Protein Transport in Plant Cells: In and Out of the Golgifb/annals of botany.pdfProtein Transport in...

doi:10.1093/aob/mcg134, available online at www.aob.oupjournals.org

INVITED REVIEW

Protein Transport in Plant Cells: In and Out of the Golgi²

ULLA NEUMANN, FEDERICA BRANDIZZI and CHRIS HAWES*

Research School of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane Campus,

Oxford OX3 0BP, UK

Received: 3 March 2003 Returned for revision: 8 April 2003 Accepted: 6 May 2003

In plant cells, the Golgi apparatus is the key organelle for polysaccharide and glycolipid synthesis, protein glyco-sylation and protein sorting towards various cellular compartments. Protein import from the endoplasmic reticu-lum (ER) is a highly dynamic process, and new data suggest that transport, at least of soluble proteins, occursvia bulk ¯ow. In this Botanical Brie®ng, we review the latest data on ER/Golgi inter-relations and the modelsfor transport between the two organelles. Whether vesicles are involved in this transport event or if direct ER±Golgi connections exist are questions that are open to discussion. Whereas the majority of proteins pass throughthe Golgi on their way to other cell destinations, either by vesicular shuttles or through maturation of cisternaefrom the cis- to the trans-face, a number of membrane proteins reside in the different Golgi cisternae.Experimental evidence suggests that the length of the transmembrane domain is of crucial importance for theretention of proteins within the Golgi. In non-dividing cells, protein transport out of the Golgi is either directedtowards the plasma membrane/cell wall (secretion) or to the vacuolar system. The latter comprises the lyticvacuole and protein storage vacuoles. In general, transport to either of these from the Golgi depends on differentsorting signals and receptors and is mediated by clathrin-coated and dense vesicles, respectively. Being at theheart of the secretory pathway, the Golgi (transiently) accommodates regulatory proteins of secretion (e.g.SNAREs and small GTPases), of which many have been cloned in plants over the last decade. In this context,we present a list of regulatory proteins, along with structural and processing proteins, that have been located tothe Golgi and the `trans-Golgi network' by microscopy. ã 2003 Annals of Botany Company

Key words: Review, Golgi, endoplasmic reticulum, prevacuolar compartment, vacuole, plasma membrane, proteintransport, protein sorting, vesicles, SNAREs, small GTPases.

INTRODUCTION

In plants, the Golgi apparatus is central to the synthesis ofcomplex cell wall polysaccharides and of glycolipids for theplasma membrane, as well as the addition of oligosacchar-ides to proteins destined to reach the cell wall, plasmamembrane or storage vacuoles. The Golgi apparatus is alsothe key organelle in sorting proteins, sending them to theirvarious destinations within the cell. The majority of theseproteins are imported into the Golgi from the endoplasmicreticulum (ER), a major organelle of the endomembranesystem involved in the folding, processing, assembly andstorage of proteins, as well as in lipid biosynthesis andstorage (Vitale and Denecke, 1999). The relative import-ance of the two major Golgi functions in a plant cell, theassembly and processing of oligo- and polysaccharides onthe one hand and protein sorting on the other, depends on thecell type and its developmental and physiological state(Juniper et al., 1982). Nevertheless, the two functionscannot be regarded as completely unrelated processes;newly synthesized cell wall polysaccharides have to reachthe correct target destination and must therefore be appro-priately sorted.

The plant Golgi apparatus shares many features with itsanimal counterpart, but also has unique characteristics. Themost important difference concerns its structure. Whereas inanimal cells the Golgi apparatus occupies a rather stationaryperinuclear position, in plant cells the Golgi is divided intoindividual Golgi stacks, which are generally considered tobe functionally independent (Staehelin and Moore, 1995)(Fig. 1). The number of Golgi stacks per cell and the numberof cisternae per stack vary with the species and cell type, butalso re¯ect the physiological conditions, the developmentalstage and the functional requirement of a cell (reviewed byStaehelin and Moore, 1995; Andreeva et al., 1998b).Despite these variations, each individual Golgi stack canbe described as a polarized structure with its cisternalmorphology and its enzymatic activities changing graduallyfrom the ER-adjacent cis-face to the trans-face (Fitchetteet al., 1999). Proteins destined for secretion enter the Golgiat the cis-face and subsequently move towards the trans-face where the majority of proteins exit the stack en route tothe plasma membrane or vacuolar system (for exceptionssee below). In between the cisternae of the Golgi stack,intercisternal elements can be observed, mainly towards thetrans-face (Ritzenthaler et al., 2002) (Fig. 1A). Although nomatrix proteins surrounding the plant Golgi stack have yetbeen identi®ed, the existence of a matrix has been predictedfrom the appearance on micrographs from ultra-rapidlyfrozen root cells of a clear zone, excluding ribosomes,around each Golgi (Staehelin et al., 1990). The Golgi matrix

* For correspondence. Fax +44 1865 483955, e-mail [email protected]

² In memorium of Jean-Claude Roland who, as an expert in plant cellwalls, would always have appreciated the importance of the Golgiapparatus.

Annals of Botany 92/2, ã Annals of Botany Company 2003; all rights reserved

Annals of Botany 92: 167±180, 2003

has been suggested to play an important role in themaintenance of stack organization against the shearingforces during cytoplasmic streaming (Staehelin and Moore,1995).

Confocal microscopy of Golgi-targeted proteins orpeptides fused to the green ¯uorescent protein (GFP) hasrevealed that individual stacks are highly mobile within theplant cell, moving over the ER on an actin-network (Boevinket al., 1998; NebenfuÈhr et al., 1999; Brandizzi et al., 2002b)(Fig. 2); this has resulted in them being christened `stacks ontracks' (Boevink et al., 1998) or `mobile factories'(NebenfuÈhr and Staehelin, 2001). The fact that the plantGolgi apparatus is divided into highly mobile biosyntheticsubunits certainly poses major problems when trying toelucidate mechanisms for controlled protein import into andtargeted product export out of the stack.

In this Botanical Brie®ng, we summarize recent ®ndingsregarding protein transport from the ER to the Golgiapparatus and sorting of proteins and membranes as theyexit the Golgi.

ENTERING THE GOLGI:IMPORT FROM THE ER

Towards a more dynamic model of ER-to-Golgi proteintransport

The discovery that Golgi stacks move in close associationwith the ER and that this movement is actin-dependent(Boevink et al., 1998) led to the question of whether Golgi

movement is necessary for ER-to-Golgi transport. The`vacuum cleaner model' (Boevink et al., 1998) suggestedthat Golgi stacks move over the surface of the ER picking upproducts, similar to a vacuum cleaner picking up dust whilstmoving over a carpet (Fig. 3A). This implies that the wholesurface of the ER is capable of protein export, and thatcontinued formation of cargo vectors occurs. In contrast, thehypothesis underlying the `stop-and-go' or `recruitmentmodel' (NebenfuÈhr et al., 1999) is that Golgi stacks receivecargo from the ER at de®ned export sites, which produce alocal stop signal that transiently halts stack movement(Fig. 3B). This model is supported by the observation thatactin-based Golgi movement is not necessary for ER-to-Golgi membrane protein transport (Brandizzi et al., 2002b).Although more attractive, as protein transport out of the ERwould be restricted to con®ned areas, the recruitment modelsuggests a rather stationary image of the ER surface. A thirdmodel might therefore propose that protein export from theER is restricted to speci®c export sites, which could beeither highly mobile within the ER membrane or mobile dueto the movement of the ER surface (Fig. 3C). Thus, Golgistacks and ER export sites may move together in an actin-dependent fashion, forming discrete `secretory units'(Brandizzi et al., 2002b).

Receptor-mediated protein transport or bulk ¯ow?

Regardless of the physical model of ER-to-Golgi trans-port, export from the ER and import into the Golgi are twointimately related processes. How proteins are sorted and

F I G . 1. Transmission electron micrographs of Golgi stacks in tobacco (A) and maize roots (B). A, Cross-section of a Golgi stack in a tobacco root capcell. High-pressure freezing and freeze-substitution improves the ultrastructural preservation of intercisternal ®laments towards the trans-face of theGolgi stack (arrows). B, Face view of a Golgi cisternum in a maize root meristematic cell. Zinc iodide and osmium tetroxide impregnation selectivelystains the ER and the Golgi and clearly shows the fenestrated margins of the Golgi cisternum. ER, Endoplasmic reticulum; M, mitochondrion; V,

vesicle. Bars = 200 nm.

168 Neumann et al. Ð Protein Transport in Plant Cells

concentrated at sites of ER export is therefore a keyquestion. Are they actively sorted with the help of speci®cER export signals linking with differential af®nity to acommon ER export receptor, or is there bulk ¯ow of productwith sorting occurring via retention signals by whichproteins are deviated from the default route at differentlevels of the pathway? Support for the second model, at leastfor soluble proteins, came from the cloning of an ERD2homologue from Arabidopsis thaliana (Lee et al., 1993).This protein acts as a transmembrane receptor that binds to aspeci®c sorting tetrapeptide (H/KDEL) at the carboxylterminus of ER-resident proteins in yeast and mammals(Lewis and Pelham, 1990; Lewis et al., 1990). Thearabidopsis ERD2 is capable of functionally complementinga yeast null mutant (Lee et al., 1993), and its GFP fusionlocates to the ER and to Golgi stacks (Boevink et al., 1998).Recent data from quantitative biochemical in vivo assaysmeasuring ER export of well-characterized cargo moleculesin Nicotiana tabacum protoplasts provided good evidencefor the bulk ¯ow theory (Phillipson et al., 2001).

In vivo observations of membrane protein transport fromER to Golgi have recently been facilitated by the use of¯uorescent protein chimeras. Selective photobleachingexperiments using two ¯uorescent marker proteins locatingto the Golgi (ST-YFP and ERD2-GFP) have permittedconfocal imaging of ER-to-Golgi protein transport(Brandizzi et al., 2002b). Fluorescence recovery in individ-ual Golgi stacks after photobleaching of ST-YFP or ERD2-GFP reached 80±90 % of the pre-bleach level after only5 min in cells treated with latrunculin B alone to disruptactin ®laments or in conjunction with colchicine to affect

microtubule integrity. This indicated a rapid exchange withpools of the fusion protein from other parts of the cell,independent from an intact actin or microtubule cytoskele-ton. The rapid cycling of both fusion proteins was dependenton energy, as could be shown by loss of ¯uorescencerecovery in cells after ATP depletion.

COPII vesicles vs. direct connections

In animal cells, protein transport between the ER and theGolgi apparatus occurs through intermediate compartmentsknown as vesicular±tubular clusters (VTCs) (or the ER±Golgi intermediate compartment, ERGIC). These compart-ments represent the ®rst site of segregation of anterograde(forward from the ER to the Golgi) and retrograde(backwards from the Golgi to ER) protein transport(reviewed by Klumperman, 2000). Transport between theER and VTCs is supposedly mediated by specializedprotein-coated vesicles, the so-called COPII vesicles.Cargo packaging and vesicle formation require sequentialbinding to the ER membrane of the GTPase Sar1p and twoheterodimeric coat protein complexes, Sec23/24p andSec13/31p (reviewed by Barlowe, 2002). Transport betweenthe VTCs and the Golgi apparatus seems not to be mediatedby distinct vesicles but by the fusion of peripheral VTCs toform the cis-Golgi cisternae (Klumperman, 2000). Theinvolvement of another set of coated vesicles (COPIvesicles formed under the in¯uence of the small GTPaseArf1p), normally thought to act as retrograde proteintransporters, between the Golgi cisternae and from VTCsback to the ER (see below), in anterograde protein transport

F I G . 2. Confocal laser scanning micrographs showing the spatial relationship between Golgi stacks and ER (A) and between Golgi stacks and actin®laments (B). A, 3D-reconstruction (Velocityâ) of serial optical sections through the cortical cytoplasm of a tobacco leaf epidermal cell transientlytransformed with a GFP-fusion targeted to the ER (GFP-HDEL in green) and a YFP-fusion labelling the Golgi (ST-YFP in red). Golgi stacks are inclose association with the ER network. B, Optical section through the cortical cytoplasm of a tobacco BY2 cell stably transformed with ST-GFP (ingreen). Af®nity labelling of actin by rhodamine-phalloidine (in red) reveals that Golgi stacks are aligned with actin ®laments (arrows). Bars = 10 mm.

Neumann et al. Ð Protein Transport in Plant Cells 169

is still hotly debated (Klumperman, 2000; Spang, 2002).Finally, delivery of cargo to the Golgi complex involves thesmall GTPase Rab1, which controls tethering and fusionevents at the Golgi level (Allan et al., 2000).

In plant cells, ER-to-Golgi protein transport might followa simpler route. To date, no equivalent of the VTCs of animalcells has been identi®ed. On the contrary, recent ®ndingsbased on GFP expression and transmission electron micro-scopy (TEM) indicate that direct connections in the form oftubular extensions may exist between the ER and the cis-faceof Golgi stacks in tobacco leaf cells (Brandizzi et al., 2002b),suggesting that vesicles might not mediate protein transportbetween the two organelles. At ®rst glance, this ®ndingseems to contradict the fact that components of the COPIImachinery have been identi®ed in plants by EST databasesearches (Andreeva et al., 1998a), are associated with the ER(Bar-Peled and Raikhel, 1997; Movafeghi et al., 1999), andare necessary for transport of proteins to the Golgi (Andreevaet al., 2000; Phillipson et al., 2001). This has been interpretedas evidence that ER±Golgi protein transport shows structuraland functional similarities between animal and plant cells.However, it is conceivable that COPII components simply

determine the site of formation of ER-to-cis-Golgi-connec-tions, and that vesicle vectors are not a prerequisite oftransport (Hawes et al., 1999). Considering that proteintransport in mammalian cells between the VTCs and the cis-Golgi seems to be by fusion of peripheral VTCs to form thecis-cisternae, and that VTCs have not been identi®ed in plantcells to date, direct fusion events between the ER and theGolgi are not implausible.

Similar to its mammalian counterpart, the A. thalianasmall GTPase AtRab1b, seems to be involved in proteintransport at early steps of the secretory pathway. Adominant-inhibitory mutant of AtRab1b inhibited thesecretion of a ¯uorescent marker protein as well as theGolgi localization of a Golgi-targeted ¯uorescent marker,leading to ¯uorescence accumulating in the ER in both cases(Batoko et al., 2000).

Retention and distribution of proteins in the Golgi

The means by which Golgi-resident proteins are retainedin the stack is still largely a matter of debate. Whilst many ofthe ER-resident processing proteins are soluble, Golgi-

F I G . 3. Models of ER-to-Golgi protein transport. A, The `vacuum cleaner model' (Boevink et al., 1998) suggests that Golgi stacks move over the ERconstantly picking up cargo. According to this model, the whole ER surface is capable of forming export sites, resulting in their random distribution.In contrast, the `stop-and-go' model (B) hypothesizes that Golgi stacks stop at ®xed ER export sites to take up cargo from the ER, before moving ontothe next stop. In the more dynamic `mobile export sites' model (C), Golgi stacks and ER export sites move together as `secretory units' (Brandizzi

et al., 2002b) allowing cargo to be transported from the ER towards the Golgi at any time during movement.


F I G . 4. Confocal laser scanning micrographs showing the location of different regulatory and structural proteins of the secretory pathway in relation toGolgi stacks. A, Optical section through the cortical cytoplasm of a tobacco BY2 cell stably transformed with the Golgi marker GmMan1-GFP(Glycine max a-mannosidase 1-GFP, green channel) after ®xation and immunolocalization with anti-AtArf1p antibodies (red channel). The mergedimage clearly reveals that anti-AtArf1p labelling is associated with the Golgi, forming a ring-shaped pattern con®ned to the periphery of each stack(Ritzenthaler et al., 2002). B, Optical section through the centre of a transgenic GmMan1-GFP BY2 cell after ®xation (GFP signal in green channel)and immunolocalization with anti-Atg-COP antibodies (red channel). As can be seen in the merged image, the COPI coatomer subunit co-localizeswith Golgi-associated GFP-¯uorescence. As for Arf1p, anti-Atg-COP ¯uorescence is restricted to the margins of the Golgi stacks (Ritzenthaler et al.,2002). C, Projection of 15 optical sections (1 mm per section) through a pea root tip cell after ®xation and double-immunolabelling with anti-VSRantibodies (17F9, green channel) and JIM 84, a trans-Golgi marker (red channel). As shown in the merged image, more than 90 % of the prevacuolarorganelles labelled by 17F9 are separate from Golgi stacks (Li et al., 2002). Occasionally, ¯uorescence labelling by the two antibodies co-localizes(merged image, open arrow). D, Optical section through the cortical cytoplasm of a leaf epidermal cell of a transgenic ST-GFP tobacco plant (GFPsignal in green channel) transiently transformed with YFP-AtRab2a (YFP signal in red channel). As can be seen in the merged image, both ¯uorescentfusion proteins co-localize in Golgi stacks. In addition to the Golgi, YFP-AtRab2a labels small spherical structures (sometimes measuring up to 3 mmin diameter) in which no GFP signal can be detected (merged image, arrows). Bars = 5 mm (A±C, insert D) and 20 mm (D). Micrographs for A and B

kindly provided by Christophe Ritzenthaler and that for C by Liwen Jiang.


Table

1.

Exa

mple

sof

pro

tein

sm

icro

scopic

all

ylo

cate

dto

the

Golg

iand

the

`tra

ns-

Golg

inet

work

'in

cell

sof

hig

her

pla

nts

Pro

tein

(sp

ecie

so

fo

rigin

)E

xp

erim

enta

lsy

stem

Mic

rosc

opic

alte

chniq

ue

Loca

liza

tion

Puta

tive

funct

ion

Ori

gin

alre

fere

nce

(pro

vid

ing

loca

tion

dat

a)

Reg

ula

tory

pro

tein

sE

RD

2(A

rabid

op

sis

tha

lia

na)

Nic

oti

an

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estr

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ytr

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ith

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sion

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len

gth

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GF

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agin

g,

TE

Mim

munogold

label

ling

wit

han

ti-G

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anti

bodie

s

ER

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lG

olg

ici

ster

nae

H/K

DE

Lre

cepto

rfo

rre

trie

val

of

esca

ped

solu

ble

ER

-res

iden

tpro

tein

s

Boev

ink

etal.

(1998)

AtR

ER

1B

(Ara

bid

op

sis

thali

an

a)

Nic

oti

an

ata

ba

cum

BY

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lls

stab

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edw

ith

aG

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sion

of

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ng

th)

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bound

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al.

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lled

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ling

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ng

anti

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s

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pto

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rth

ely

tic

vac

uole

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al.

(1997)*

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a)

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etal.

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bodie

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and

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all

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etal.

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in(N

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oti

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mal

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etal.

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(2003)


TABLE

1.

Conti

nued

Pro

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(sp

ecie

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fo

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in)

Ex

per

imen

tal

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tive

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Ori

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(pro

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ing

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tion

dat

a)

AD

L6

(Ara

bid

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tha

lia

na)

Ara

bid

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sis

tha

lia

na

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tti

ps;

A.

tha

lia

na

pro

top

last

str

ansf

orm

edw

ith

aG

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-fu

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no

fA

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6(f

ull

len

gth

)

Imm

uno¯

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scen

ceusi

ng

anti

-AD

L6

anti

bodie

s(r

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ing

(pro

topla

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in-l

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edin

ves

icle

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cat

the

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etal.

(2001)*

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(Ara

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tha

lia

na

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llen

gra

ins

TE

Mim

munogold

label

ling

wit

han

ti-

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H3P

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tibodie

s

PM

and

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cent

ves

icle

s,ves

icle

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the

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N',

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Pro

tein

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edin

the

®ss

ion

and

unco

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go

fcl

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n-c

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icle

s

Lam

etal.

(2001)*

a-ac

tin

in-l

ike

pro

tein

(N/A

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uno¯

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ce,

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ling

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ng

com

mer

cial

lyav

aila

ble

anti

-a-a

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tibodie

s

Mem

bra

nes

of

Golg

i-as

soci

ated

ves

icle

s

Buddin

gan

dso

rtin

gof

Golg

i-as

soci

ated

ves

icle

sL

ian

dY

en(2

001)*

Str

uct

ura

lp

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ins

Cla

thri

n(N

/A)

N/A

Ele

ctro

nm

icro

scopy

(iden

ti®

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ions)

Golg

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tial

lyco

ated

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mult

ives

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mem

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ne,

clat

hri

n-c

oat

edves

icle

s

Coat

pro

tein

of

clat

hri

nco

ated

ves

icle

sfo

rori

gin

alre

fere

nce

sse

eC

ole

man

etal.

(1988)*

Atg

-CO

P(A

rab

ido

psi

sth

ali

an

a)

Zm

d-C

OP

,Z

me-

CO

P(Z

eam

ays

)A

rabid

op

sis

tha

lia

na

and

Zea

ma

ysro

ots

TE

Mim

munogold

label

ling

usi

ng

anti

bodie

sra

ised

agai

nst

the

dif

fere

nt

CO

Pco

mponen

ts

Rim

sof

Golg

ici

ster

nae

and

CO

PI

ves

icle

s

Coat

om

ersu

bunit

of

CO

PI

ves

icle

sP

impl

etal.

(2000)

SN

AR

Es

AtV

TI1

a(n

ow

AtV

TI1

1)

(Ara

bid

op

sis

thali

an

a)

Ara

bid

op

sis

tha

lia

na

roo

tsfr

om

pla

nts

stab

lytr

ansf

orm

edw

ith

T7

-ta

gged

AtV

TI1

a

TE

Mim

munogold

label

ling

usi

ng

anti

-T7

anti

bodie

s

`Tra

ns-

Golg

inet

work

',den

seves

icle

s,P

VC

SN

AR

Ein

volv

edin

pro

tein

tran

sport

from

the

Golg

ito

the

PV

C

Zhen

get

al.

(1999)*

AtV

PS

45

,A

tTL

G2

a,-b

(no

wA

tSY

P41

,A

tSY

P4

2)

(Ara

bid

op

sis

thali

an

a)

Ara

bid

op

sis

tha

lia

na

roo

ts:

wil

dty

pe

or

from

pla

nts

tran

sfo

rmed

wit

hH

A-t

agg

edA

tTL

G2

aan

dT

7-t

agg

edA

tTL

G2

b

TE

Mim

munogold

label

ling

usi

ng

anti

-A

tVP

S45,

anti

-A

tTL

G2a,

anti

-HA

and/o

ran

ti-T

7an

tibodie

s

`Tra

ns-

Golg

inet

work

'S

NA

RE

sB

assh

amet

al.

(2000)*

AtS

YP

51

,A

tSY

P61

(Ara

bid

op

sis

tha

lia

na)

Ara

bid

op

sis

tha

lia

na

roo

ts:

wil

dty

pe

or

from

pla

nts

tran

sfo

rmed

wit

hH

A-A

tSY

P41

and

T7

-AtS

YP

42

TE

Mim

munogold

label

ling

AtS

YP

51:

`TG

N',

PV

C;

AtS

YP

61:

`TG

N'

Synta

xin

s(S

NA

RE

s)S

ander

foot

etal.

(2001)*


TABLE

1.

Conti

nued

Pro

tein

(sp

ecie

so

fo

rig

in)

Ex

per

imen

tal

syst

emM

icro

scopic

alte

chniq

ue

Loca

liza

tion

Puta

tive

funct

ion

Ori

gin

alre

fere

nce

(pro

vid

ing

loca

tion

dat

a)

AtS

ed5

(no

wca

lled

AtS

YP

31

)(A

rab

ido

psi

sth

ali

an

a)

Nic

oti

an

ata

ba

cum

BY

2ce

lls

tran

sfo

rmed

wit

ha

GF

P-f

usi

on

of

AtS

ed5

(fu

llle

ng

th)

GF

Pim

agin

gG

olg

iS

ynta

xin

(SN

AR

E)

Tak

euch

iet

al.

(2002)

Pro

cess

ing

pro

tein

sa-

2,6

-sia

lyl

tran

sfer

ase

(rat

)N

ico

tia

na

clev

ela

nd

iile

aves

tran

sien

tly

tran

sfo

rmed

wit

ha

GF

P-f

usi

on

of

the

52

N-t

erm

inal

amin

oac

ids

of

sial

yl

tran

sfer

ase

GF

Pim

agin

g,

TE

Mim

munogold

label

ling

wit

han

ti-G

FP

anti

bodie

s

Tra

ns-

face

of

the

Golg

iG

lyco

sylt

ransf

eras

eB

oev

ink

etal.

(1998)

a-2,6

-sia

lyl

tran

sfer

ase

(rat

)A

rabis

op

sis

tha

lia

na

(ro

ots

and

call

us)

stab

lytr

ansf

orm

edw

ith

My

c-ta

gged

sial

yl

tran

sfer

ase

(full

len

gth

)

Imm

uno¯

uore

scen

cean

dT

EM

imm

unogold

label

ling

usi

ng

9E

10

and

A14

anti

-Myc

anti

bodie

s

Tra

ns-

face

of

the

Golg

iG

lyco

sylt

ransf

eras

eW

eeet

al.

(1998)*

N-a

cety

lglu

coso

-am

inylt

ran

sfer

ase

I(G

nT

I)(N

ico

tia

na

taba

cum

)N

ico

tia

na

ben

tha

mia

na

leav

estr

ansi

entl

ytr

ansf

orm

edw

ith

aG

FP

-fu

sion

of

the

Gn

TI

cyto

pla

smic

tran

smem

bra

ne

stem

(CT

S)

do

mai

n

GF

Pim

agin

gG

olg

iG

lyco

sylt

ransf

eras

eE

ssl

etal.

(1999)

a-1,2

man

no

sid

ase

Iso

ybea

n(G

lyci

ne

max)

Nic

oti

an

ata

ba

cum

BY

2ce

lls

stab

lytr

ansf

orm

edw

ith

aG

FP

-fu

sio

no

fm

ann

osi

das

eI

(del

etio

no

fC

-ter

min

al1

1am

ino

acid

s)

GF

Pim

agin

g,

TE

Mim

munogold

label

ling

wit

han

ti-G

FP

anti

bodie

s

Cis

-fac

eof

the

Golg

iN

-lin

ked

oli

gosa

cchar

ide

pro

cess

ing

enzy

me

Neb

enfuÈ

hr

etal.

(1999)

GO

NS

T1

(Ara

bid

op

sis

tha

lia

na)

On

ion

epid

erm

alce

lls

tran

sien

tly

tran

sfo

rmed

wit

ha

YF

P-f

usi

on

of

GO

NS

T1

(full

len

gth

)

YF

Pim

agin

gG

olg

iN

ucl

eoti

de

sugar

(GD

P-m

annose

)tr

ansp

ort

erB

aldw

inet

al.

(2001)*

b1

,2-x

ylo

sylt

ran

sfer

ase

(Ara

bid

op

sis

thali

an

a)

Nic

oti

an

ab

enth

am

ian

ale

aves

tran

sien

tly

tran

sfo

rmed

wit

hG

FP

-fu

sio

ns

of

dif

fere

nt

b1

,2-x

ylo

sylt

ran

sfer

ase

dom

ains

(CT

S,

CT

,T

,C

)

GF

Pim

agin

gG

olg

iG

lyco

sylt

ransf

eras

eD

irnber

ger

etal.

(2002)

Xy

lan

syn

thas

e(P

ha

seo

lus

vulg

ari

s)P

ha

seo

lus

vulg

ari

sh

yp

oco

tyl

TE

Mim

munogold

label

ling

wit

han

ti-b

ean

xyla

nsy

nth

ase

anti

bodie

s

Golg

ian

dpost

-Golg

ives

icle

sof

dev

elopin

gxyle

mce

lls

Synth

esis

of

seco

ndar

yw

all

xyla

nG

regory

etal.

(2002)*


TABLE

1.

Conti

nued

Pro

tein

(sp

ecie

so

fo

rig

in)

Ex

per

imen

tal

syst

emM

icro

scopic

alte

chniq

ue

Loca

liza

tion

Puta

tive

funct

ion

Ori

gin

alre

fere

nce

(pro

vid

ing

loca

tion

dat

a)

b1

,3-g

luca

n(c

allo

se)

syn

thas

e(P

ha

seo

lus

vulg

ari

s)P

ha

seo

lus

vulg

ari

sh

yp

oco

tyls

and

roo

tti

ps

TE

Mim

munogold

label

ling

wit

han

ti-b

1,3

-glu

can

synth

ase

anti

bodie

s

Golg

iof

root

tip

mer

iste

mat

icce

lls

duri

ng

cell

pla

tefo

rmat

ion,

surf

ace

of

seco

ndar

yw

all

thic

ken

ings

and

PM

inpit

sin

dev

elopin

gxyle

mce

lls

Cal

lose

synth

esis

Gre

gory

etal.

(2002)*

b-1

,4-g

alac

tosy

ltr

ansf

eras

e(h

um

an)

Nic

oti

ana

tab

acu

mle

aves

tran

sien

tly

tran

sfo

rmed

wit

ha

GF

P-f

usi

on

of

gal

acto

syl

tran

sfer

ase

(60

C-t

erm

inal

amin

oac

ids)

GF

Pim

agin

gE

Ran

dG

olg

iG

lyco

sylt

ransf

eras

eS

aint-

Jore

etal.

(2002)

MU

R4

(Ara

bid

op

sis

thali

an

a)

Ro

ot

pro

top

last

so

fA

rab

ido

psi

sth

ali

an

ap

lants

stab

lytr

ansf

orm

edw

ith

aG

FP

-fu

sio

no

fM

UR

4(f

ull

len

gth

)

GF

Pim

agin

gG

olg

iU

DP

-D-X

yl

4-e

pim

eras

eB

urg

etet

al.

(2003)*

*L

iter

atu

reci

ted

inth

ista

ble

on

ly,

no

tin

the

mai

nte

xt:

Bald

win

TC

,H

an

dfo

rdM

G,

Yu

seff

MI,

Ore

lla

na

A,

Du

pre

eP

.2001.

Iden

ti®

cati

on

and

char

acte

riza

tion

of

GO

NS

T1,

aG

olg

i-lo

cali

zed

GD

P-m

annose

tran

sport

erin

Ara

bid

opsi

s.T

he

Pla

nt

Cel

l13:

22

83±

22

95

.B

ass

ha

mD

C,

Sa

nd

erfo

ot

AA

,K

ov

ale

va

V,

Zh

eng

H,

Ra

ikh

elN

V.

2000.

AtV

PS

45

com

ple

xfo

rmat

ion

atth

etr

ans-

Golg

inet

work

.M

ole

cula

rB

iolo

gy

of

the

Cel

l11:

2251±2265.

Bu

rget

EG

,V

erm

aR

,M

ùlh

ùj

M,

Rei

ter

W-D

.2

00

3.

Th

eb

iosy

nth

esis

of

L-a

rabin

ose

inpla

nts

:m

ole

cula

rcl

onin

gan

dch

arac

teri

zati

on

of

aG

olg

i-lo

cali

zed

UD

P-D

-xylo

se4-e

pim

eras

een

coded

by

the

MU

R4

gen

eo

fA

rabid

op

sis.

Th

eP

lan

tC

ell

15

:5

23

±5

31.

Ch

eun

gA

Y,

Ch

enC

YH

,T

ao

LZ

,A

nd

rey

eva

T,

Tw

ell

D,

Wu

HM

.2003.

Reg

ula

tion

of

poll

entu

be

gro

wth

by

Rac

-lik

eG

TP

ases

.Jo

urn

al

of

Exp

erim

enta

lB

ota

ny

54

:73±81.

Co

lem

an

J,

Eva

ns

H,

Ha

wes

C.

19

88.

Pla

nt

coat

edv

esic

les.

Pla

nt,

Cel

land

Envi

ronm

ent

11

:669±684.

Co

uch

yI,

Min

icZ

,L

ap

ort

eJ

,B

row

nS

,S

ati

at-

Jeu

nem

ait

reB

.1998.

Imm

unodet

ecti

on

of

Rho-l

ike

pla

nt

pro

tein

sw

ith

Rac

1an

dC

dc4

2H

san

tibodie

s.Jo

urn

al

of

Exp

erim

enta

lB

ota

ny

49:

1647±1659.

Gre

go

ryA

CE

,S

mit

hC

,K

erry

ME

,W

hea

tley

ER

,B

olw

ell

GP

.2002.

Com

par

ativ

esu

bce

llula

rim

munolo

cati

on

of

poly

pep

tides

asso

ciat

edw

ith

xyla

nan

dca

llose

synth

ases

inF

rench

bea

n(P

hase

olu

svu

lga

ris)

du

rin

gse

con

dar

yw

all

form

atio

n.

Ph

yto

chem

istr

y5

9:

249±259.

Inab

aT

,N

ag

an

oY

,N

ag

asa

ki

T,

Sa

sak

iY

.2

00

2.

Dis

tin

ctlo

cali

zati

on

of

two

close

lyre

late

dY

pt3

/Rab

11

pro

tein

son

the

traf

®ck

ing

pat

hw

ayin

hig

her

pla

nts

.Jo

urn

al

of

Bio

logic

al

Chem

istr

y277

:9

18

3±

91

88

.J

inJ

B,

Kim

YA

,K

imS

J,

Lee

SH

,K

imD

H,

Ch

eon

gG

W,

Hw

an

gI.

2001.

Anew

dynam

in-l

ike

pro

tein

,A

DL

6,

isin

volv

edin

traf

®ck

ing

from

the

trans-

Golg

inet

work

toth

ece

ntr

alvac

uole

inA

rabid

op

sis.

Th

eP

lan

tC

ell

13

:1

51

1±

152

5.

Lam

BC

H,

Sa

ge

TL

,B

ian

chi

F,

Blu

mw

ald

E.

20

01.

Ro

leo

fS

H3-c

onta

inin

gpro

tein

sin

clat

hri

n-m

edia

ted

ves

icle

traf

®ck

ing

inA

rabid

opsi

s.T

he

Pla

nt

Cel

l13:

2499±2512.

Li

Y,

Yen

LF

.2

00

1.

Pla

nt

Go

lgi-

asso

ciat

edv

esic

les

con

tain

anovel

alpha-

acti

nin

-lik

epro

tein

.E

uro

pea

nJo

urn

al

of

Cel

lB

iolo

gy

80

:703±710.

Pa

ris

N,

Ro

ger

sS

W,

Jia

ng

L,

Kir

sch

T,

Bee

ver

sL

,P

hil

lip

sT

E,

Roger

sJC

.1997.

Mole

cula

rcl

onin

gan

dfu

rther

char

acte

riza

tion

of

apro

bab

lepla

nt

vac

uola

rso

rtin

gre

cepto

r.P

lant

Phys

iolo

gy

115:

29

±39

.S

an

der

foo

tA

A,

Ah

med

SU

,M

art

y-M

aza

rsD

,R

ap

op

ort

I,K

irch

hau

sen

T,

Mart

yF

,R

aik

hel

NV

.1998.

Aputa

tive

vac

uola

rca

rgo

rece

pto

rpar

tial

lyco

loca

lize

sw

ith

AtP

EP

12p

on

apre

vac

uola

rco

mp

artm

ent

inA

rabid

op

sis

roo

ts.

Pro

ceed

ings

of

the

Nati

on

al

Aca

dem

yof

Sci

ence

sof

the

USA

95:

9920±9925.

Sa

nd

erfo

ot

AA

,K

ov

ale

va

V,

Ba

ssh

am

DC

,R

aik

hel

NV

.2

001.

Inte

ract

ions

bet

wee

nsy

nta

xin

sid

enti

fyat

leas

t®

ve

SN

AR

Eco

mple

xes

wit

hin

the

Golg

i/pre

vac

uola

rsy

stem

of

the

Ara

bid

opsi

sce

ll.

Mo

lecu

lar

Bio

log

yo

fth

eC

ell

12:

37

33±

37

43

.T

ak

euch

iM

,U

eda

T,

Sa

toK

,A

be

H,

Na

ga

taT

,N

ak

an

oA

.2000.

Adom

inan

tneg

ativ

em

uta

nt

of

Sar

1p

GT

Pas

ein

hib

its

pro

tein

tran

spo

rtfr

om

the

endopla

smic

reti

culu

mto

the

Golg

iap

par

atus

into

bac

coan

dA

rabid

op

sis

cult

ure

dce

lls.

Th

eP

lan

tJo

urn

al

23:

517±525.

Wee

EG

T,

Sh

erri

erJ

,P

rim

eT

A,

Du

pre

eP

.1

99

8.

Tar

get

ing

of

acti

ve

sial

ylt

ransf

eras

eto

the

pla

nt

Golg

iap

par

atus.

The

Pla

nt

Cel

l10:

175

9±1768.

Zh

eng

H,

Fis

cher

vo

nM

oll

ard

G,

Ko

va

lev

aV

,S

tev

ens

TH

,R

aik

hel

NV

.1999.

The

pla

nt

ves

icle

-ass

oci

ated

SN

AR

EA

tVT

I1a

likel

ym

edia

tes

ves

icle

tran

sport

from

the

trans-

Golg

inet

work

toth

ep

revac

uo

lar

com

par

tmen

t.M

ole

cula

rB

iolo

gy

of

the

Cel

l1

0:

2251±2264.


resident enzymes are transmembrane proteins (Table 1).Information regarding the retention of membrane proteins atvarious points of the secretory pathway has recently beenrevealed by a study investigating the default pathway of typeI membrane-bound proteins (Brandizzi et al., 2002c). In thisstudy, GFP was fused to proteins with transmembranedomains of variable length and the fusion proteins wereshown to be distributed along the organelles of the secretorypathway in a transmembrane length-dependent manner. Asfor animal cells (Munro, 1995), it is expected that themembrane thickness increases in plant cells from the ER tothe Golgi and ®nally to the plasma membrane/tonoplastowing to a sterol gradient in the lipid composition (Moreauet al., 1998). Accumulation in the Golgi occurred when GFPwas fused to transmembrane domains of 19 or 20 aminoacids (Brandizzi et al., 2002c). Whether this retentionmechanism is universally valid for true Golgi residentsremains to be elucidated, although the signal anchorsequence of a rat sialyl transferase targets GFP to the plantGolgi (Boevink et al., 1998; Saint-Jore et al., 2002), andequivalent sequences of plant glycosyltransferases give thesame result (Essl et al., 1999; Dirnberger et al., 2002; Pagnyet al., 2003). Phe residues, suggested to play a role in Golgiretention in mammalian cells, only seem to play a minor rolein plants, as indicated by comparison of the number andposition of Phe residues of the transmembrane domain ofproteins within the BP80 family (Brandizzi et al., 2002c).

As mentioned earlier, enzymatic activities change grad-ually from the cis-face to the trans-face of the Golgi stack,re¯ecting a sequential processing down the stack. Therefore,a logical question is what controls the distribution of residentenzymes across the different cisternae. This has only beenaddressed experimentally a couple of times in plant cells(Fitchette et al., 1999), and models were ®rst formulated formammalian Golgi enzymes. Again, the bilayer/membranethickness model provides one possible explanation.Alternatively, as in mammalian cells, retrograde transportof processing machinery in vesicle vectors may both reduceloss and control positioning of enzymes whilst cisternaecontinually mature from the cis- to the trans-face (Opat et al.,2001). More evidence is needed before ruling out one orother model for plant Golgi enzymes.

Intra-Golgi transport and retrograde transport (COPImachinery)

The fact that each Golgi stack is divided into a number ofcisternae leads to the question of how cargo is transporteddown the stack. In analogy to mammalian cells, two modelshave been proposed, the `vesicle shuttle' and the `cisternalmaturation' models (reviewed by Hawes and Satiat-Jeunemaitre, 1996; NebenfuÈhr and Staehelin, 2001).According to the vesicle shuttle model, Golgi cisternae arestable entities with a speci®c set of processing proteins, andcargo is sequentially transported down the stack in vesicularshuttles. The second model suggests that cisternae progres-sively move down the stack and mature from the cis-face tothe trans-face of the Golgi. New cis-cisternae are formed byfusion of ER-to-Golgi transport intermediates and retro-grade transport vesicles, which shuttle back the processing

enzymes from the trans-cisternum. Experimental evidenceexists for both models and it cannot be ruled out that eithermechanism or a mixture of both is active. In addition to thealgal scale argument (Becker et al., 1995), recent data on theultrastructural morphology of Golgi stacks in BY2 cells andthe redistribution of a ¯uorescent cis-Golgi marker proteinafter brefeldin A treatment (a fungal toxin widely used tostudy protein transport along the secretory pathway;NebenfuÈhr et al., 2002) seem to favour the cisternalmaturation model (Ritzenthaler et al., 2002). Finally, itcannot be excluded that cisternae of the stack are all joinedby interconnecting tubules, necessitating a different modelto explain the regulation of cis-to-trans-transport.

In addition to anterograde protein transport from the ERto the Golgi and down the Golgi stack, retrograde proteintransport occurs between the Golgi and the ER (e.g.transport of escaped ER residents) as well as inside theGolgi stack, from the trans- towards the cis-face. Inmammalian cells, retrograde transport is likely to bemediated by COPI vesicles and regulated by the Arf1pGTPase (Spang, 2002). Components of the COPI machineryhave been located to the Golgi by means of immuno¯uor-escence (Ritzenthaler et al., 2002) (Fig. 4A and B). Moreprecisely, a study combining biochemistry with cryo-section immunogold labelling techniques, revealed that inplant cells, components of the COPI coat as well as Arf1pmainly locate to the cis-half of Golgi stacks and that COPIcoat proteins are present on small vesicles budding off fromcis-cisternae (Pimpl et al., 2000). In addition, in vitro COPIvesicle induction from ER/Golgi membranes of transgenictobacco plants overproducing the soluble secretory markera-amylase fused to HDEL (the C-terminal sorting peptideof ER residents), showed that COPI vesicles contained themodi®ed secretory marker as well as the ER-residentcalreticulin. This was the ®rst indication in plants thatCOPI vesicles might be involved in retrograde transportfrom the Golgi (Pimpl et al., 2000). The involvement ofAtArf1 in this particular transport event was also deducedfrom the effect of two dominant negative mutant forms ofAtArf1 on the distribution of three ¯uorescent Golgimarkers (Takeuchi et al., 2002). To date, the demonstrationof the role of COPI vesicles in intra-Golgi retrogradetransport remains a challenge, mainly owing to the lack ofmarker molecules for this speci®c transport event.

Recycling through the Golgi: protein import fromdestinations other than the ER

During secretion, vesicle-derived membrane is continu-ously added to the plasma membrane. To balance thisincrease in membrane surface area, clathrin-coated endo-cytic vesicles pinch off from the plasma membrane towardsthe inside of the cell (reviewed by Holstein, 2002). Thedifferent endocytic compartments in plant cells are still notfully characterized. In analogy to mammalian cells, the term`endosome' is often found in the plant literature indicating acompartment containing endocytosed material (JuÈrgens andGeldner, 2002). Ultrastructurally, the `plant endosome'most likely corresponds to the partially coated reticulum(PCR), a compartment that may originate from the `trans-


Golgi network' (Hillmer et al., 1988). From this compart-ment, protein might be recycled towards the Golgi ortransported towards the lytic vacuole via multi-vesicularbodies (MVBs) for degradation. This was shown by eleganttime-course studies following the uptake of cationizedferritin (CF) in soybean protoplasts (Fowke et al., 1991),re¯ecting vesicle-mediated plasma membrane recycling(and not receptor-mediated endocytosis). This marker isquickly endocytosed through coated pits into coatedvesicles. Within the cytoplasm, CF sequentially labelledthe following organelles: tubular elements of the PCR;periphery of Golgi cisternae; MVBs; and ®nally the centralvacuole. Labelling of the Golgi by CF almost certainlyre¯ects membrane recycling from the plasma membrane,presumably through the PCR (Fowke et al., 1991). Whetherdirect plasma membrane recycling to the Golgi can occur inplant cells still has to be elucidated.

In addition to maintaining the plasma membrane surface,protein import into the Golgi through the `plant endosome'could also be important for regulating, by endocytosis at theplasma membrane, the number of ion channels, protonpumps (Crooks et al., 1999) and carrier proteins, such asPIN1 (auxin ef¯ux carrier; see Geldner et al., 2001).Remodelling the distribution of such proteins at the plasmamembrane via vesicle traf®cking must certainly be regardedas an important way in which a plant can react to changes inenvironmental conditions (Levine, 2002).

OUT OF THE GOLGI

The major destinations for proteins exiting the Golgiapparatus are the plasma membrane and the vacuolarsystem. During cytokinesis, another protein traf®ckingroute leads towards the developing cell plate (reviewed byBednarek and Falbel, 2002). Here we focus on how sortingof proteins to the plasma membrane or the vacuolar systemoccurs and where this sorting takes place.

As to the place of sorting, the term `trans-Golgi network'(TGN) from the mammalian `endomembrane nomenclat-ure' is becoming more common in the plant literature.However, in plant cells, no discrete protein-sorting com-partment with a characteristic set of proteins, downstreamfrom the trans-face of the Golgi has yet been described. Inaddition, in plants, protein sorting and secretory vesicleproduction can take place as early as at the cis-cisternae ofthe Golgi stack (see below). Therefore, it might be better tosuggest that production of clathrin-coated vesicles destinedfor the lytic vacuolar system is limited to the trans-face ofthe Golgi. Whether this face of the Golgi, which certainlycan comprise a network of tubules and vesicles, ishomologous to the mammalian TGN is open for debate.

Secretion: transport towards the plasma membrane and thecell wall

The default destination of soluble proteins and complexcarbohydrates has been suggested to be the plasma mem-brane (Denecke et al., 1990). Secretory proteins usuallycarry an amino terminal signal peptide for insertion into theER, which is clipped off upon translocation across the ER

membrane (Vitale and Denecke, 1999). Soluble secretoryproteins are not known to carry any positive targetinginformation, which would divert them from the defaultpathway to the plasma membrane, either to the vacuolarsystem or back to the ER (Hadlington and Denecke, 2000).

In contrast, the situation with membrane proteins is lessclear. Until recently, the tonoplast had been regarded as thedefault destination (Barrieu and Chrispeels, 1999).However, a VSR-based (vacuolar sorting receptor) GFP-fusion with a lengthened transmembrane domain accumu-lated on the plasma membrane, indicating that positivesorting information might be necessary to direct membraneproteins from the Golgi towards the tonoplast (Brandizziet al., 2002c). Nevertheless, transport of proteins to speci®careas of the plasma membrane, nicely illustrated by proteinssuch as the auxin ef¯ux carrier PIN1, which locates to thedistal part of the plasma membrane in root cells ofarabidopsis seedlings (Geldner et al., 2001; JuÈrgens andGeldner, 2002), is dif®cult to imagine without some form ofeither positive targeting or retention mechanism.

Transport towards the vacuolar system: different types ofvacuoles, vesicles, sorting signals, receptors and sortingsites

As mentioned earlier, sorting signals are necessary fortransport to the vacuolar system. In plant cells, two maintypes of vacuoles [distinguishable by different sets of TIPs(tonoplast intrinsic proteins) and lumenal contents] may co-exist, and sorting towards either of them depends ondifferent peptide targeting signals and is mediated bydifferent sets of transport vesicles (Paris et al., 1996; Jiangand Rogers, 1998; Hinz et al., 1999).

Transport to the lytic vacuole, characterized by g-TIPs,occurs via an intermediate compartment known as theprevacuolar compartment (PVC) and relies on amino-terminal, sequence-speci®c propeptides (NPIR or equiva-lent), which are recognized by VSRs. The ®rst VSR to beidenti®ed was BP80 from Pisum sativum (now calledVSRPS-1; reviewed by Paris and Neuhaus, 2002). VCRs arethought to cycle between the Golgi apparatus and the PVC(Mitsuhashi et al., 2000) where they are preferentiallylocated, as was shown by confocal immuno¯uorescence forseveral VSRPS-1 homologues (Li et al., 2002) (Fig. 4C). It isthought that VSRs mediate the packaging of cargo destinedto the lytic vacuole in trans-Golgi-located clathrin-coatedvesicles (Hinz et al., 1999).

In contrast, proteins delivered to the protein storagevacuole (a-TIP vacuoles) reach their destination in vesiclesapparently devoid of any speci®c protein coating and, inlegume seeds, have been termed dense vesicles (Hohl et al.,1996). Sorting relies on a carboxyl-terminal propeptide andan as yet unidenti®ed sorting receptor, the existence ofwhich is deduced from the observation that transporttowards the storage vacuole can be saturated (Frigerioet al., 1998). Immunogold labelling indicates that sorting ofsome storage proteins can occur as early as the cis-cisternaeof the Golgi in pea cotyledons (Hillmer et al., 2001). Otherstorage vacuole proteins as well as integral membraneproteins are sorted even earlier, at the ER level, where they


are packed into so-called precursor-accumulating (PAC)vesicles and transported to the storage vacuole via a routebypassing the Golgi (Mitsuhashi et al., 2001). A vacuolarsorting receptor for this pathway has been identi®ed inpumpkin seeds (Shimada et al., 2002).

REGULATORY PROTEINS AT THE HEARTOF THE SECRETORY PATHWAY

In recent years, it has become apparent that the differenttransport events along the secretory/biosynthetic andendocytic pathway are orchestrated by a plethora ofproteins. The functional importance of the plant Golgiapparatus in the secretory pathway is re¯ected by the factthat numerous regulatory proteins are structurally andfunctionally linked to the Golgi and the `trans-Golginetwork' (Hawes et al., 1999; Sanderfoot and Raikhel,1999; NebenfuÈhr, 2002; Rutherford and Moore, 2002; seeTable 1). For example, small GTPases act as molecularswitches involved in the formation, transport and fusion oftransport vesicles, while SNAREs are integral membraneproteins involved in determining the speci®city of fusionevents along the endomembrane system, residing ontransport vesicles (R-SNAREs) and target membranes (Q-SNAREs). Considerable effort has been put into comparingvarious genomes to identify plant homologues of proteinsthat have been shown to regulate various traf®cking eventsalong the secretory pathway in yeast and animals (Andreevaet al., 1998a; Sanderfoot et al., 2000; JuÈrgens and Geldner,2002). Statements such as `Rab functions are conservedacross eukaryotes, such that their subcellular localisationcan be inferred from known localisations of members of thesame subfamily in other species' (JuÈrgens and Geldner,2002) may be correct as to the functional aspect. However,even though homologous Rab proteins may show the samesubcellular location, it cannot be necessarily concluded thatthey exert the same function. For instance, despite theGolgi-location of two mammalian splice-variants of Rab6,which differ only in three amino acid residues, Rab6A andRab6A¢ seem to function in different membrane-traf®ckingevents (Echard et al., 2000). Likewise, two plant Rab2isoforms have been found to locate to Golgi stacks (Fig. 4D)but seem to regulate quite different transport steps. Intobacco pollen tubes, NtRab2 seems to sustain ER-to-Golgitraf®c (Cheung et al., 2002), while studies conducted in ourgroup seem to indicate that an arabidopsis Rab2 isoformregulates vesicle traf®c between the Golgi and a post-Golgicompartment (U. Neumann, I. Moore, C. Hawes and H.Batoko, unpubl. res.).

It is outside the scope of this current Brie®ng to considerthe putative roles of the various small GTPases andSNAREs that have been identi®ed in plants. A summaryof the major regulatory proteins locating to the plant Golgiidenti®ed to date is given in Table 1.

CONCLUSIONS

Immense progress has been made in recent years toelucidate the various transport events along the secretoryand endocytic pathways in eukaryotic cells. This is certainly

true for the Golgi apparatus, which has seen a renaissance inresearch popularity since the 100th anniversary of itsdiscovery by Camillo Golgi in 1898. New data regardingthe molecular machinery that drives and regulates traf®ck-ing events at the Golgi level are published almost weekly. Inthis context, comparative genomic analyses have helped toidentify plant homologues of yeast and animal proteinsregulating Golgi-related transport steps. One of the mainchallenges of the post-genomic era is to provide functionaland structural evidence for the speci®c roles of plantproteins putatively playing a role in the secretory/endocyticpathway. As to structural data at the subcellular level,technological progress in light microscopy such as confocalmicroscopy and deconvolution technology for improvingimages, combined with developments in immunolabellingand ¯uorescent protein technology have revolutionized thestudy of cell biology (Brandizzi et al., 2002a). However,especially with regard to the Golgi apparatus, confocalmicroscopy combined with GFP technology in order tolocate proteins is not without its pitfalls. It becomes moreand more common to assume that a punctate distribution ofGFP ¯uorescence in the cytoplasm is suf®cient to establishthat a speci®c protein is located to the Golgi withoutadditional con®rmatory evidence such as co-localizationwith a known Golgi marker at the light microscopical level(e.g. a ¯uorescent protein marker or by immunocytochem-ical labelling with `anti-Golgi' antibodies) or immunogoldlabelling at the TEM level. Cryotechniques for specimenpreparation, such as ultra-rapid freezing at ambient or highpressure and freeze-substitution, allow for excellent ultra-structural preservation, but even without these moresophisticated microscopical techniques, immunogold label-ling at the TEM level is an excellent way to localize proteinsat the subcellular level.

Despite the progress made in recent years regarding thefunctioning of the secretory/endocytic pathway in generaland the plant Golgi apparatus in particular, many aspectsstill remain to be discovered. The necessary advances willbe made only if we are able to link molecular with structuraland functional data.

ACKNOWLEDGEMENTS

We thank David Evans for critical reading of the manu-script, Barry Martin for skilful assistance with high-pressurefreezing and Liwen Jiang and Christophe Ritzenthaler forkindly providing micrographs. We acknowledge theBiotechnology and Biological Sciences Research Council,UK, for supporting the work undertaken in our laboratory.

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