Oliver DB,Deisenhofer J. 2002. Nucleotide control of interdomain interactionsin the conformational reaction cycle of SecA. Science 297:2018–2026.

Ingolia NT, Ghaemmaghami S, Newman JRS, Weissman JS. 2009.Genome-wide analysis in vivo of translation with nucleotide reso-lution using ribosome profiling. Science 324:218–223.

Ismail N, Crawshaw SG, Cross BCS, Haagsma AC, High S. 2008.Specific transmembrane segments are selectively delayed at the ERtranslocon during opsin biogenesis. Biochem J 411:495–506.

Ismail N, Hedman R, Schiller N, von Heijne G. 2012. A biphasic pullingforce acts on transmembrane helices during translocon-mediatedmembrane integration. Nat Struct Mol Biol 19:1018–1022.

Ito K, Akiyama Y. 2005. Cellular functions, mechanism of action, andregulation of FtsH protease. Annul Rev Microbiol 59:211–231.

Ito K, Wittekind M, Nomura M, Shiba K, Yura T, Miura A, NashimotoH. 1983. A temperature-sensitive mutant of E. coli exhibiting slowprocessing of exported proteins. Cell 32:789–797.

Jagath JR, Matassova NB, de Leeuw E, Warnecke JM, Lentzen G,Rodnina MV, et al. 2001. Important role of the tetraloop region of4.5S RNA in SRP binding to its receptor FtsY. RNA 7:293–301.

Janska H, Kwasniak M, Szczepanowska J. 2013. Protein quality controlin organelles – AAA/FtsH story. Biochim Biophys Acta – Mol CellRes 1833:381–387.

Jaqaman K, Grinstein S. 2012. Regulation from within: The cytoskeletonin transmembrane signaling. Trends Cell Biol 22:515–526.

Jarchow S, Luck C, Gorg A, Skerra A. 2008. Identification of potentialsubstrate proteins for the periplasmic Escherichia coli chaperone Skp.Proteomics 8:4987–4994.

Jermy AJ, Willer M, Davis E, Wilkinson BM, Stirling CJ. 2006. The Brldomain in Sec63p is required for assembly of functional endoplasmicreticulum translocons. J Biol Chem 281:7899–7906.

Jiang Y, Cheng Z, Mandon EC, Gilmore R. 2008. An interaction betweenthe SRP receptor and the translocon is critical during cotranslationalprotein translocation. J Cell Biol 180:1149–1161.

Johnson N, Habdenteufel S, Theis M, Paton AW, Paton JC, ZimmermannR, High S. 2013. The signal sequence influences post-translational ERtranslocation at distinct stages. PLoS ONE 8:e75394.

Josefsson LG, Randall LL. 1981a. Different exported proteins in E. colishow differences in the temporal mode of processing in vivo. Cell 25:151–157.

Josefsson LG, Randall LL. 1981b. Processing in vivo of precursormaltose-binding protein in Escherichia coli occurs post-translationallyas well as co-translationally. J Biol Chem 256:2504–2507.

Junne T, Schwede T, Goder V, Spiess M. 2006. The plug domain of yeastSec61p is important for efficient protein translocation, but is notessential for cell viability. Mol Cell Biol 17:4063–4068.

Kalies K-U, Rapoport TA, Hartmann E. 1998. The b subunit of theSec61 complex facilitates cotranslational protein transport andinteracts with the signal peptidase during translocation. J Cell Biol141:887–894.

Karamyshev AL, Johnson AE. 2005. Selective SecA association withsignal sequences in ribosome-bound nascent chains: A potential rolefor SecA in ribosome targeting to the bacterial membrane. J BiolChem 280:37930–37940.

78 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Karaoglu D, Kelleher DJ, Gilmore R. 1997. The highly conserved Stt3protein is a subunit of the yeast oligosaccharyltransferase and forms asubcomplex with Ost3p and Ost4p. J Biol Chem 272:32513–32520.

Keenan RJ, Freymann DM, Walter P, Stroud RM. 1998. Crystal structureof the signal sequence binding subunit of the signal recognitionparticle. Cell 94:181–191.

Kelkar A, Dobberstein B. 2009. Sec61beta, a subunit of the Sec61protein translocation channel at the endoplasmic reticulum, isinvolved in the transport of Gurken to the plasma membrane. BMCCell Biol 10:11.

Keller R, de Keyzer J, Driessen AJM, Palmer T. 2012. Co-operationbetween different targeting pathways during integration of amembrane protein. J Cell Biol 199:303–315.

Keller RCA. 2011. The prediction of novel multiple lipid-bindingregions in protein translocation motor proteins: A possible generalfeature. Cell Mol Biol Lett 16:40–54.

Kihara A, Akiyama Y, Ito K. 1995. FtsH is required for proteolyticelimination of uncomplexed forms of SecY, an essential proteintranslocase subunit. Proc Natl Acad Sci USA 92:4532–4536.

Kim S, Coulombe PA. 2010. Emerging role for the cytoskeleton as anorganizer and regulator of translation. Nat Rev Mol Cell biol 11:75–81.

Kinch LN, Saier JMH, Grishin NV. 2002. Sec61b – a component ofthe archaeal protein secretory system. Trends Biochem Sci 27:170–171.

Knoblauch NTM, Rudiger S, Schonfeld H-J, Driessen AJM, Schneider-Mergener J, Bukau B. 1999. Substrate specificity of the SecBchaperone. J Biol Chem 274:34219–34225.

Knyazev DG, Lents A, Krause E, Ollinger N, Siligan C, Papinski D,et al. 2013. The bacterial translocon SecYEG opens upon ribosomebinding. J Biol Chem 288:17941–17946.

Koch HG, Hengelage T, Neumann-Haefelin C, MacFarlane J,Hoffschulte HK, Schimz K-L, et al. 1999. In vitro studies withpurified components reveal signal recognition particle (SRP) andSecA/SecB as constituents of two independent protein-targetingpathways of Escherichia coli. Mol Cell Biol 10:2163–2173.

Koch HG, Muller M. 2000. Dissecting the translocase and integrasefunctions of the Escherichia coli SecYEG translocon. J Cell Biol 150:689–694.

Kohler R, Boehringer D, Greber B, Bingel-Erlenmeyer R, Collinson I,Schaffitzel C, Ban N. 2009. YidC and Oxa1 form dimeric insertionpores on the translating ribosome. Mol Cell 34:344–353.

Kramer G, Boehringer D, Ban N, Bukau B. 2009. The ribosome as aplatform for co-translational processing, folding and targeting ofnewly synthesized proteins. Nat Struct Mol Biol 16:589–597.

Kramer G, Rauch T, Rist W, Vorderwulbecke S, Patzelt H, Schulze-Specking A, et al. 2002. L23 protein functions as a chaperone dockingsite on the ribosome. Nature 419:171–174.

Kraut-Cohen J, Afanasieva E, Haim-Vilmovsky L, Slobodin B, Yosef I,Bibi E, Gerst JE. 2013. Translation- and SRP-independent mRNAtargeting to the endoplasmic reticulum in the yeast Saccharomycescerevisiae. Mol Cell Biol 24:3069–3084.

Kraut-Cohen J, Gerst JE. 2010. Addressing mRNAs to the ER: Cissequences act up!. Trends Biochem Sci 35:459–469.

Krehenbrink M, Edwards A, Downie JA. 2011. The superoxidedismutase SodA is targeted to the periplasm in a SecA-dependentmanner by a novel mechanism. Mol Microbiol 82:164–179.

Krieg UC, Johnson AE, Walter P. 1989. Protein translocation acrossthe endoplasmic reticulum membrane: identification by photocross-linking of a 39-kD integral membrane glycoprotein as part of aputative translocation tunnel. J Cell Biol 109:2033–2043.

Kroczynska B, Evangelista CM, Samant SS, Elguindi EC, Blond SY.2004. The SANT2 domain of the murine tumor cell DnaJ-like protein1 human homologue interacts with a1-antichymotrypsin and kinetic-ally interferes with its serpin inhibitory activity. J Biol Chem 279:11432–11443.

Kudva R, Denks K, Kuhn P, Vogt A, Muller M, Koch HG, Muller M.2013. Protein translocation across the inner membrane of Gram-negative bacteria: The Sec and Tat dependent protein transportpathways. Res Microbiol 164:505–534.

Kuhn P, Weiche B, Sturm L, Sommer E, Drepper F, Warscheid B, et al.2011. The bacterial SRP receptor, SecA and the ribosome useoverlapping binding sites on the SecY translocon. Traffic 12:563–578.

Kuhn P, Kudva R, Welte T, Sturm L, Koch HG. 2014. Targeting andintegration of bacterial membrane proteins. In: Remi Fronzes HR, ed.

Bacterial Membranes: Structural and Molecular Biology. Norwich,UK: Caister Academic Press; Horizon Scientific Press.

Kurzchalia TV, Wiedmann M, Girshovich AS, Bochkareva ES, Bielka H,Rapoport TA. 1986. The signal sequence of nascent preprolactininteracts with the 54K polypeptide of the signal recognition particle.Nature 320:634–636.

Kusters R, Dowhan W, de Kruijff B. 1991. Negatively chargedphospholipids restore prePhoE translocation across phosphatidylgly-cerol-depleted Escherichia coli inner membranes. J Biol Chem 266:8659–8662.

Lakkaraju AK, Mary C, Scherrer A, Johnson AE, Strub K. 2008. SRPkeeps polypeptides translocation-competent by slowing translation tomatch limiting ER-targeting sites. Cell 133:440–451.

Lakkaraju AKK, Abrami L, Lemmin T, Blaskovic S, Kunz B, Kihara A,et al. 2012a. Palmitoylated calnexin is a key component of theribosome-translocon complex. EMBO J 31:1823–1835.

Lakkaraju AKK, Thankappan R, Mary C, Garrison JL, Taunton J,Strub K. 2012b. Efficient secretion of small proteins in mammaliancells relies on Sec62-dependent posttranslational translocation.Mol Cell Biol 23:2712–2722.

Lam VQ, Akopian D, Rome M, Henningsen D, Shan SO. 2010. Lipidactivation of the signal recognition particle receptor provides spatialcoordination of protein targeting. J Cell Biol 190:623–635.

Lang S, Benedix J, Fedeles SV, Schorr S, Schirra C, Schauble N, et al.2012. Different effects of Sec61a, Sec62 and Sec63 depletion ontransport of polypeptides into the endoplasmic reticulum of mamma-lian cells. J Cell Sci 125:1958–1969.

Larsen N, Zwieb C. 1993. The signal recognition particle database(SRPDB). Nucleic Acids Res 21:3019–3020.

Lau JT, Welply JK, Shenbagamurthi P, Naider F, Lennarz WJ. 1983.Substrate recognition by oligosaccharyl transferase. Inhibition ofco-translational glycosylation by acceptor peptides. J Biol Chem 258:15255–15260.

Lee AH, Iwakoshi NN, Glimcher LH. 2003. XBP-1 regulates a subset ofendoplasmic reticulum resident chaperone genes in the unfoldedprotein response. Mol Cell Biol 23:7448–7459.

Lee HC, Bernstein HD. 2001. The targeting pathway of Escherichia colipresecretory and integral membrane proteins is specified by thehydrophobicity of the targeting signal. Proc Natl Acad Sci USA 98:3471–3476.

Leung E, Brown JD. 2010. Biogenesis of the signal recognition particle.Biochem Soc Trans 38:1093–1098.

Leznicki P, Clancy A, Schwappach B, High S. 2010. Bat3 promotes themembrane integration of tail-anchored proteins. J Cell Sci 123:2170–2178.

Li W, Schulman S, Boyd D, Erlandson K, Beckwith J, Rapoport TA.2007. The plug domain of the SecY protein stabilizes the closed stateof the translocation channel and maintains a membrane seal. Mol Cell26:511–521.

Liang H, VanValkenburgh C, Chen X, Mullins C, Van Kaer L, Green N,Fang H. 2003. Genetic complementation in yeast reveals functionalsimilarities between the catalytic subunits of mammalian signalpeptidase complex. J Biol Chem 278:50932–50939.

Liao S, Lin J, Do H, Johnson AE. 1997. Both lumenal andcytosolic gating of the aqueous ER translocon pore are regulatedfrom inside the ribosome during membrane protein integration. Cell90:31–41.

Lill R, Dowhan W, Wickner W. 1990. The ATPase activity of SecA isregulated by acidic phospholipids, SecY, and the leader and maturedomains of precursor proteins. Cell 60:271–280.

Lithgow T, Waksman G. 2013. Seaside transportation – from structure tofunction of translocation machines. EMBO Rep 14:585–587.

Loibl M, Wunderle L, Hutzler J, Schulz BL, Aebi M, Strahl S. 2014.Protein O-mannosyltransferases associate with the translocon tomodify translocating polypeptide chains. J Biol Chem 289:8599–8611.

Luirink J, High S, Wood H, Giner A, Tollervey D, Dobberstein B. 1992.Signal sequence recognition by an Escherichia coli ribonucleoproteincomplex. Nature 359:741–743.

Luirink J, ten Hagen-Jongman CM, van der Weijden CC, Oudega B,High S, Dobberstein B, Kusters R. 1994. An alternative proteintargeting pathway in Escherichia coli: studies on the role of ftsY.EMBO J 13:2289–2296.

Lutcke H, High S, Romisch K, Ashford A, Dobberstein B. 1992. Themethionine-rich domain of the 54 kDa subunit of signal recognition

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 79

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

particle is sufficient for the interaction with signal sequences. EMBO J11:1543–1551.

Lycklama a Nijeholt JA, de Keyzer J, Prabudiansyah I, Driessen AJM.2013. Characterization of the supporting role of SecE in proteintranslocation. FEBS Lett 587:3083–3088.

Lyman SK, Schekman R. 1995. Interaction between BiP and Sec63p isrequired for the completion of protein translocation into the ER ofSaccharomyces cerevisiae. J Cell Biol 131:1163–1171.

Lyman SK, Schekman R. 1997. Binding of secretory precursorpolypeptides to a translocon subcomplex is regulated by BiP. Cell88:85–96.

Macfarlane J, Muller M. 1995. The functional integration of a polytopicmembrane protein of Escherichia coli is dependent on the bacterialsignal-recognition particle. Eur J Biochem 233:766–771.

Mades A, Gotthardt K, Awe K, Stieler J, Doring T, Fuser S, Prange R.2012. Role of human Sec63 in modulating the steady-state levels ofmulti-spanning membrane proteins. PLoS One 7:e49243.

Maillard AP, Lalani S, Silva F, Belin D, Duong F. 2007. Deregulation ofthe SecYEG translocation channel upon removal of the plug domain.J Biol Chem 282:1281–1287.

Martoglio B, Hofmann MW, Brunner J, Dobberstein B. 1995.The protein-conducting channel in the membrane of the endoplas-mic reticulum is open laterally toward the lipid bilayer. Cell 81:207–214.

Matern Y, Barion B, Behrens-Kneip S. 2010. PpiD is a player in thenetwork of periplasmic chaperones in Escherichia coli. BMCMicrobiol 10:251.

Matlack KES, Misselwitz B, Plath K, Rapoport TA. 1999. BiP acts as amolecular ratchet during posttranslational transport of prepro-a factoracross the ER membrane. Cell 97:553–564.

Menetret J-F, Hegde RS, Heinrich SU, Chandramouli P, Ludtke SJ,Rapoport TA, Akey CW. 2005. Architecture of the ribosome-channelcomplex derived from native membranes. J Mol Biol 348:445–457.

Menetret J-F, Schaletzky J, Clemons WM, Osborne AR, Skanland SS,Denison C, et al. 2007. Ribosome binding of a single copy of the SecYcomplex: Implications for protein translocation. Mol Cell 28:1083–1092.

Menetret JF, Hegde RS, Aguiar M, Gygi SP, Park E, Rapoport TA, et al.2008. Single copies of Sec61 and TRAP associate with a nontranslat-ing mammalian ribosome. Structure 16:1126–1137.

Merdanovic M, Clausen T, Kaiser M, Huber R, Ehrmann M. 2011.Protein quality control in the bacterial periplasm. Annul RevMicrobiol 65:149–168.

Meyer H-A, Grau H, Kraft R, Kostka S, Prehn S, Kalies K-U, HartmannE. 2000. Mammalian Sec61 is associated with Sec62 and Sec63.J Biol Chem 275:14550–14557.

Mikhaleva NI, Golovastov VV, Zolov SN, Bogdanov MV, Dowhan W,Nesmeyanova MA. 2001. Depletion of phosphatidylethanolamineaffects secretion of Escherichia coli alkaline phosphatase and itstranscriptional expression. FEBS Lett 493:85–90.

Miller JD, Bernstein HD, Walter P. 1994. Interaction of E. coli Ffh/4.5Sribonucleoprotein and FtsY mimics that of mammalian signalrecognition particle and its receptor. Nature 367:657–659.

Millman JS, Qi HY, Vulcu F, Bernstein HD, Andrews DW. 2001. FtsYbinds to the Escherichia coli inner membrane via interactions withphosphatidylethanolamine and membrane proteins. J Biol Chem 276:25982–25989.

Mircheva M, Boy D, Weiche B, Hucke F, Graumann P, Koch HG. 2009.Predominant membrane localization is an essential feature of thebacterial signal recognition particle receptor. BMC Biol 7:76.

Missiakas D, Betton J-M, Raina S. 1996. New components of proteinfolding in extracytoplasmic compartments of Escherichia coli SurA,FkpA and Skp/OmpH. Mol Microbiol 21:871–884.

Mitra K, Schaffitzel C, Shaikh T, Tama F, Jenni S, Brooks CL, et al.2005. Structure of the E. coli protein-conducting channel bound to atranslating ribosome. Nature 438:318–324.

Montero Llopis P, Jackson AF, Sliusarenko O, Surovtsev I, Heinritz J,Emonet T, Jacobs-Wagner C. 2010. Spatial organization of the flowof genetic information in bacteria. Nature 466:77–81.

Mori H, Ito K. 2006. Different modes of SecY-SecA interactionsrevealed by site-directed in vivo photo-cross-linking. Proc Natl AcadSci USA 103:16159–16164.

Morita K, Tokuda H, Nishiyama K-i. 2012. Multiple SecA moleculesdrive protein translocation across a single translocon with SecGinversion. J Biol Chem 287:455–464.

Moser M, Nagamori S, Huber M, Tokuda H, Nishiyama K-i. 2013.Glycolipozyme MPIase is essential for topology inversion of SecGduring preprotein translocation. Proc Natl Acad Sci USA 110:9734–9739.

Muller L, de Escauriaza MD, Lajoie P, Theis M, Jung M, Muller A, et al.2010. Evolutionary gain of function for the ER membrane proteinSec62 from yeast to humans. Mol Cell Biol 21:691–703.

Muller M, Koch HG, Beck K, Schafer U. 2001. Protein traffic inbacteria: Multiple routes from the ribosome to and across themembrane. Prog Nucleic Acid Res Mol Biol 66:107–157.

Nagamori S, Smirnova IN, Kaback HR. 2004. Role of YidC in folding ofpolytopic membrane proteins. J Cell Biol 165:53–62.

Nakashima K, Ishida H, Nakatomi A, Yazawa M. 2012. Specificconformation and Ca2+-binding mode of yeast calmodulin: Insightinto evolutionary development. J Bacteriol 152:27–35.

Nam S-E, Paetzel M. 2013. Structure of signal peptide peptidaseA with C-termini bound in the active sites: Insights intospecificity, self-processing, and regulation. Biochemistry 52:8811–8822.

Neumann-Haefelin C, Schafer U, Muller M, Koch HG. 2000. SRP-dependent co-translational targeting and SecA-dependent transloca-tion analyzed as individual steps in the export of a bacterial protein.EMBO J 19:6419–6426.

Nevo-Dinur K, Nussbaum-Shochat A, Ben-Yehuda S, Amster-Choder O.2011. Translation-independent localization of mRNA in E. coli.Science 331:1081–1084.

Ng DT, Brown JD, Walter P. 1996. Signal sequences specify thetargeting route to the endoplasmic reticulum membrane. J Cell Biol134:269–278.

Nicchitta CV, Blobel G. 1993. Lumenal proteins of the mammalianendoplasmic reticulum are required to complete protein translocation.Cell 73:989–998.

Nilsson I, Ohvo-Rekila H, Slotte JP, Johnson AE, von Heijne G. 2001.Inhibition of protein translocation across the endoplasmic reticulummembrane by sterols. J Biol Chem 276:41748–41754.

Nishiyama K-i, Suzuki T, Tokuda H. 1996. Inversion of the membranetopology of SecG coupled with SecA-dependent preprotein transloca-tion. Cell 85:71–81.

Nishiyama K, Hanada M, Tokuda H. 1994. Disruption of the geneencoding p12 (SecG) reveals the direct involvement and importantfunction of SecG in the protein translocation of Escherichia coli atlow temperature. EMBO J 13:3272–3277.

Nishiyama K, Maeda M, Yanagisawa K, Nagase R, Komura H,Iwashita T, et al. 2012. MPIase is a glycolipozyme essential formembrane protein integration. Nat Commun 3:1260.

Nohara T, Nakai M, Goto A, Endo T. 1995. Isolation and characteriza-tion of the cDNA for pea chloroplast SecA. Evolutionary conservationof the bacterial-type SecA-dependent protein transport within chloro-plasts. FEBS Lett 364:305–308.

Nothaft H, Szymanski CM. 2010. Protein glycosylation in bacteria:Sweeter than ever. Nat Rev Miocobiol 8:765–778.

Nouwen N, de Kruijff B, Tommassen J. 1996. DmH+ dependency ofin vitro protein translocation into Escherichia coli inner-membranevesicles varies with the signal-sequence core-region composition.Mol Microbiol 19:1205–1214.

Nouwen N, Driessen AJM. 2002. SecDFyajC forms a heterotetramericcomplex with YidC. Mol Microbiol 44:1397–1405.

Nyathi Y, Wilkinson BM, Pool MR. 2013. Co-translational targetingand translocation of proteins to the endoplasmic reticulum. BiochimBiophys Acta 1833:2392–2402.

O’Neil K, DeGrado WF. 1990. How calmodulin binds its targets:Sequence independent recognition of amphiphilic alpha-helices.Trends Biochem Sci 15:59–64.

Ogg SC, Poritz MA, Walter P. 1992. Signal recognition particle receptoris important for cell growth and protein secretion in Saccharomycescerevisiae. Mol Cell Biol 3:895–911.

Ogg SC, Walter P. 1995. SRP samples nascent chains for the presenceof signal sequences by interacting with ribosomes at a discrete stepduring translation elongation. Cell 81:1075–1084.

Oh E, Becker AH, Sandikci A, Nichols RJ, Typas A, Gross CA, et al.2011. Selective ribosome profiling reveals the cotranslationalchaperone action of trigger factor in vivo. Cell 147:1295–1308.

Ojemalm K, Botelho SC, Studle C, von Heijne G. 2013. Quantitativeanalysis of SecYEG-mediated insertion of transmembrane a-helicesinto the bacterial inner membrane. J Mol Biol 425:2813–2822.

80 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Ojemalm K, Halling KK, Nilsson I, von Heijne G. 2012. Orientationalpreferences of neighboring helices can drive ER insertion of amarginally hydrophobic transmembrane helix. Mol Cell 45:529–540.

Oliver DB, Beckwith J. 1981. E. coli mutant pleiotropically defectivein the export of secreted proteins. Cell 25:765–772.

Osborne AR, Rapoport TA. 2007. Protein translocation is mediated byoligomers of the SecY complex with one SecY copy forming thechannel. Cell 129:97–110.

Osborne R, Silhavy TJ. 1993. PrlA suppressor mutations cluster inregions corresponding to three distinct topological domains. EMBO J12:3391–3398.

Paetzel M, Dalbey RE, Strynadka NCJ. 1998. Crystal structure of abacterial signal peptidase in complex with a [beta]-lactam inhibitor.Nature 396:186–190.

Palacios IM. 2007. How does an mRNA find its way? Intracellularlocalisation of transcripts. Semin Cell Develop Biol 18:163–170.

Palmer T, Berks BC. 2012. The twin-arginine translocation (Tat) proteinexport pathway. Nat Rev Miocobiol 10:483–496.

Panzner S, Dreier L, Hartmann E, Kostka S, Rapoport TA. 1995.Posttranslational protein transport in yeast reconstituted with apurified complex of Sec proteins and Kar2p. Cell 81:561–570.

Papanikou E, Karamanou S, Economou A. 2007. Bacterial proteinsecretion through the translocase nanomachine. Nat Rev Miocobiol 5:839–851.

Park E, Menetret J-F, Gumbart JC, Ludtke SJ, Li W, Whynot A, et al.2014. Structure of the SecY channel during initiation of proteintranslocation. Nature 506:102–106.

Park E, Rapoport TA. 2012. Mechanisms of Sec61/SecY-mediatedprotein translocation across membranes. Annu Rev Biophys 41:21–40.

Parlitz R, Eitan A, Stjepanovic G, Bahari L, Bange G, Bibi E, Sinning I.2007. Escherichia coli signal recognition particle receptor FtsYcontains an essential and autonomous membrane-binding amphipathichelix. J Biol Chem 282:32176–32184.

Pfeffer S, Brandt F, Hrabe T, Lang S, Eibauer M, Zimmermann R,Forster F. 2012. Structure and 3D arrangement of endoplasmicreticulum membrane-associated ribosomes. Structure 20:1508–1518.

Pfeffer S, Dudek J, Gogala M, Schorr S, Linxweiler J, Lang S, et al.2014. Structure of the mammalian oligosaccharyl-transferase complexin the native ER protein translocon. Nat Commun 5:3072.

Plath K, Mothes W, Wilkinson BM, Stirling CJ, Rapoport TA. 1998.Signal sequence recognition in posttranslational protein transportacross the yeast ER membrane. Cell 94:795–807.

Plath K, Wilkinson BM, Stirling CJ, Rapoport TA. 2004. Interactionsbetween Sec complex and prepro-alpha-factor during posttranslationalprotein transport into the endoplasmic reticulum. Mol Cell Biol 15:1–10.

Pogliano JA, Beckwith J. 1994. SecD and SecF facilitate protein exportin Escherichia coli. EMBO J 13:554–561.

Pohlschroder M, Hartmann E, Hand NJ, Dilks K, Haddad A. 2005.Diversity and evolution of protein translocation. Annu Rev Microbiol59:91–111.

Pool MR. 2009. A trans-membrane segment inside the ribosome exittunnel triggers RAMP4 recruitment to the Sec61p translocase. J CellBiol 185:889–902.

Poritz M, Bernstein H, Strub K, Zopf D, Wilhelm H, Walter P. 1990. AnE. coli ribonucleoprotein containing 4.5S RNA resembles mammaliansignal recognition particle. Science 250:1111–1117.

Poritz MA, Siegel V, Hansen W, Walter P. 1988a. Small ribonucleopro-teins in Schizosaccharomyces pombe and Yarrowia lipolyticahomologous to signal recognition particle. Proc Natl Acad Sci USA85:4315–4319.

Poritz MA, Strub K, Walter P. 1988b. Human SRP RNA and E. coli 4.5SRNA contain a highly homologous structural domain. Cell 55:4–6.

Potter MD, Nicchitta CV. 2000. Regulation of ribosome detachmentfrom the mammalian endoplasmic reticulum membrane. J Biol Chem275:33828–33835.

Potter MD, Nicchitta CV. 2002. Endoplasmic reticulum-bound ribo-somes reside in stable association with the translocon followingtermination of protein synthesis. J Biol Chem 277:23314–23320.

Powers T, Walter P. 1997. Co-translational protein targeting catalyzedby the Escherichia coli signal recognition particle and its receptor.EMBO J 16:4880–4886.

Prilusky J, Bibi E. 2009. Studying membrane proteins through the eyesof the genetic code revealed a strong uracil bias in their codingmRNAs. Proc Natl Acad Sci USA 106:6662–6666.

Prinz A, Behrens C, Rapoport TA, Hartmann E, Kalies K-UU.2000. Evolutionarily conserved binding of ribosomes to the trans-location channel via the large ribosomal RNA. EMBO J 19:1900–1906.

Raden D, Song W, Gilmore R. 2000. Role of the cytoplasmic segmentsof Sec61a in the ribosome-binding and translocation-promotingactivities of the Sec61 complex. J Cell Biol 150:53–64.

Raetz CR. 1978. Enzymology, genetics, and regulation of membranephospholipid synthesis in Escherichia coli. Microbiol Rev 42:614–659.

Randall LL, Hardy SJS. 1977. Synthesis of exported proteins bymembrane-bound polysomes from Escherichia coli. Eur J Biochem75:43–53.

Randall LL, Topping TB, Smith VF, Diamond DL, Hardy SJ 1998.SecB: A chaperone from Escherichia coli. Methods Enzymol 290:444–459.

Rapiejko PJ, Gilmore R. 1997. Empty site forms of the SRP54 and SRalpha GTPases mediate targeting of ribosome-nascent chain com-plexes to the endoplasmic reticulum. Cell 89:703–713.

Rapp M, Granseth E, Seppala S, von Heijne G. 2006. Identification andevolution of dual-topology membrane proteins. Nat Struct Mol Biol13:112–116.

Raue U, Oellerer S, Rospert S. 2007. Association of protein biogenesisfactors at the yeast ribosomal tunnel exit is affected by thetranslational status and nascent polypeptide sequence. J Biol Chem282:7809–7816.

Reid DW, Nicchitta CV. 2012. Primary role for endoplasmic reticulum-bound ribosomes in cellular translation identified by ribosomeprofiling. J Biol Chem 287:5518–5527.

Reithinger JH, Kim JEH, Kim H. 2013. Sec62 protein mediatesmembrane insertion and orientation of moderately hydrophobicsignal anchor proteins in the endoplasmic reticulum (ER). J BiolChem 288:18058–18067.

Rensing SA, Maier UG. 1994. The SecY protein family: Comparativeanalysis and phylogenetic relationships. Mol Phylogen Evol 3:187–191.

Rietveld AG, Koorengevel MC, de Kruijff B. 1995. Non-bilayer lipidsare required for efficient protein transport across the plasmamembrane of Escherichia coli. EMBO J 14:5506–5513.

Romisch K, Ribes V, High S, Lutcke H, Tollervey D, Dobberstein B.1990. Structure and function of signal recognition particle (SRP). MolBiol Rep 14:71–72.

Saaf A, Andersson H, Gafvelin G, Heijnet Gv. 2009. SecA-dependenceof the translocation of a large periplasmic loop in the Escherichia coliMalF inner membrane protein is a function of sequence context. MolMembr Biol 12:209–215.

Sachelaru I, Petriman NA, Kudva R, Kuhn P, Welte T, Knapp B, et al.2013. YidC occupies the lateral gate of the SecYEG translocon and issequentially displaced by a nascent membrane protein. J Biol Chem288:16295–16307.

Sadlish H, Pitonzo D, Johnson AE, Skach WR. 2005. Sequential triageof transmembrane segments by Sec61alpha during biogenesis of anative multispanning membrane protein. Nat Struct Mol Biol 12:870–878.

Samuelson JC, Jiang F, Yi L, Chen M, de Gier JW, Kuhn A, Dalbey RE.2001. Function of YidC for the insertion of M13 procoat protein inEscherichia coli: Translocation of mutants that show differencesin their membrane potential dependence and Sec requirement. J BiolChem 276:34847–34852.

Sandikci A, Gloge F, Martinez M, Mayer MP, Wade R, Bukau B, KramerG. 2013. Dynamic enzyme docking to the ribosome coordinatesN-terminal processing with polypeptide folding. Nat Struct Mol Biol20:843–850.

Satoh Y, Matsumoto G, Mori H, Ito K. 2003. Nearest neighbor analysisof the SecYEG complex. 1. Identification of a SecY-SecG interface.Biochemistry 42:7434–7441.

Saurı A, McCormick PJ, Johnson AE, Mingarro I. 2007. Sec61a andTRAM are sequentially adjacent to a nascent viral membrane proteinduring its ER integration. J Mol Biol 366:366–374.

Schachter H, Freeze HH. 2009. Glycosylation diseases: Quo vadis?Biochim Biophys Acta 1792:925–930.

Schafer U, Beck K, Muller M. 1999. Skp, a molecular chaperone ofgram-negative bacteria, is required for the formation of solubleperiplasmic intermediates of outer membrane proteins. J Biol Chem274:24567–24574.

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 81

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Schaffitzel C, Oswald M, Berger I, Ishikawa T, Abrahams JP, KoertenHK, et al. 2006. Structure of the E. coli signal recognition particlebound to a translating ribosome. Nature 444:503–506.

Schaletzky J, Rapoport TA. 2006. Ribosome binding to and dissociationfrom translocation sites of the endoplasmic reticulum membrane.Mol Cell Biol 17:3860–3869.

Schatz PJ, Beckwith J. 1990. Genetic analysis of protein export inEscherichia coli. Annu Rev Genet 24:215–248.

Schatz PJ, Bieker KL, Ottemann KM, Silhavy TJ, Beckwith J. 1991.One of three transmembrane stretches is sufficient for the functioningof the SecE protein, a membrane component of the E. coli secretionmachinery. EMBO J 10:1749–1757.

Schauble N, Lang S, Jung M, Cappel S, Schorr S, Ulucan O, et al. 2012.BiP-mediated closing of the Sec61 channel limits Ca2+ leakage fromthe ER. EMBO J 31:3282–3296.

Schiebel E, Driessen AJM, Hartl F-U, Wickner W. 1991. mH+ and ATPfunction at different steps of the catalytic cycle of preproteintranslocase. Cell 64:927–939.

Schlenker O, Hendricks A, Sinning I, Wild K. 2006. The structure of themammalian signal recognition particle (SRP) receptor as prototype forthe interaction of small GTPases with Longin domains. J Biol Chem281:8898–8906.

Schlunzen F, Wilson DN, Tian P, Harms JM, McInnes SJ, Hansen HA,et al. 2005. The binding mode of the trigger factor on the ribosome:implications for protein folding and SRP interaction. Structure 13:1685–1694.

Schroder K, Martoglio B, Hofmann M, Holscher C, Hartmann E,Prehn S, et al. 1999. Control of glycosylation of MHC class II-associated invariant chain by translocon-associated RAMP4. EMBO J18:4804–4815.

Schwartz T, Blobel G. 2003. Structural basis for the function of the bsubunit of the eukaryotic signal recognition particle receptor. Cell112:793–803.

Schwarz F, Aebi M. 2011. Mechanisms and principles of N-linkedprotein glycosylation. Curr Opin Struct Biol 21:576–582.

Seitl I, Wickles S, Beckmann R, Kuhn A, Kiefer D. 2013. TheC-terminal regions of YidC from Rhodopirellula baltica andOceanicaulis alexandrii bind to ribosomes and partially substitutefor SRP receptor function in Escherichia coli. Mol Microbiol 91:408–421.

Shao S, Hegde RS. 2011. Membrane protein insertion at the endoplasmicreticulum. Annu Rev Cell Dev Biol 27:25–56.

Shaw AS, Rottier PJ, Rose JK. 1988. Evidence for the loop model ofsignal-sequence insertion into the endoplasmic reticulum. Proc NatlAcad Sci USA 85:7592–7596.

Shibatani T, David LL, McCormack AL, Frueh K, Skach WR. 2005.Proteomic analysis of mammalian oligosaccharyltransferase revealsmultiple subcomplexes that contain Sec61, TRAP, and two potentialnew subunits. Biochemistry 44:5982–5992.

Shiozuka K, Tani K, Mizushima S, Tokuda H. 1990. The proton motiveforce lowers the level of ATP required for the in vitro translocationof a secretory protein in Escherichia coli. J Biol Chem 265:18843–18847.

Siegel V, Walter P. 1985. Elongation arrest is not a prerequisite forsecretory protein translocation across the microsomal membrane.J Cell Biol 100:1913–1921.

Siegel V, Walter P. 1986. Removal of the Alu structural domain fromsignal recognition particle leaves its protein translocation activityintact. Nature 320:81–84.

Skowronek MH, Rotter M, Haas IG. 1999. Molecular characterizationof a novel mammalian DnaJ-like Sec63p homolog. Biol Chem 380:1133–1138.

Smith MA, Clemons WM, DeMars CJ, Flower AM. 2005. Modeling theeffects of prl mutations on the Escherichia coli SecY complex.J Bacteriol 187:6454–6465.

Sommer N, Junne T, Kalies KU, Spiess M, Hartmann E. 2013. TRAPassists membrane protein topogenesis at the mammalian ER mem-brane. Biochim Biophys Acta 1833:3104–3111.

Song W, Raden D, Mandon EC, Gilmore R. 2000. Role of Sec61a in theregulated transfer of the ribosome-nascent chain complex from thesignal recognition particle to the translocation channel. Cell 100:333–343.

Soromani C, Zeng N, Hollemeyer K, Heinzle E, Klein M-C, Tretter T,et al. 2012. N-acetylation and phosphorylation of Sec complexsubunits in the ER membrane. BMC Cell Biol 13:34.

Stephens SB, Dodd RD, Brewer JW, Lager PJ, Keene JD, Nicchitta CV.2005. Stable ribosome binding to the endoplasmic reticulum enablescompartment-specific regulation of mRNA translation. Mol Cell Biol16:5819–5831.

Sugai R, Takemae K, Tokuda H, Nishiyama K-i. 2007. Topologyinversion of SecG is essential for cytosolic SecA-dependent stimu-lation of protein translocation. J Biol Chem 282:29540–29548.

Tai VW, Imperiali B. 2001. Substrate specificity of the glycosyl donorfor oligosaccharyl transferase. J Org Chem 66:6217–6228.

Tamborero S, Vilar M, Martinez-Gil L, Johnson AE, Mingarro I. 2011.Membrane insertion and topology of the translocating chain-associat-ing membrane protein (TRAM). J Mol Biol 406:571–582.

Terzi L, Pool MR, Dobberstein B, Strub K. 2004. Signal recognitionparticle Alu domain occupies a defined site at the ribosomalsubunit interface upon signal sequence recognition. Biochemistry43:107–117.

Tian H, Beckwith J. 2002. Genetic screen yields mutations in genesencoding all known components of the Escherichia coli signalrecognition particle pathway. J Bacteriol 184:111–118.

Tjalsma H, Bolhuis A, van Roosmalen ML, Wiegert T, Schumann W,Broekhuizen CP, et al. 1998. Functional analysis of the secretoryprecursor processing machinery of Bacillus subtilis: Identification of aeubacterial homolog of archaeal and eukaryotic signal peptidases.Genes Develop 12:2318–2331.

Tomkiewicz D, Nouwen N, Driessen AJM. 2007. Pushing, pulling andtrapping – modes of motor protein supported protein translocation.FEBS Lett 581:2820–2828.

Tomkiewicz D, Nouwen N, van Leeuwen R, Tans S, Driessen AJM.2006. SecA supports a constant rate of preprotein translocation. J BiolChem 281:15709–15713.

Tong J, Dolezal P, Selkrig J, Crawford S, Simpson AGB, Noinaj N, et al.2011. Ancestral and derived protein import pathways in themitochondrion of Reclinomonas americana. Mol Biol Evol 28:1581–1591.

Trager C, Rosenblad MA, Ziehe D, Garcia-Petit C, Schrader L, Kock K,et al. 2012. Evolution from the prokaryotic to the higher plantchloroplast signal recognition particle: The signal recognition particleRNA is conserved in plastids of a wide range of photosyntheticorganisms. Plant Cell 24:4819–4836.

Tseng TT, Gratwick KS, Kollman J, Park D, Nies DH, Goffeau A, SaierMH. 1999. The RND permease superfamily: An ancient, ubiquitousand diverse family that includes human disease and developmentproteins. J Mol MicroBio Biotechnol 1:107–125.

Tsukazaki T, Mori H, Echizen Y, Ishitani R, Fukai S, Tanaka T, et al.2011. Structure and function of a membrane component SecDF thatenhances protein export. Nature 474:235–238.

Tsukazaki T, Mori H, Fukai S, Ishitani R, Mori T, Dohmae N, et al.2008. Conformational transition of Sec machinery inferred frombacterial SecYE structures. Nature 455:988–991.

Tyedmers J, Lerner M, Bies C, Dudek J, Skowronek MH, Haas IG, et al.2000. Homologs of the yeast Sec complex subunits Sec62p andSec63p are abundant proteins in dog pancreas microsomes. Proc NatlAcad Sci USA 97:7214–7219.

Tyedmers J, Lerner M, Wiedmann M, Volkmer J, Zimmermann R. 2003.Polypeptide-binding proteins mediate completion of co-translationalprotein translocation into the mammalian endoplasmic reticulum.EMBO Rep 4:505–510.

Tyson JR, Stirling CJ. 2000. LHS1 and SIL1 provide a lumenal functionthat is essential for protein translocation into the endoplasmicreticulum. EMBO J 19:6440–6452.

Ullers RS, Houben EN, Raine A, ten Hagen-Jongman CM, Ehrenberg M,Brunner J, et al. 2003. Interplay of signal recognition particle andtrigger factor at L23 near the nascent chain exit site on the Escherichiacoli ribosome. J Cell Biol 161:679–684.

Urbanus ML, Scotti PA, Froderberg L, Saaf A, de Gier JW, Brunner J,et al. 2001. Sec-dependent membrane protein insertion: sequentialinteraction of nascent FtsQ with SecY and YidC. EMBO Rep 2:524–529.

Valencia-Burton M, McCullough RM, Cantor CR, Broude NE. 2007.RNA visualization in live bacterial cells using fluorescent proteincomplementation. Nat Meth 4:421–427.

Valent Q, Kendall D, High S, Kusters R, Oudega B, Luirink J. 1995.Early events in preprotein recognition in E. coli: interaction ofSRP and trigger factor with nascent polypeptides. EMBO J 14:5494–5505.

82 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Valent QA, Scotti PA, High S, de Gier JW, von Heijne G, Lentzen G,et al. 1998. The Escherichia coli SRP and SecB targeting pathwaysconverge at the translocon. EMBO J 17:2504–2512.

van Bloois E, Dekker HL, Froderberg L, Houben EN, Urbanus ML,de Koster CG, et al. 2008. Detection of cross-links between FtsH,YidC, HflK/C suggests a linked role for these proteins in qualitycontrol upon insertion of bacterial inner membrane proteins. FEBSLett 582:1419–1424.

van Dalen A, de Kruijff B. 2004. The role of lipids in membraneinsertion and translocation of bacterial proteins. Biochim BiophysActa – Mol Cell Res 1694:97–109.

Van den Berg B, Clemons WM, Collinson I, Modis Y, Hartmann E,Harrison SC, Rapoport TA. 2004. X-ray structure of a protein-conducting channel. Nature 427:36–44.

van der Does C, Swaving J, van Klompenburg W, Driessen AJM. 2000.Non-bilayer lipids stimulate the activity of the reconstituted bacterialprotein translocase. J Biol Chem 275:2472–2478.

van der Laan M, Nouwen N, Driessen AJM. 2004. SecYEG proteolipo-somes catalyze the Deltaphi-dependent membrane insertion of FtsQ.J Biol Chem 279:1659–1664.

van der Sluis EO, Driessen AJM. 2006. Stepwise evolution of the Secmachinery in proteobacteria. Trends Microbiol 14:105–108.

van der Sluis EO, Nouwen N, Driessen AJM. 2002. SecY–SecY andSecY–SecG contacts revealed by site-specific crosslinking. FEBS Lett527:159–165.

van der Sluis EO, van der Vries E, Berrelkamp G, Nouwen N,Driessen AJM. 2006. Topologically fixed SecG is fully functional.J Bacteriol 188:1188–1190.

van Meer G, Voelker DR, Feigenson GW. 2008. Membrane lipids:Where they are and how they behave. Nat Rev Mol Cell biol 9:112–124.

VanValkenburgh C, Chen X, Mullins C, Fang H, Green N. 1999.The catalytic mechanism of endoplasmic reticulum signal peptidaseappears to be distinct from most eubacterial signal peptidases. J BiolChem 274:11519–11525.

Vassylyev DG, Mori H, Vassylyeva MN, Tsukazaki T, Kimura Y,Tahirov TH, Ito K. 2006. Crystal structure of the translocation ATPaseSecA from Thermus thermophilus reveals a parallel, head-to-headdimer. J Mol Biol 364:248–258.

Vitrac H, Bogdanov M, Heacock P, Dowhan W. 2011. Lipids andtopological rules of membrane protein assembly: balance betweenlong and short range lipid-protein interactions. J Biol Chem 286:15182–15194.

von Heijne G. 1984. Analysis of the distribution of charged residuesin the N-terminal region of signal sequences: Implications forprotein export in prokaryotic and eukaryotic cells. EMBO J 3:2315–2318.

von Heijne G. 1985. Signal sequences. The limits of variation. J Mol Biol184:99–105.

von Heijne G. 1986. A new method for predicting signal sequencecleavage sites. Nucleic Acids Res 14:4683–4690.

von Heijne G. 1990. The signal peptide. J Membr Biol 115:195–201.von Heijne G, Gavel Y. 1988. Topogenic signals in integral membrane

proteins. Eur J Biochem 174:671–678.Voss M, Schroder B, Fluhrer R. 2013. Mechanism, specificity, and

physiology of signal peptide peptidase (SPP) and SPP-like proteases.Biochim Biophys Acta – Biomembr 1828:2828–2839.

Walter P, Blobel G. 1980. Purification of a membrane-associated proteincomplex required for protein translocation across the endoplasmicreticulum. Proc Natl Acad Sci USA 77:7112–7116.

Walter P, Blobel G. 1981. Translocation of proteins across theendoplasmic reticulum III. Signal recognition protein (SRP) causessignal sequence-dependent and site-specific arrest of chain elong-ation that is released by microsomal membranes. J Cell Biol 91:557–561.

Walter P, Blobel G. 1982. Signal recognition particle contains a 7S RNAessential for protein translocation across the endoplasmic reticulum.Nature 299:691–698.

Walter P, Ibrahimi I, Blobel G. 1981. Translocation of proteins across theendoplasmic reticulum. I. Signal recognition protein (SRP) binds toin-vitro-assembled polysomes synthesizing secretory protein. J CellBiol 91:545–550.

Wang L, Dobberstein B. 1999. Oligomeric complexes involved intranslocation of proteins across the membrane of the endoplasmicreticulum. FEBS Lett 457:316–322.

Wang P, Shim E, Cravatt B, Jacobsen R, Schoeniger J, Kim AC, et al.2008. Escherichia coli signal peptide peptidase A is a serine-lysine protease with a lysine recruited to the nonconserved amino-terminal domain in the S49 protease family. Biochemistry 47:6361–6369.

Watanabe M, Blobel G. 1989. SecB functions as a cytosolic signalrecognition factor for protein export in E. coli. Cell 58:695–705.

Weiche B, Burk J, Angelini S, Schiltz E, Thumfart JO, Koch HG. 2008.A cleavable N-terminal membrane anchor is involved in membranebinding of the Escherichia coli SRP receptor. J Mol Biol 377:761–773.

Welte T, Kudva R, Kuhn P, Sturm L, Braig D, Muller M, et al. 2012.Promiscuous targeting of polytopic membrane proteins to SecYEG orYidC by the Escherichia coli signal recognition particle. Mol CellBiol 23:464–479.

Whitehouse S, Gold VAM, Robson A, Allen WJ, Sessions RB, CollinsonI. 2012. Mobility of the SecA 2-helix-finger is not essential forpolypeptide translocation via the SecYEG complex. J Cell Biol 199:919–929.

Wild K, Sinning I, Cusack S. 2001. Crystal structure of an early protein-RNA assembly complex of the signal recognition particle. Science294:598–601.

Wirth A, Jung M, Bies C, Frien M, Tyedmers J, Zimmermann R, WagnerR. 2003. The Sec61p complex is a dynamic precursor activatedchannel. Mol Cell 12:261–268.

Wittke S, Dunnwald M, Johnsson N. 2000. Sec62p, a component of theendoplasmic reticulum protein translocation machinery, containsmultiple binding sites for the Sec-complex. Mol Cell Biol 11:3859–3871.

Wu ZC, de Keyzer J, Kedrov A, Driessen AJM. 2012. Competitivebinding of the SecA ATPase and ribosomes to the SecYEGtranslocon. J Biol Chem 287:7885–7895.

Xing L, Bassell GJ. 2013. mRNA localization: An orchestration ofassembly, traffic and synthesis. Traffic 14:2–14.

Xu Z, Knafels JD, Yoshino K. 2000. Crystal structure of thebacterial protein export chaperone SecB. Nat Struct Mol Biol 7:1172–1177.

Yamaguchi A, Hori O, Stern DM, Hartmann E, Ogawa S, Tohyama M.1999. Stress-associated endoplasmic reticulum protein 1 (Serp1)/ribosome-associated membrane protein 4 (Ramp4) stabilizes mem-brane proteins during stress and facilitates subsequent glycosylation.J Cell Biol 147:1195–1204.

Yamamoto H, Fujita H, Kida Y, Sakaguchi M. 2012. Pleiotropic effectsof membrane cholesterol upon translocation of protein across theendoplasmic reticulum membrane. Biochemistry 51:3596–3605.

Yamamoto H, Kida Y, Sakaguchi M. 2013. Phosphatidylserine-bindingprotein lactadherin inhibits protein translocation across the ERmembrane. Biochem Biophys Res Commun 434:620–626.

Young BP. 2001. Sec63p and Kar2p are required for the translocation ofSRP-dependent precursors into the yeast endoplasmic reticulumin vivo. EMBO J 20:262–271.

Yuan J, Zweers J, Dijl J, Dalbey R. 2010. Protein transport across andinto cell membranes in bacteria and archaea. Cell Mol Life Sci 67:179–199.

Zafar S, Nasir A, Bokhari H. 2011. Computational analysis revealsabundance of potential glycoproteins in Archaea, Bacteria andEukarya. Bioinformation 6:352–355.

Zhang X, Rashid R, Wang K, Shan So. 2010. Sequential checkpointsgovern substrate selection during cotranslational protein targeting.Science 328:757–760.

Zhang X, Schaffitzel C, Ban N, Shan S-o. 2009a. Multiple conform-ational switches in a GTPase complex control co-translational proteintargeting. Proc Natl Acad Sci USA 106:1754–1759.

Zhang Y-J, Tian H-F, Wen J-F. 2009b. The evolution of YidC/Oxa/Alb3family in the three domains of life: A phylogenomic analysis. BMCEvol Biol 9:137.

Zhao X, Jantti J. 2009. Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocationcomplex beta-subunit. BMC Cell Biol 10:76.

Zheng N, Gierasch LM. 1997. Domain interactions in E. coli SRP:stabilization of M domain by RNA is required for effective signalsequence modulation of NG domain. Mol Cell 1:79–87.

Zhou Y, Xue S, Yang JJ. 2013. Calciomics: Integrative studies of Ca2+-binding proteins and their interactomes in biological systems.Metallomics 5:29–42.

DOI: 10.3109/09687688.2014.907455 Eukaryotic and prokaryotic Sec translocon 83

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018

Zimmer J, Nam Y, Rapoport TA. 2008. Structure of a complex of theATPase SecA and the protein-translocation channel. Nature 455:936–943.

Zimmermann R, Eyrisch S, Ahmad M, Helms V. 2011. Proteintranslocation across the ER membrane. Biochim Biophys Acta1808:912–924.

Zimmermann R, Sagstetter M, Lewis M, Pelham HR. 1988. Seventy-kilodalton heat shock proteins and an additional component fromreticulocyte lysate stimulate import of M13 procoat protein intomicrosomes. EMBO J 7:2875–2880.

Zopf D, Bernstein H, Johnson A, Walter P. 1990. The methionine-richdomain of the 54 kd protein subunit of the signal recognition particle

contains an RNA binding site and can be crosslinked to a signalsequence. EMBO J 9:4511–4517.

Zopf D, Bernstein HD, Walter P. 1993. GTPase domain of the 54-kDsubunit of the mammalian signal recognition particle is required forprotein translocation but not for signal sequence binding. J Cell Biol120:1113–1121.

Zwieb C, Bhuiyan S. 2010. Archaea signal recognition particle shows theway. Archaea 2010:485051.

Zwizinski C, Wickner W. 1980. Purification and characterization ofleader (signal) peptidase from Escherichia coli. J Biol Chem 255:7973–7977.

84 K. Denks et al. Mol Membr Biol, 2014; 31(2–3): 58–84

Dow

nloa

ded

by [S

mith

soni

an A

strop

hysic

s Obs

erva

tory

] at 1

3:05

10

Janu

ary

2018