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.
170 Neumann et al. Ð Protein Transport in Plant Cells
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.
Neumann et al. Ð Protein Transport in Plant Cells 171
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
acl
evel
an
dii
leav
estr
ansi
entl
ytr
ansf
orm
edw
ith
aG
FP
-fu
sion
of
ER
D2
(full
len
gth
)
GF
Pim
agin
g,
TE
Mim
munogold
label
ling
wit
han
ti-G
FP
anti
bodie
s
ER
,al
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
2ce
lls
stab
lytr
ansf
orm
edw
ith
aG
FP
-fu
sion
of
AtR
ER
1B
(fu
llle
ng
th)
GF
Pim
agin
gG
olg
iR
ecycl
ing
rece
pto
rfo
rm
embra
ne-
bound
ER
pro
tein
sTak
euch
iet
al.
(2000)
BP
-80
(als
oca
lled
VS
RP
S-1
)(P
isu
msa
tivu
m)
Pis
um
sati
vum
roo
tti
ps
Imm
uno¯
uore
scen
cean
dT
EM
imm
unogold
label
ling
usi
ng
anti
-V
SR
PS
-1an
tibodie
s
Golg
i,P
VC
Sort
ing
rece
pto
rfo
rth
ely
tic
vac
uole
Par
iset
al.
(1997)*
AtE
LP
(Ara
bid
op
sis
thali
an
a)
Ara
bid
op
sis
tha
lia
na
roots
TE
Mim
munogold
label
ling
wit
han
ti-
AtE
LP
anti
bodie
s
Golg
i,`t
rans-
Golg
inet
work
'.P
VC
Sort
ing
rece
pto
rfo
rth
ely
tic
vac
uole
San
der
foot
etal.
(1998)*
AtA
rf1
p(A
rabid
op
sis
tha
lia
na)
Ara
bid
op
sis
tha
lia
na
and
Zea
ma
ysro
ots
TE
Mim
munogold
label
ling
wit
han
ti-
AtA
rf1p
anti
bodie
s
Rim
sof
Golg
ici
ster
nae
and
CO
PI
ves
icle
s
Sm
all
GT
Pas
ein
volv
edin
CO
PI
ves
icle
form
atio
n
Pim
pl
etal.
(2000)
Rho
-lik
ep
rote
in(N
/A)
Nic
oti
an
ata
ba
cum
BY
2ce
lls
Imm
uno¯
uore
scen
ceusi
ng
anti
-hum
anR
ac1
anti
bodie
s
Golg
iS
mal
lG
TP
ase
involv
edin
regula
ting
the
cyto
skel
eton
org
aniz
atio
n
Couch
yet
al.
(1998)*
NtR
ab2
(Nic
oti
an
ata
ba
cum
)N
ico
tia
na
tab
acu
mp
oll
entu
bes
tran
sien
tly
and
stab
lytr
ansf
orm
edw
ith
aG
FP
-fu
sion
of
NtR
ab2
(full
len
gth
)
GF
Pim
agin
g,
TE
Mim
munogold
label
ling
wit
han
ti-G
FP
anti
bodie
s
Golg
ist
ack
per
ipher
yS
mal
lG
TP
ase
involv
edin
ER
-to-G
olg
itr
af®
cC
heu
ng
etal.
(2002)
AtR
ab2
a(A
rabid
op
sis
tha
lia
na)
Nic
oti
an
ata
ba
cum
leav
estr
ansi
entl
ytr
ansf
orm
edw
ith
aG
FP
-fu
sio
no
fA
tRab
2a
(full
len
gth
)
GF
Pim
agin
g,
TE
Mim
munogold
label
ling
wit
han
ti-G
FP
anti
bodie
s
Golg
i,cy
toso
lan
dsm
all
spher
ical
bodie
s
Sm
all
GT
Pas
ein
volv
edin
post
-Golg
itr
af®
ckin
gU
.N
eum
ann,
I,M
oore
,C
.H
awes
and
H.
Bat
oko
(unpubl.
res.
)
Pra
2,
Pra
3(P
isu
msa
tivu
m)
Nic
oti
an
ata
ba
cum
BY
2ce
lls
stab
lytr
ansf
orm
edw
ith
aG
FP
-fu
sion
of
Pra
2an
dP
ra3
(fu
llle
ng
th)
GF
Pim
agin
gP
ra2:
Golg
ian
d`e
ndoso
mes
';P
ra3:
`TG
N'
and/o
rP
VC
Rab
GT
Pas
es(R
ab11
hom
olo
gues
)In
aba
etal.
(2002)*
AtR
ac7
(Ara
bid
op
sis
tha
lia
na)
Nic
oti
an
ata
ba
cum
po
llen
tubes
tran
sfo
rmed
wit
ha
GF
P-f
usi
on
of
AtR
ac7
(full
len
gth
)
GF
Pim
agin
gG
olg
i,pla
sma
mem
bra
ne
(not
atth
epoll
entu
be
tip)
Rac
-lik
eG
TP
ase
Cheu
ng
etal.
(2003)
172 Neumann et al. Ð Protein Transport in Plant Cells
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)
AD
L6
(Ara
bid
op
sis
tha
lia
na)
Ara
bid
op
sis
tha
lia
na
roo
tti
ps;
A.
tha
lia
na
pro
top
last
str
ansf
orm
edw
ith
aG
FP
-fu
sio
no
fA
DL
6(f
ull
len
gth
)
Imm
uno¯
uore
scen
ceusi
ng
anti
-AD
L6
anti
bodie
s(r
oot
tips)
;G
FP
imag
ing
(pro
topla
sts)
Golg
iD
ynam
in-l
ike
pro
tein
involv
edin
ves
icle
form
atio
nfo
rvac
uola
rtr
af®
cat
the
`TG
N'
Jin
etal.
(2001)*
AtS
H3
P1
(Ara
bid
op
sis
tha
lia
na)
Ara
bid
op
sis
tha
lia
na
po
llen
gra
ins
TE
Mim
munogold
label
ling
wit
han
ti-
AtS
H3P
1an
tibodie
s
PM
and
adja
cent
ves
icle
s,ves
icle
sof
the
`TG
N',
PC
R
Pro
tein
involv
edin
the
®ss
ion
and
unco
atin
go
fcl
athri
n-c
oat
edves
icle
s
Lam
etal.
(2001)*
a-ac
tin
in-l
ike
pro
tein
(N/A
)L
iliu
md
avi
dii
,p
oll
enan
dp
oll
entu
bes
Imm
uno¯
uore
scen
ce,
TE
Mim
munogold
label
ling
usi
ng
com
mer
cial
lyav
aila
ble
anti
-a-a
ctin
inan
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
rote
ins
Cla
thri
n(N
/A)
N/A
Ele
ctro
nm
icro
scopy
(iden
ti®
cati
on
of
clat
hri
nin
terl
ock
ing
tris
kel
ions)
Golg
i,par
tial
lyco
ated
reti
culu
m,
mult
ives
icula
rbodie
s,pla
sma
mem
bra
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)*
Neumann et al. Ð Protein Transport in Plant Cells 173
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)*
174 Neumann et al. Ð Protein Transport in Plant Cells
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.
Neumann et al. Ð Protein Transport in Plant Cells 175
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-
176 Neumann et al. Ð Protein Transport in Plant Cells
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
Neumann et al. Ð Protein Transport in Plant Cells 177
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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