Tracing the Retrograde Route in Protein Trafficking · 1176 Cell 135, December 26, 2008 ©2008...

Leading Edge

Review

Tracing the Retrograde Route in Protein TraffickingLudger Johannes1,2,* and Vincent Popoff1,2

1CNRS UMR1442Institut CurieCentre de Recherche, Traffic, Signaling, and Delivery Laboratory, 75248 Paris Cedex 05, France*Correspondence: [email protected] 10.1016/j.cell.2008.12.009

Retrograde transport, in which proteins and lipids are shuttled between endosomes and biosyn-thetic/secretory compartments such as the Golgi apparatus, is crucial for a diverse range of cellular functions. Mechanistic studies that explore the molecular machinery involved in this retrograde trafficking route are shedding light on the functions of transport proteins and are providing fresh insights into possible new therapeutic directions.

IntroductionEndocytosis at the plasma membrane and endosomal pro-tein sorting are critical events for diverse cellular functions such as signaling, nutrient uptake, and development. Of these processes, the mechanisms underlying clathrin-dependent and -independent internalization (Conner and Schmid, 2003; Kirkham and Parton, 2005; Mayor and Pagano, 2007), recep-tor recycling to the plasma membrane (Maxfield and McGraw, 2004), and membrane trafficking to compartments of the late endocytic pathway (Gruenberg and Stenmark, 2004) have been studied in some detail. In contrast, much less is known about endosomal trafficking to the biosynthetic/secretory compart-ments, which is known as retrograde transport (Figure 1). Exit from endosomal compartments is termed retrograde sorting and occurs in different parts of the endocytic pathway. Retro-grade sorting acts on cargo molecules targeted to endosomes from the Golgi apparatus or the plasma membrane. These cargo proteins or lipids are then transported to the trans-Golgi network (TGN), Golgi membranes, or, in some cases, the endo-plasmic reticulum (ER) via the retrograde route (Bonifacino and Rojas, 2006; Lamaze and Johannes, 2006; Sandvig and van Deurs, 2005). This transport route is also co-opted by patho-gens during their entry into cells. Thus, structural and mecha-nistic studies on pathogen products and cellular factors have led to the first molecular models for trafficking between endo-somes and the TGN. Distinct features of trafficking by the ret-rograde route are providing fresh avenues for developing new therapeutic strategies in immunotherapy and drug delivery.

Cellular Functions of Retrograde TransportThe list of proteins that traffic between endosomes and the TGN is expanding rapidly, as is the number of their functions (Table 1). The best-studied examples of retrograde cargo pro-teins include acid hydrolase sorting receptors such as Vps10 from the budding yeast Saccharomyces cerevisae and the two types of mannose 6-phosphate receptors (MPRs) in mammals (cation-dependent and cation-independent). Sorting recep-tors mediate membrane exchanges between the biosyn-

Figure 1. Transport Factors in Retrograde TraffickingRetrograde trafficking can occur at different levels of the endosome (early, late, recycling) to the trans-Golgi network (TGN) and the Golgi apparatus. With the aid of adaptor proteins such as AP-1 and epsinR, membrane coat proteins such as clathrin, the retromer complex, and TIP47 promote the formation of trans-port intermediates containing cargo proteins. The membrane fission GTPase dynamin and lipid biosynthesis proteins such as OCRL1 have functions during this process. The retrograde transport of cargo proteins can take different routes through the endosomal compartments. Tethering, docking, and fusion of retro-grade transport intermediates with the TGN depend on a wide range of regulatory factors. Tethering factors such as golgin-97, golgin-245, GCC88, and GCC185 mediate the initial step of vesicle-organelle interaction. SNARE (soluble N-ethyl-maleimide-sensitive fusion factor attachment receptor) complexes are required for membrane fusion. Tethering and fusion factors can vary between cargos and at different membrane interfaces. For example, SNARE complexes around syn-taxin 5 (Syn5) and syntaxin 6/syntaxin 16 (Syn6/Syn16) are specifically required for retrograde transport of STxB from early endosomes, while a SNARE complex around syntaxin 10 drives fusion for retrograde transport intermediates transport-ing MPRs from late endosomes.

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Table 1. Cargo Proteins of the Retrograde Route

Cargo Cellular Function Role of Retrograde Transport in Cargo Function

Mammalian Proteins

MPR Lysosomal enzyme trafficking Receptor recycling from endosomes to the TGN

Sortilin, SorLAs, SorCSs Multiligand carriers Receptor recycling from endosomes to the TGN

Wntless Wingless (morphogen)-specific transport factor Receptor recycling from endosomes to the TGN

VAMP4 SNARE Steady-state localization

GS15 SNARE Steady-state localization

Furin Proenzyme maturation Enzyme recycling from endosomes to the TGN

MT-MMP Cleavage of extracellular matrix components Enzyme recycling from endosomes to the TGN

P-selectin Cell adhesion Recycling to TGN for repackaging into new secretory granules

GLUT4 Glucose transporter Recycling to TGN for repackaging into new storage vesicles

BACE1 Processing of beta amyloid precursor protein Steady-state localization

BACE2 Processing of beta amyloid precursor protein Steady-state localization

Menkes protein Copper transporter Functional localization to cellular compartments

Atg9 Autophagy Functional localization to cellular compartments

EGFR Signaling receptor Membrane translocation

Soluble antigens Crosspresentation Membrane translocation

SUMF1 Posttranslational modification Functional recycling to the ER

TGN38/46 Unknown Unknown

Yeast Proteins

Vps10 Vacuolar enzyme trafficking Receptor recycling from endosomes to the TGN

Kex2 Proenzyme maturation (furin ortholog) Enzyme recycling from endosomes to the TGN

Chs3 Chitin synthesis Recycling to TGN, possibly for polarized secretion

Snc1 SNARE Steady-state localization

Tlg1 SNARE Steady-state localization

Pep12 SNARE Steady-state localization

Fet3/Ftr1 Iron transporter Functional localization to cellular compartments

Plant Proteins

VSR1/ELP Seed storage protein Receptor recycling from storage vacuoles to TGN

Pathogen-Produced Proteins

Shiga toxin, Cholera toxin, ricin Toxins Access to cytosol

HIV-1 Nef Immune evasion Downregulation of MHC-I, CD80, CD86

HIV-1 Env Virus envelope Virus assembly, surface depletion of adhesion factor

HSV-1 gM Virus envelope Surface depletion of adhesion factor

AAV5 Virus Unknown

Retrograde transport is required for a wide range of processes such as receptor trafficking, cell signaling, transporter activity control, antigen cross-presentation, and pathogen entry. The transit of cargo (proteins or lipids) through the retrograde route can allow targeting of cargo to specific biosyn-thetic/secretory compartments, promote steady-state localization of cargo, or serve as a step in a regulatory circuit mediating cargo function. TGN, trans-Golgi network; MPR, mannose 6-phosphate receptor; MT-MMP, membrane-type matrix metalloproteases; SNARE, soluble N-ethylmaleimide-sensitive fusion factor attachment receptor; SUMF1, sulfatase modifying factor 1.

thetic/secretory and endocytic pathways. Newly synthesized acid hydrolase precursor proteins are captured by sorting receptors in TGN membranes and transferred to endosomes. Once at the endosome, the acidic pH of the endosomal com-partment results in the dissociation of the receptor-ligand complexes (Munier-Lehmann et al., 1996) and the transport of the hydrolase precursors by general membrane flow to late endosomes/lysosomes for processing into mature enzymes. The receptors, on the other hand, are recycled to the TGN. This housekeeping mechanism appears to be conserved for the mammalian family of multiligand type-I receptors (such as sortilin, sorLA, sorCS) (Mari et al., 2008) that have homology

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to Vps10, as well as plant seed storage proteins such as the VSR1/ELP protein of the plant Arabidopsis thaliana (Yamazaki et al., 2008).

Cycling between endosomes and the TGN has also been described for a number of other proteins such as the mam-malian membrane fusion receptors or SNAREs (soluble N-ethylmaleimide-sensitive fusion factor attachment recep-tors) VAMP4 and GS15, the yeast SNAREs Tlg1p, Pep12p, and Snc1, the precursor protein processing convertase furin, and the transmembrane cell adhesion protein P-selectin (Straley and Green, 2000; Bonifacino and Rojas, 2006). In some cases, the cycling of specific cargo through the retrograde route is a

crucial part of a regulatory circuit (Table 1). For example, the trafficking of a specific sorting receptor through the retrograde route was recently proposed to be required in the establish-ment of gradients of Wnt family morphogens, which contrib-ute to tissue patterning in many organisms (reviewed in Eaton, 2008). The secretion of Wnt proteins from Wnt-producing cells requires a sorting receptor, Wntless. In the absence of a func-tional retromer, a key component of the retrograde trafficking machinery, Wntless fails to be recycled from the plasma mem-brane to perinuclear TGN membranes. This defect in recycling destabilizes the Wntless protein and results in diminished Wnt secretion from cells. These studies strongly suggest that endo-some-to-TGN trafficking is an integral part of the functional Wntless cycle.

Menkes proteins, components of the mammalian copper transport pathway, also rely on continuous cycling between the plasma membrane and Golgi apparatus for regulation. In cells subjected to low extracellular copper concentrations, exit of Menkes proteins from the TGN is slower than retrograde retrieval from the plasma membrane. Thus, Menkes proteins accumulate at steady state in the TGN. However, when extra-cellular copper concentrations are increased, the rate of Men-kes protein exocytosis is increased and leads to redistribution of Menkes proteins to the plasma membrane. This presumably increases the efficiency of copper removal from cells (Petris and Mercer, 1999). Similar cycles depending on cell stimulation by a specific trafficking signal have also been described in yeast for the reductive iron transporter Fet3p/Ftr1p, whose trans-port is triggered by changes in iron concentration (Strochlic et al., 2007), and the mammalian glucose transporter GLUT4, which uses the hormone insulin as its trafficking signal in adi-pocytes and skeletal muscle cells (Shewan et al., 2003). The yeast autophagy protein 9 (Atg9) also appears to be regulated by cycling through the retrograde transport route, although its precise trafficking inducers have not yet been identified. Under conditions of nutrient abundance, the Atg9 is localized at the TGN. Upon starvation, Atg9 relocalizes to autophagosomes (Young et al., 2006).

Protein transport by the retrograde route to the TGN can also provide a mechanism to establish pools of cargo for “polarized” secretion from specific regions of the cell. The membrane-type matrix metalloproteases (MT-MMPs), which mediate the irre-versible cleavage of extracellular matrix components during angiogenesis or tissue remodeling, are rapidly endocytosed and appear to be recycled to the TGN for packaging into new vesicles. Thus, a dynamic pool of MT-MMP enzymes can be maintained at the TGN for ready deployment to the cell surface (Wang et al., 2004). The localization of these proteases in the invadopodia of motile cells indicates that their secretion from storage vesicles may be polarized. The yeast chitin synthase-III (Chs3) is another protein whose cycling between the plasma membrane, early endosomes, and the TGN likely ensures its polarized localization at the mother cell-bud junction (Valdivia et al., 2002). The findings that the retrograde transport ret-romer complex is required for selective targeting of specific membrane domains is also consistent with a possible link between retrograde retrieval and polarized secretion. In mam-malian polarized epithelial cells, the retromer is required for

the transport of polymeric immunoglobin receptors from the basolateral surface to the apical surface (Verges et al., 2004), whereas in the plant Arabidopsis thaliana, the retromer func-tions in the relocalization of PIN1 proteins from the basal pole to the lateral face of stele cells during the establishment of an auxin gradient (Jaillais et al., 2007). However, there is no direct evidence for the functional involvement of retrograde transport in either of these cases, suggesting that the retromer complex may participate in nonretrograde trafficking events.

Retrograde transport can also promote the transport of some proteins to ER membranes. This has been shown for the mam-malian protein sulfatase modifying factor 1 (SUMF1) in mor-phological and biochemical studies (Zito et al., 2007). SUMF1 requires targeting to the ER membranes in order to interact with its substrates. A similar trafficking pattern has also been proposed for the autocrine motility factor receptor (Benlimame et al., 1998). Retrograde targeting to ER membranes also pro-vides access to the cellular ER-associated protein degradation (ERAD) machinery for protein retrotranslocation to the cytosol. Retrotranslocation has been implicated in specific signaling responses (such as cyclin D expression) induced by the EGF receptor (Liao and Carpenter, 2007). Additionally, retrograde trafficking of soluble exogenous antigenic proteins to the ER in dendritic cells might be important for antigen crosspresen-tation (Ackerman et al., 2005). Following retrotranslocation to the cytosol, antigenic proteins are proteolytically processed by the proteasome. The antigenic peptides are loaded, in a TAP-transporter-dependent manner, onto newly synthesized major histocompatibility complex (MHC) class I proteins in the ER lumen. Whether these retrograde cargo proteins traffic through Golgi membranes to the ER (as has been shown for protein toxins) or whether they bypass Golgi cisternae altogether has not yet been studied directly. It should be noted that alternative routes for the crosspresentation of soluble antigens have also been proposed (Ackerman et al., 2006).

Retrograde Transport and DiseaseEndosome-to-Golgi trafficking is crucial to the cellular entry of certain pathogens or pathogenic products. For example, proper targeting of the Shiga toxin molecule produced by the bacterium Shigella dysenteriae within the host cell requires ret-rograde transport. The nontoxic B-subunit (STxB) of Shiga toxin binds the host cell toxin receptor, the glycosphingolipid Gb3. Following receptor binding, Shiga toxin is endocytosed into early/recycling endosomes and transported to the ER by way of the TGN/Golgi membranes (Lamaze and Johannes, 2006; Sandvig and van Deurs, 2005). In the lumen of the ER, the cata-lytic A-subunit of Shiga toxin exploits the ERAD machinery for its retrotranslocation into the cytosol where it induces cleav-age of ribosomal RNA and halts protein synthesis. Other toxins such as Cholera toxin (CTx) from the bacterium Vibrio cholera, exotoxin from the bacterium Pseudomonas aeruginosa, Shiga toxin-related verotoxins (or Shiga-like toxins) from enterohem-orrhagic strains of the bacterium Escherichia coli, and the plant toxin ricin all show a qualitatively similar trafficking pattern.

Viruses can also rely on retrograde trafficking during the infection of host cells. The HIV-1 envelope protein undergoes retrograde transport, and its interaction with the retrograde


sorting machinery appears to play a role in virus assembly and in the removal of excess fusogenic viral envelope proteins from the plasma membrane (Lopez-Verges et al., 2006). Strikingly, a recent functional genomics screen has uncovered require-ments for several retrograde trafficking factors in HIV infection (Brass et al., 2008). Another HIV-1 protein, nef, promotes virus immune evasion by inducing retrograde trafficking of MHC-I molecules (Blagoveshchenskaya et al., 2002) and costimula-tory molecules (Chaudhry et al., 2008) from the host cell surface to the TGN. Such virus protein-induced transport has further-more been observed for the alphavirus glycoprotein M, which promotes the relocalization of other alphavirus proteins from the plasma membrane to the TGN (Crump et al., 2004). Adeno-associated virus particles have also been detected in the Golgi compartment following their uptake into cells, although the sig-nificance of this localization for infection has yet to be deter-mined (Bantel-Schaal et al., 2002). In contrast, it is clear that trafficking of SV40 virus from the plasma membrane to the ER is critical for its interaction with the ERAD machinery, a prereq-uisite for productive infection (Schelhaas et al., 2007).

Retrograde transport, when disrupted, may play a role in Alzheimer’s disease as well. Genetic variants in the sorting receptor sorLA (a member of the family of multiligand type-I receptors with homology to Vps10) have been found to be linked to late-onset forms of Alzheimer’s disease (Rogaeva et al., 2007). Expression studies have revealed that retromer levels are reduced in certain brain regions vulnerable to the develop-ment of the disease. Cell biology studies further demonstrated that the disruption of retromer activity affects the production of Aß peptide, the core component of amyloid plaques (reviewed in Small, 2008). The retrograde route has also been implicated in the trafficking of the amyloid precursor protein (APP) pro-cessing β-secretases BACE1 (Wahle et al., 2005) and BACE2 (He et al., 2005).

Retrograde Transport ProteinsMechanistic studies using innovative tools (Table S1 available online) have identified elements of the molecular machinery at the endosome-TGN interface involved in diverse processes such as cargo selection, budding, translocation of transport interme-diates across the cytoplasm, vesicle docking, and fusion (Table 2 and Figure 1). We focus here on recent contributions that have allowed the first molecular models for retrograde transport at the endosome-TGN interface to be developed.Clathrin Membrane CoatsClathrin is a widely studied molecular membrane coat protein with a well-established role in the endocytic uptake of plasma membrane receptors. The presence of clathrin on endosomes was debated for some time. A major step toward resolving this issue was the detection of clathrin on pit-like structures on early endosomal membranes using electron microscopy and the whole-mount technique (Table S1) (Stoorvogel et al., 1996). Ultrastructural studies also revealed flat endosomal lattices composed of clathrin on maturing vacuolar endosomal struc-tures called coated endosomes (Burke et al., 1982).

The role of clathrin in retrograde transport between endo-somes and the TGN has been addressed using the B-subunit of Shiga toxin as a tracer. These experiments revealed that


the disruption of clathrin activity by various means inhibits STxB from exiting early/recycling endosomes, indicating that clathrin and its binding partner epsinR are required for retro-grade trafficking (Lauvrak et al., 2004; Saint-Pol et al., 2004). STxB has become a versatile tool for use in morphological and biochemical studies of the retrograde route (Amessou et al., 2006) (Table S1). Its principal strengths as a tool reside in its unidirectional trafficking pattern (unlike endogenous cargo molecules that undergo both anterograde and retrograde transport) and its large number of cellular binding sites that ensure sufficient signal strength in experiments. Although a majority of retrograde factors identified using STxB have sub-sequently been shown to be required for retrograde traffick-ing of endogenous proteins (Table 2), the fact that STxB is not a passive membrane cargo must be taken into consideration (Römer et al., 2007). Indeed, when bound to its glycolipid receptor Gb3, STxB can induce plasma membrane curvature and may exert effects on transport machinery to modulate its own transport.

The requirement for clathrin-binding proteins in retrograde transport from the early endosome to the TGN has been demonstrated for endogenous cargos as well. The retrograde transport of the β-secretases BACE1 and BACE2 requires the monomeric clathrin adaptors of the GGA (Golgi-localized, gamma-ear-containing, ARF-binding protein) family, which are present on early endosomal membranes and the TGN (He et al., 2005; Wahle et al., 2005). Furthermore, in cells defi-cient for the µ1A subunit of the clathrin adaptor AP-1, MPRs are no longer recycled to the TGN and accumulate in early endosomal structures (Meyer et al., 2000). Membrane prepa-rations from cells lacking AP-1 also did not support efficient retrograde MPR transport in a cell-free assay (Medigeshi and Schu, 2003). However, other experiments suggest that the requirement for clathrin may differ depending on the route of retrograde transport. Indeed, studies performed in an in vitro system that specifically reconstitutes retrograde MPR traf-ficking between late endosomes and the TGN showed that an interfering antibody against clathrin had no effect (Draper et al., 1990).

All of these clathrin adaptors have been also implicated in vesicle formation at the TGN, often in the transport of the same cargo proteins such as the MPRs (Bonifacino and Lip-pincott-Schwartz, 2003). These apparent contradictions in the requirement for clathrin for the same cargos at both sides of the endosome-TGN interface need to be elucidated by further study. Clathrin coats may recognize patterns on both cargo proteins (interaction motifs) and the membranes (auxiliary factors and lipids), defining sorting modules that play a role in cargo inclusion into transport intermediates within differ-ent membrane environments. These membrane coat adap-tors would then be expected to have cargo-specific roles and may not be generally required for the formation of retrograde transport intermediates. Indeed, the clathrin adaptor AP-1 is involved in retrograde trafficking of MPRs (Meyer et al., 2000) but not trafficking of STxB (Saint-Pol et al., 2004) despite the fact that both cargos depend on the same syntaxins for dock-ing and fusion with TGN membranes upon being transported from early endosomes (Amessou et al., 2007) (Table 2).

Table 2. Retrograde Transport at the Endosome-TGN Interface

Trafficking Factor Localization

Proposed Function

Retrograde Cargo

Clathrin Plasma membrane, endosomes, TGN

Nanodomain organization

STxB

AP-1 Golgi, endosomes, CCV

Adaptor MPR, furin

AP-3 Endosomes Adaptor MPR

EpsinR CCV, endosomes, TGN

Adaptor STxB, MPR, TGN38/46, Vti1b

GGA TGN, endosomes, CCV

Adaptor BACE1, BACE2

PACS-1 Golgi, endosomes Adaptor MPR, furin, MHC-I

Dynamin Endosomes Scission MPR, STxB, ricin

TIP47 Endosomes, lipid droplets

Coat MPR, HIV-I env

Vps26/29/35 (Retromer)

Early endosomes Coat MPR, STxB, VSR1/ELP, Fet3-Ftr1, Wntless

SNX1 Early endosomes Coat MPR, STxB, sortilin

SNX2 Early endosomes Coat STxB, ricin

SNX4 Endosomes Coat Ricin

SNX5 Early endosomes Coat MPR

SNX6 Early endosomes Coat MPR

EHD1 Early endosomes Unknown MPR, TGN38/46

Cholesterol Late Golgi/TGN, PM, endosomes

Membrane lipid

MPR, STxB, ricin

Golgin-97 Golgi Tethering STxB, CTxB

Golgin-245 Golgi Tethering STxB

GCC185 TGN Tethering MPR, STxB

GCC88 Golgi Tethering TGN38/46

COG Golgi Tethering STxB

GARP Golgi Tethering STxB, MPR

TRAPP-II Golgi Tethering Snc1, Chs3

Arl1 Golgi Tethering factor recruitment

STxB, MPR

Syntaxin 5, Ykt6, GS15, GS28 (SNARE complex)

Golgi Fusion STxB, CTxB, ricin, MPR

Syntaxin 6, syntaxin 16, Vti1a, VAMP3/4 (SNARE complex)

Golgi Fusion STxB, CTxB, ricin, MPR, TGN38/46

Syntaxin 10, syntaxin 16, Vti1a, VAMP3 (SNARE complex)

Golgi Fusion MPR

Table 2. Continued

Trafficking Factor Localization

Proposed Function

Retrograde Cargo

Rab6a′ Golgi Docking and fusion

STxB

Rab6IP2 Golgi Rab GAP STxB

Rab9 Late endosome, Golgi

Docking and fusion

MPR

p40 Unknown Unknown MPR

Rab11 Early/recycling endosomes, Golgi

Docking and fusion

STxB, TGN38/46

Rab22a Early endosomes Docking and fusion

MPR

SMAP2 Early endosomes Arf GAP TGN38/46

BIG1 TGN Arf GEF TGN38/46, furin

BIG2 TGN, recycling endosomes

Arf GEF TGN38/46, furin

EVI5 Cytosol Rab GAP STxB

RN-tre Cell surface Rab GAP STxB

TBC1D10A Cell surface Rab GAP STxB

TBC1D10B Cell surface Rab GAP STxB

TBC1D10C Cell surface Rab GAP STxB

TBS1D17 Cytosol Rab GAP STxB

Syk Unknown Kinase STxB

PKA type II α Golgi Kinase Ricin

p38 Unknown Kinase STxB

PKCδ Unknown Kinase STxB

CK2 kinase Unknown Kinase MPR, furin

OCRL1 Golgi, early endosomes, CCV

PI metabolism

STxB, MPR, TGN38/46

WIPI49 Golgi, endosomes Unknown MPR

PIKfyve Early endosomes PI metabolism

MPR

Vps34 Early endosomes PI metabolism

Ricin

Dynein Microtubles Motor STxB

Microtubules Cytoplasm/membranes

Cytoskeleton STxB

TMF Golgi Unknown STxB

A large number of factors have been implicated in retrograde traf-ficking and are found at different compartments along the retrograde route. Some factors are not required for the transport of all cargos. Clathrin function is not required for MPR trafficking between late endo-somes and the trans-Golgi network (TGN). STxB sorting to retrograde transport intermediates is independent of AP-1. TGN38/46 does not require PACS-1 as it does not possess an acidic cluster PACS-1 in-teraction motif. TGN38/46 requires GCC88 and not GCC185, whereas STxB requires GCC185 but not GCC88. Syntaxin 10 regulates retro-grade trafficking from late endosomes and is therefore not needed for cargos such as TGN38/46, STxB, and CTxB, which exclusively use the early endosome-TGN interface. Ricin does not require Rab9 as it can probably use multiple trafficking receptors and pathways between endosomes and the TGN, thus in part bypassing the late endosomal pathway. CCV, clathrin-coated vesicles; CTxB, Cholera toxin B-subunit; MPR, mannose 6-phosphate receptor; STxB, Shiga toxin B-subunit.


The mechanisms underlying how directionality is established in retrograde transport still need to be elucidated. One possible mechanism involves the specific interaction of membrane coat components with the SNARE fusion receptors. This hypothesis is supported by recent findings regarding the clathrin adaptor epsinR. EpsinR is required for the efficient retrograde trans-port of MPRs and STxB (Saint-Pol et al., 2004). It interacts with clathrin, AP-1, GGAs, and, through its N-terminal epsin homology domain, phosphoinositol-4-phosphate molecules in membranes. Recent studies demonstrated that epsinR inter-acts with the SNARE protein Vti1b and may mediate, through a specific interaction motif, the post-Golgi dynamics of this and other SNARE proteins (Miller et al., 2007).

Another important question in clathrin-mediated membrane coating concerns the communication between the different coat components. These modules may either act in parallel or sequentially. Several studies argue in favor of sequential action. An in vitro assay using membranes prepared from AP-1-defi-cient cells or cytosol from AP-3-deficient cells demonstrated that MPR retrograde transport is inefficient in the absence of AP-1 or AP-3 (Medigeshi and Schu, 2003). Moreover, MPR retrograde transport efficiency was not further reduced when both AP-1 and AP-3 were depleted from the cell, thus sug-gesting some integrated function between AP-1 and AP-3. It should be noted, however, that in vivo observations from yeast and mouse knockout studies are generally not consistent with the hypothesis that AP-1 and AP-3 coat components function within the same sorting pathway (reviewed in Boehm and Boni-facino, 2002). Further studies are required to settle this case for the retrograde pathway.

An alternative mechanism for the transfer of cargo from one coat component to another has been suggested by studies of the mammalian phosphofurin acidic-cluster sorting protein-1 (PACS-1). This protein is involved in the efficient retrograde traf-ficking of MPRs and furin convertase but is not necessary for the transport of TGN46 (and its rat ortholog TGN38), a cycling protein of unknown function that lacks the PACS-1 interaction motif (Table 2). PACS-1 binds to the γ subunit of AP-1 and the phosphorylated acidic clusters on the cytosolic tails of MPR and furin. Following binding, PACS-1 recruits and activates the CK2 kinase and forms a ternary complex with the clath-rin adaptor GGA3 (Scott et al., 2006). CK2 phosphorylation of PACS-1 promotes cargo binding, whereas CK2 phosphoryla-tion of GGA3 inhibits cargo binding. PACS-1 could thus inter-act with acidic cluster motifs of retrograde cargos and induce coat component switching by simultaneously promoting cargo release of GGA3 through CK2 phosphorylation and recruiting AP-1. This mechanism may be specific for higher eukaryotes as no ortholog of PACS-1 has been found in simpler organisms such as yeast.

Additional factors regulate the recruitment of adaptors and clathrin assembly and provide intriguing links to specific lipids. The oculocerebrorenal syndrome of Lowe protein 1 (OCRL1) is a phosphatidylinositol (PtdIns) (4,5)P2 5-phosphatase that catalyzes the production of PtdIns(4)P lipids at Golgi and early endosomal membranes. PtdIns(4)P lipids recruit the clathrin adaptors AP-1 (Wang et al., 2003) and epsinR (Mills et al., 2003). OCRL1 overexpression blocks retrograde STxB transport,


whereas its depletion causes partial redistribution of MPR and TGN46 to endosomes (Choudhury et al., 2005). OCRL1 may also have a direct effect on the formation of retrograde trans-port intermediates. Indeed, OCRL1 localizes to clathrin-coated vesicles and stimulates clathrin polymerization in vitro. OCRL1 may therefore modulate phosphoinositide levels for accessory factor recruitment and promote clathrin assembly.

Various GTPases and GTPase-regulating proteins also are involved in clathrin-mediated membrane coat formation. SMAP2 is an Arf1-specific GAP (GTPase-activating protein) that directly interacts with clathrin and colocalizes with AP-1 and epsinR (Natsume et al., 2006). Overexpression of SMAP2 impairs the transport of TGN38 from early endosomes to the TGN. BIG1 and BIG2 are two Arf GEFs (guanine nucleotide exchange factors) that are mainly localized to the TGN. BIG2 also associates with recycling endosomes where it recruits AP-1. Retrograde transport of furin is affected in cells depleted for BIG1 and BIG2, similar to that observed when AP-1 is depleted (Ishizaki et al., 2008). The GTPase dynamin is involved in membrane scission and plays a role in endosome-to-TGN transport of several cargo proteins such as MPRs, STxB, and the plant toxin ricin (reviewed in Lamaze and Johannes, 2006; Sandvig and van Deurs, 2005).Nonclathrin Membrane CoatsThe tail-interacting 47 kDa protein (TIP47) functions inde-pendently of clathrin (Draper et al., 1990) as a cargo-specific coat-like selection device for MPR retrograde trafficking from late endosomes to the TGN (Carroll et al., 2001). TIP47 selec-tively binds the cytoplasmic domain of both MPRs and furin convertase (Diaz and Pfeffer, 1998). Its interaction with MPR cargo protein is stimulated by the small GTPase Rab9. As is the case for PACS-1, TIP47 is not conserved in simple eukary-otes such as yeast. This again suggests the existence of spe-cialized requirements for MPR trafficking. TIP47 has also been observed to associate with lipid droplets (Wolins et al., 2001), pointing to a possible link between TIP47 function in retrograde trafficking and lipid droplet dynamics.

The retromer is another coat-like protein complex with an established role in retrograde trafficking. It is a heteropentameric complex that was first discovered in budding yeast (reviewed in Bonifacino and Hurley, 2008; Collins, 2008). The retromer complex can be divided into a sorting nexin (SNX) dimer and a Vps26-Vps29-Vps35 trimer. In yeast, the SNX dimer consists of Vps5 and Vps17. In mammals, SNX1, SNX2, SNX5, and SNX6 bind in different combinations to Vps26-Vps29-Vps35, although some controversy still exists in the understanding of strength and cell type variations for some of these interactions (Bonifacino and Hurley, 2008). SNX proteins bind high-curvature regions of early endosomal membranes through their Phox homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. PX domains interact with phosphoinositides such as the endosomal PtdIns(3)P and PtdIns(3,5)P, whereas BAR domains are able to sense and, in some cases, to induce membrane curvature. PtdIns(3)P and PtdIns(3,5)P2 lipids are generated by the enzymes Vps34 and PIKfyve, respectively. Both of these enzymes are also required for retrograde trafficking (Rutherford et al., 2006; Skanland et al., 2007). Membrane recruitment of the SNX dimer is independent of Vps26-Vps35-Vps29 (Bonifacino and Hurley, 2008).

The Vps26-Vps35-Vps29 trimer binds to cytosolic tails of several retrograde cargos such as MPRs and sortilin. It is there-fore referred to as the cargo recognition complex (Bonifacino and Hurley, 2008). The elongated structure of Vps35 serves as a scaffold for Vps26 and Vps29 binding (Hierro et al., 2007). Vps26 exhibits structural homologies with arrestins, a family of adaptors that link G protein-coupled receptors to clathrin, although this G protein-coupled receptor binding activity has not yet been shown for Vps26. Whether Vps26 undergoes con-formational changes like arrestins as part of its functional cycle remains to be defined. Vps29 possesses a metalloprotease fold and can bind to divalent metal ions in vitro, but no cata-lytic activity has been detected. The assembled Vps26-Vps35-Vps29 cargo recognition complex likely adapts to the shape of curved tubulovesicular structures. Retromer decorates highly dynamic tubules emanating proximal to clathrin flat lattices on vacuolar early endosomes in vivo (Bonifacino and Hurley, 2008). Current models suggest that the SNX dimer partici-pates in tubule formation on early endosomal membranes and

Figure 2. Models of Sequential Clathrin and Retromer Function on Early Endosomes(A) Retromer binds to the edge of a flat clathrin lattice. The BAR domain-dependent membrane deformation activity of a retromer subunit allows it to induce membrane curvature, eventually resulting in the formation of a retro-grade tubule. (B) Clathrin molecules within the flat lattice at the membrane surface dynami-cally organize into nanodomains containing both clathrin and other lipid-bind-ing proteins such as AP-1, epsinR, or possibly also SNX components of the retromer complex. The clathrin nanodomain may then undergo membrane deformation due to the presence of those lipid-binding proteins. The retromer may be involved in the subsequent processing of retrograde tubules.

that the cargo recognition complex drives the incorporation of cargos into these structures (Collins, 2008). Some SNX pro-teins are involved in retrograde transport independent of the retromer complex. The yeast-sorting nexins 4/41/42 mediate a distinct retrieval pathway to the Golgi (Hettema et al., 2003). In mammalian cells, SNX2 appears to operate independently of the SNX4 and SNX1/retromer in the retrograde trafficking of ricin (Skanland et al., 2007).

Retromer activity has been shown to be critical for the ret-rograde transport of several cargo proteins, including sortil-ins, Wntless, MPRs, and Shiga toxin (Bonifacino and Hurley, 2008). For some of these cargos, clathrin or clathrin-interacting proteins have also been implicated in their retrograde sorting on early endosomes. This raises the question of whether the clathrin and retromer membrane coats function in parallel or in sequential steps. A proteomics study showed that the retromer coat is lost from a vesicle preparation upon clathrin depletion (Borner et al., 2006), thus strongly suggesting that some inter-action exists between these two protein complexes. Electron microscopical analysis revealed the localization of retromer on tubules emanating from coated endosomes (Arighi et al., 2004). Although retromer-specific immunolabeling tends to accumulate at the base of protruding tubules, the retromer is not excluded from flat clathrin lattices. Moreover, whole-mount analysis revealed the colocalization of clathrin and the retromer protein Vps26 on the early endosome (Popoff et al., 2007). The quantitative analysis of retrograde transport using methods exploiting STxB trafficking (Table S1) showed a pronounced inhibition of transport in cells depleted for either clathrin (Saint-Pol et al., 2004) or Vps26 (Popoff et al., 2007). These obser-vations strongly suggest that clathrin and retromer activities are tightly linked, as only partial retrograde transport inhibi-tion would be observed if the two membrane coats functioned independent of one another.

The analysis of endosomes with an accumulation of STxB has allowed the link between retromer and clathrin to be exam-ined in further detail. These studies have provided evidence for sequential roles for both coat protein complexes. In clathrin-depleted cells, STxB colocalized with the transferrin receptor, a marker of conventional early/recycling endosomes (Popoff et al., 2007; Saint-Pol et al., 2004). In contrast, STxB was no longer found in the conventional early endosome in Vps26-depleted cells lacking retromer function. Instead, STxB was found in transferrin-free tubular structures (termed retrograde tubules) connected to transferrin receptor endosomes at their base (Popoff et al., 2007). These data strongly suggest that clathrin acts before the retromer in the retrograde transport of STxB on the early endosome.

Two possible models can be drawn to explain the respec-tive roles of clathrin and the retromer in retrograde sorting on the early endosome (Figure 2). Based on the BAR domain-dependent membrane deformation activity of the SNX sub-unit, the retromer could induce curvature changes that lead to retrograde tubule formation (Figure 2A). In this model, clathrin would be necessary at a predeformation step before retromer binding, possibly to organize a sorting domain. Although this model would not appear to fit with the observation of STxB-containing retrograde tubules in Vps26-depleted cells (Popoff


et al., 2007), this inconsistency could be explained if the mem-brane deformation observed in those experiments is the con-sequence of spontaneous membrane bending induced by the dynamic construction of STxB-Gb3 nanodomains (Römer et al., 2007). Alternatively, clathrin could dynamically organize nano-domains encompassing lipid-binding proteins such as epsinR and AP-1 (and possibly also SNX proteins) in the endosomal membrane (Figure 2B). Retrograde tubules would emerge from these nanodomains, and the retromer complex could then be involved in the processing of these tubules.

Major aspects of both of these models still need to be tested and represent fruitful ground for future mechanistic explora-tion. Features of the models to be examined include their gen-eral applicability to endogenous cargo proteins of the retro-grade route, the exact nature of the clathrin nanodomain, the molecules involved in coupling clathrin and retromer activities, and the hypothesized role for the retromer in retrograde tubule processing. The identification of retromer-interacting proteins such as EHD1 (Eps15 homology domain-containing protein 1) (Gokool et al., 2007) may provide clues to conceptualizing the multiple activities of this protein complex. How toxin mol-ecules that are associated with the exoplasmic membrane lipid leaflet communicate with the cytosolic sorting machinery also remains to be determined. The reduction of free cholesterol levels in membranes prevents STxB, ricin, and MPRs from reaching the Golgi (Lamaze and Johannes, 2006; Sandvig and van Deurs, 2005), suggesting a possible role for dynamic mem-brane nanocompartmentalization in communication across the membrane.Mediators of Cytoplasmic TranslocationThe mechanisms that underlie the microtubule-mediated trans-location of retrograde transport intermediates across the cyto-plasm remain to be determined. However, it is known that bind-ing of STxB to the Gb3 receptor induces intracellular signaling, which leads to an increased rate of microtubule polymerization and results in the remodeling of the cytoskeleton (Hehnly et al., 2006; Takenouchi et al., 2004). Moreover, STxB requires the molecular motor dynein for efficient retrograde transport (Hehnly et al., 2006). Whether these requirements are shared by endogenous cargo is not known.Tethering, Docking, and Fusion ProteinsSmall GTPases of the Rab family localize to defined organ-elles and recruit specific sets of effectors. Rab proteins thus coordinate the different steps of intracellular traffick-ing, notably that of docking and fusion. Rab6a′ regulates retrograde trafficking between early endosomes and the TGN (Mallard et al., 2002). A recent study demonstrated a potential link between Rab6 and Rab11 proteins mediated by the Rab6-interacting protein Rab6IP1 (Miserey-Lenkei et al., 2007). Another study of 39 predicted Rab GTPase-activating proteins (Rab GAPs) revealed the involvement of six of these proteins in retrograde transport to the TGN; the GAP of Rab6a′ was not identified in this study (Fuchs et al., 2007). As previously mentioned, Rab9 plays a role in MPR trafficking between late endosomes and the TGN. Interest-ingly, Rab9 does not regulate the retrograde trafficking of ricin (Table 2) (Iversen et al., 2001). This likely reflects the fact that ricin can use several retrograde trafficking routes,


including that from the early endosomes to the TGN, through its ability to bind to a multitude of terminal galactosylated receptor molecules (Table S2).

Tethering factors are coiled-coil domain-containing pro-teins or multisubunit complexes that link organelles and trans-port intermediates in the initial step of the molecular events that lead to membrane fusion. Several members of the golgin family of tethering factors are involved in endosome-to-TGN transport. Golgin-97 and golgin-245 (also called p230 or tGol-gin-1) are targeted through their GRIP domains by the Arl1 GTPase to Golgi membranes and are required for efficient ret-rograde trafficking of STxB (Table 2) (reviewed in Bonifacino and Rojas, 2006; Lamaze and Johannes, 2006). Depletion of tethering protein GCC185 from mammalian cells disrupts the Golgi structure. Under these conditions, anterograde transport is not affected but retrograde trafficking of STxB and MPRs is inhibited (Derby et al., 2007; Reddy et al., 2006). Interest-ingly, TGN38 retrograde transport is not affected by GCC185 depletion (Derby et al., 2007). However, it is affected by GCC88 depletion, which does not disrupt STxB transport (Lieu et al., 2007). The physiological significance of this tethering factor specificity for STxB and TGN38 is not currently understood.

Additional tethering factors continue to be identified in ret-rograde transport. Recently, TMF (TATA element modulatory factor) has been implicated in endosome-to-TGN transport in mammalian cells (Yamane et al., 2007). In yeast, three teth-ering complexes, GARP/VFT (Golgi-associated retrograde transport/Vps fifty three), COG (conserved oligomeric Golgi), and TRAPP-II (transport particle-II), have been observed to play roles in retrograde trafficking (reviewed in Bonifacino and Rojas, 2006); similar observations have since been made in mammalian cells (Table 2).

It remains unclear why so many factors mediate the tether-ing of retrograde transport intermediates to TGN membranes. Although some cargo specificity exists, many of these factors are generally required for efficient trafficking of the same cargo molecules (Table 2). Further complexity is added by recent findings (Burguete et al., 2008; Ganley et al., 2008) revealing that GCC185 interacts not only with Rab9, a GTPase that regu-lates late endosome-to-TGN trafficking (Lombardi et al., 1993), but also with Rab6 and syntaxin 16, factors involved in early endosome-to-TGN trafficking (Mallard et al., 2002). Clearly, additional studies are required to elucidate the underlying prin-ciples that determine the identity of the tethering.

SNARE proteins constitute a large protein superfamily with more than 60 members in yeast and mammals. The primary role of SNARE proteins is to mediate the fusion of cellular transport intermediates with the plasma membrane or with a target com-partment. STxB transport from endosomes to the TGN requires two SNARE complexes whose members are specifically local-ized on early endosomes and the TGN. One of these complexes is composed of VAMP4 or VAMP3/cellubrevin, syntaxin 6, syn-taxin 16, and Vti1a, whereas the other contains GS15, syntaxin 5, Ykt6, and GS28 (Table 2) (reviewed in Bonifacino and Rojas, 2006; Lamaze and Johannes, 2006). Efficient retrograde trans-port of MPRs requires syntaxin 16 and Vti1a (Amessou et al., 2007; Medigeshi and Schu, 2003; Saint-Pol et al., 2004), in addi-tion to syntaxin 10 (Ganley et al., 2008). The differential SNARE

requirements of STxB and MPRs could reflect a difference in the routes taken by the two cargo proteins in retrograde transport. Unlike STxB, MPRs can also traffic between late endosomes and the TGN, a transport step that appears to be specifically regulated by syntaxin 10 (Table 2).KinasesStudies using techniques ranging from pharmacological dis-ruption to RNA interference suggest that several kinases, including CK2 kinase (Scott et al., 2006), protein kinase C delta (Torgersen et al., 2007), protein kinase A type II alpha (Birkeli et al., 2003), mitogen-activated protein kinase p38, and syk (Wälchli et al., 2008), are involved in the regulation of retro-grade trafficking. However, in most cases the molecular targets of the kinases in the context of retrograde transport are not yet known.

Retrograde PathwaysThe large number of trafficking factors at the endosome-TGN interface raises questions about how their functions are con-nected. Here, we address the apparent complexity of traffick-ing proteins and discuss potential conceptual explanations. It should be noted that the biochemical properties of many of these factors, such as that of the retromer, have not yet been fully elucidated. Thus, the interpretation of the functions of these poorly understood factors often remains somewhat speculative.

Certain proteins in retrograde trafficking, such as clath-rin and the retromer, appear to act sequentially at the endo-some-TGN interface (Figure 2). The mode of function of other transport factors seems to depend on cell type and possibly on culture conditions. For example, SNX2 is sufficient for ret-romer function in some instances (Rojas et al., 2007) but not in others (Carlton et al., 2005) and may be replaced by SNX5 or SNX6 (Wassmer et al., 2007). The potential dependence of retrograde transport on cargo concentration is another aspect that has been little studied. Shiga toxin enters cells through clathrin-dependent and -independent pathways (Lamaze and Johannes, 2006; Römer et al., 2007; Sandvig and van Deurs, 2005). In this case, it was found that the balance between these two modes of cellular entry is regulated by toxin concentration (Torgersen et al., 2005). Whether and how this phenomenon may relate to retrograde sorting of cargo on endosomes has not yet been addressed directly.

The diversity of factors that regulate the retrograde traffick-ing of some cargo proteins also suggests the possibility of parallel pathways for transport of the same cargos between endosomes and the TGN (Figure 1). The well-studied MPR proteins are one such example. MPRs are trafficked from late endosomes to the TGN. Recent evidence suggests that it can also undergo retrograde sorting at earlier stages of the endo-cytic pathway. The MPR retrograde route from the late endo-some to the TGN is the first retrograde transport pathway to be described and requires the GTPase Rab9 and its effectors TIP47, GCC185, and p40 (Carroll et al., 2001; Diaz and Pfeffer, 1998; Lombardi et al., 1993; Reddy et al., 2006). The trans-port route from early endosomes to the TGN was first dem-onstrated for the Shiga toxin and TGN38 retrograde cargo molecules, which could not be detected in compartments of

the late endocytic pathway upon their retrograde passage to TGN/Golgi membranes (Ghosh et al., 1998; Mallard et al., 1998). Disruption of components of molecular machinery at the early endosome-TGN interface—Rab6a′ (Mallard et al., 2002), SNARE complexes involving the TGN-localized SNARE chains syntaxin 5 (Amessou et al., 2007) and syntaxin 16 (Mallard et al., 2002), tGolgin-1, and golgin-97—induced retention of Shiga toxin in early endosomal membranes (reviewed in Bonifacino and Rojas, 2006; Lamaze and Johannes, 2006). Many of these factors involved in the early endosome-to-TGN trafficking of Shiga toxin also play a role in the retrograde transport of MPRs, supporting the notion that MPRs can follow a similar traffick-ing route. Consistent with this, MPRs relocalize to early endo-somes in cells depleted of retromer components (reviewed in Bonifacino and Rojas, 2006), syntaxin 10 (Ganley et al., 2008), AP-1 (Meyer et al., 2000), or PIKfyve (Rutherford et al., 2006). The combined evidence from these studies strongly suggests that MPRs may be specialized for cargo retrieval at early and late stages of the endocytic membrane system. It should how-ever be noted that other interpretations have been suggested for these studies (Ganley et al., 2008). How the relatively mild acidic conditions of early endosomes can induce the release of MPRs from their receptors for maturation has also remained a matter of controversy. Further investigation is clearly required to advance our understanding of parallel retrograde pathways. Interestingly, some studies have suggested that recycling endosomes can also support retrograde sorting (reviewed in Bonifacino and Rojas, 2006), although this remains to be con-firmed by the visualization of bona fide retrograde cargo exit from the recycling endosome.

Applications of Retrograde Transport ResearchUnderstanding retrograde trafficking provides the obvious benefit of information that could be used to disrupt toxin ret-rograde transport to intracellular targets (Saenz et al., 2007). However, retrograde trafficking-competent factors such as these pathogenic toxins may also be exploited as drug delivery tools. These proteins have the capacity to transport extracel-lular proteins—in the case of holotoxins, the catalytic A-sub-units—to membrane translocation-competent compartments and to become resident proteins of biosynthetic/secretory pathway membranes. Thus, they can be used for specific tar-geting of therapeutic molecules to cells and are indeed cur-rently being explored for the development of innovative immu-notherapy strategies (Moron et al., 2004; Tartour et al., 2002). For example, antigens that are fused or chemically coupled to STxB selectively target dendritic cells expressing the Gb3 receptor and stimulate MHC class I-restricted antigen presen-tation using a proteasome- and TAP transporter-dependent mechanism. This strongly suggests that exogenous antigen is indeed translocated to the cytosolic compartment by STxB (reviewed in Johannes and Decaudin, 2005). In mice, this deliv-ery of exogenous antigens to dendritic cells induces a potent cytotoxic T-lymphocyte response and protects the animals against experimentally induced tumor growth (Vingert et al., 2006). Similar strategies are currently being explored for other protein toxins (Moron et al., 2004). Some of these toxins use the retrograde route, such as ricin (Noakes et al., 1999) and the


B-subunit of the E. coli bacterial heat-labile enterotoxin (Hearn et al., 2004). In other cases, the intracellular trafficking route taken by the toxins remains to be determined. The effective antigen delivery tool of the adenylate cyclase toxin from the bacterium Bordetella pertussis is one such example (reviewed in Moron et al., 2004). Cell-penetrating peptides (CPPs) that are characterized by their capacity to spontaneously cross mem-branes can also deliver antigens into the cell cytosol by retro-grade transport (Fischer et al., 2004) and might be coupled to cell type-specific ligands to generate selectivity.

The ability of retrograde transport-competent factors to form stable associations with cells and avoid recycling to the plasma membrane or degradation in the late endocytic path-way can also be exploited to target tumors (reviewed in Tar-rago-Trani and Storrie, 2007). Indeed, the host STxB receptor Gb3 is overexpressed by many human tumors (Gariepy, 2001; Johannes and Decaudin, 2005) and intratumoral injection of Shiga toxin can inhibit tumor growth (Arab et al., 1999; Ishitoya et al., 2004). STxB has also been shown to target spontaneous digestive tract adenocarcinomas in mice following intravenous injection or oral application (Janssen et al., 2006). Recently, a prodrug composition using the topoisomerase I inhibitor SN38 has been developed that is specifically activated in biosyn-thetic/secretory pathway membranes (El Alaoui et al., 2007). Exploitation of this retrograde delivery route may be particu-larly beneficial in therapeutic strategies requiring prolonged association of the prodrug with the tumor cells so as to ensure efficient conversion of the prodrug to the active form.

Future PerspectivesThe recognition of the central role played by the retrograde route and the availability of increasingly more power tools for analysis (Table S1) have made this transport pathway an area of particular interest in cell biology research. The complexity of trafficking mechanisms involved in retrograde transport between endosomes and the TGN is surprising. In this Review, we have discussed several factors that may account for some of this complexity, such as the existence of sequential transport steps within the same pathway, the existence of parallel transport pathways (i.e., from early or late endosomes), the presence of different receptor mole-cules for the same cargo (as in the case of ricin), and dif-ferential requirements for sorting into retrograde transport intermediates. It may be possible to classify cargo mole-cules into pathways as a function of the transport factors involved. However, the current data are too limited to offer a satisfactory solution (Table S2), and systems-level analyses of transport factor-cargo relationships are required to obtain a complete picture. It should also be considered that path-way models may not be the best way to conceptualize mem-brane exchanges at the endosome-TGN interface. It may be more suitable to consider trafficking events at this interface as reactions in which overlapping sets of molecular machin-ery are shared between cargos. The use of these machin-ery may depend on molecular structure (cargo recognition signals), concentration, context (membrane composition), and topology (curved or planar membranes). Further studies such as the high-resolution imaging and biochemical purifi-


cation of bona fide retrograde transport intermediates and reconstitution of retrograde sorting on model membranes will be needed to explore this possibility.

Many questions also remain unanswered regarding the roles of clathrin and the retromer in endosomal retrograde sorting. Current evidence suggests that clathrin and retromer have integrated functions in this process, but it is not known how their activities may be coordinated. The molecular composi-tion of clathrin nanodomains involved in retrograde sorting and their function also remain a mystery. If these nanodomains are involved in generating curvature, what are the underlying mechanisms and what is their relationship with flat clathrin lat-tices on coated endosomes? Assays that allow rigorous dis-section of the molecular events at the endosome-TGN inter-face are necessary to answer these questions. These studies are likely to make broad conceptual contributions beyond ret-rograde sorting to the understanding of membrane mechanics. The study of retrograde trafficking not only has revealed unex-pected complexities in the regulation of protein transport but also may lead to the development of new therapeutic strategies for the clinical management of infectious disease and cancer.

Supplemental DataSupplemental Data include two tables and can be found with this article on-line at http://www.cell.com/supplemental/S0092-8674(08)01570-5.

ACknowlEdgMEnTS

We thank C. Lamaze for critical reading of the manuscript. L.J. is supported by grants from Human Frontier Science Program Organization, 6th European Framework Program CancerImmunotherapy, and the Curie Institute (PIC Vec-torization).

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