Protein Secretion in Plants: from the Trans-Golgi Network to the Outer Space

Protein Secretion in Plants: from the trans-Golgi Network to the Outer Space

Gerd Jürgens* and Niko Geldner cells suggests that the basic machinery was invented by a unicellular eukaryote and subsequently adapted to the needs ZMBP, Entwicklungsgenetik, Universität Tübingen, Auf der of multicellular life. For example, targeted secretion to the Morgenstelle 3, D-72076 Tübingen, Federal Republic of apical vs. baso-lateral surface of polarised epithelial cells re- Germany quires sorting of proteins destined to specific subdomains * Corresponding author: Gerd Jürgens, within the plasma membrane and uses modified subsets of [email protected] trafficking components already present in yeast. Compared Functional analysis of exocytosis in yeast and animal to yeast and animals, much less is known about mechanisms cells has led to the identification of conserved ele- of protein secretion in plants (2). Considering that plants and ments and mechanisms of the trafficking machinery animals display different cellular organisations and also have over the last decade. Although functional studies of achieved multicellularity independently during evolution, it protein secretion in plants are still fairly limited, the seems likely that protein secretion has undergone specific Arabidopsis genome sequence provides an opportunity modifications in the plant lineage. to identify key players of vesicle trafficking that are conserved across the eukaryotic kingdoms. Here, we A few examples from plant development may illustrate re- review and add to recent genome analyses of traffick- quirements that have to be met by the protein secretory sys- ing components and highlight some plant-specific modifications of the common eukaryotic machinery. tem. Vegetative pollen cells secrete a peptide ligand (SCR) Furthermore, we discuss the evidence for targeted, into their cell wall that interacts with a Ser/Thr kinase receptor polarised secretion in plant cells, and speculate about complex (SRK-SLG) in the plasma membrane of stigmatic possible underlying cargo sorting processes at the cells, ensuring recognition of matching partners in the self- trans-Golgi network and endosomes, based on what is incompatibility response (3). Similarly, signaling between the known in animals and yeast. stem cells and the organising centre of the shoot meristem requires interaction between a secreted peptide ligand Key words: Arabidopsis genome, ARF, endosomes, in- (CLV3) and a Ser/Thr kinase receptor complex (CLV1-CLV2) tracellular trafficking, plasma membrane, polarised se- for continual reassessment of cell fate and maintenance of cretion, rab, SNARE complex, trans-Golgi network, shoot meristem organisation (4). Whereas secretion of the vesicle budding, vesicle fusion ligand into the apoplast has been demonstrated, internalis- Received 3 June 2002, revised and accepted for publi- ation of the ligand-receptor complex and potential recycling cation 14 June 2002 of the receptor complex to the plasma membrane have not been studied. Root hairs and pollen tubes grow by delivery of transport vesicles to their tips (5). Moreover, pollen tubes Protein delivery to the cell surface, via the endomembrane grow directionally towards the ovules for fertilisation, which system, is a common feature of eukaryotic cells. Integral pro- requires continual changes of vesicle targeting to the plasma teins of the plasma membrane as well as secreted proteins membrane in response to as yet unknown chemotactic sig- are synthesised at the endoplasmic reticulum (ER) and are nals (6). Plant cells are able to elongate directionally up to inserted into or translocated across the ER membrane. All 200 times their original length (7), which requires differential subsequent steps of protein trafficking involve transport ves- insertion of membrane material into subdomains of the icles that bud from a donor membrane and fuse with a target plasma membrane. Some cell types such as trichomes and membrane. Most vesicles that leave the ER are trafficked to root hairs undergo localised outgrowth from specific sites the Golgi complex, although some vesicles bypass the Golgi within the plasma membrane (8,9). Finally, the cells of the on their way to the vacuole (1). The Golgi complex is a major endodermis partition their surface into two domains, with an sorting station where trafficking routes diverge to the plasma inner surface separated from an outer surface by the Casp- membrane, the endosome, the vacuole or the cell plate (2) arian strip (10). This is formally similar to the zonula adherens (Figure 1). Post-Golgi sorting occurs in the endosome which border between apical and baso-lateral domains of animal is also involved in recycling of vesicles to the plasma mem- epithelial cells. brane. The cellular organisation of plants may entail specific modi- Extensive analysis of protein secretion in yeast and animal fications of protein secretion. Not only does an ER network

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Figure1: Model of the trafficking routes in the late secretory system. Schematic of an Arabidopsis root meristem cell expressing plasma membrane markers located to distinct subdomains (marker names are coloured and in italic). Coloured arrows mark the respective possible pathways to different plasma membrane compartments from the TGN and/or the endosome. The size of the arrows indicates the possible differences in relative contributions to the total transport of these plasma membrane markers. Names of membrane compartments are in bold. Reported locations of proteins of the transport machinery mentioned are in plain text. The circle of dotted lines represents the ill-defined plant endosome, whose structure and subcompartments (early endosomes, sorting endosomes, etc.) will have to be defined in the future.

606 Traffic 2002; 3: 605–613 Late Secretory Pathway in Plants occur at the cell periphery, but also the Golgi complex is dis- the animal ECM is mainly proteinaceous. Finally, due to the persed into many stacks whose numbers vary from about 20 absence of cell movement, plant organs are shaped by to 400 in plant cells (11). Furthermore, the endomembrane oriented cell division and regulated cell expansion, with the organisation is highly dynamic, due to extensive cytoplasmic latter resulting from targeted protein secretion. streaming, which moves organelles about the cell and may bring Golgi stacks and ER in close proximity (12,13). As plant This review serves two purposes. First, we will exploit the cells enlarge, they often contain a large central vacuole that Arabidopsis genome sequence to provide an overview of leaves a thin layer of cytoplasm underneath the plasma mem- some central players of the plant secretion machinery by brane. Specific cell types contain two different kinds of vacu- comparison with the components that have been functionally ole: a lytic vacuole akin to those in yeast or the lysosome of characterised in animals and yeast. Second, we will summar- animal cells and a storage vacuole from which reserve ma- ise recent findings that may shed light on protein trafficking terials can be mobilised (14). The plant cytoskeleton also has between the trans-Golgi network (TGN) and the plasma unique features. Actin filaments mediate cell growth, cyto- membrane, discussing the role of the endosomal system in plasmic streaming, ER–Golgi association and organelle protein sorting. Trafficking from the TGN to the vacuole has movement. Microtubules are not nucleated from localised been reviewed recently (17) and will not be covered here. MTOCs (centrosomes) and form particular arrays such as in- terphase cortical hoops that maintain cell shape, and prepro- phase band and phragmoplast that mediate oriented cell divi- Plant Proteins Involved in Vesicle Trafficking sion and the execution of cytokinesis, respectively (15). How- ever, MTs do not seem to be necessary for basic cell growth, Studies in yeast and animals have characterised components as protein trafficking to the plasma membrane occurs in MT- of an ever-growing molecular machinery that ensures the deficient cells (16). Moreover, the extracellular matrix (ECM) budding of vesicles with defined sets of cargo proteins from of plant cells is a largely polysaccharidic cell wall, whereas the donor membrane as well as their recognition by, and fu-

Table1: Overview of Arabidopsis thaliana protein families involved in vesicle trafﬁcking. Asterisks indicate searches done by the authors Protein group Subgroup/class Proteins/ AGI numbers Ref. protein numbers ARF class I ARF1 – ARF6 At3g62290; At2g47170; At1g23490; * G-proteins At1g70490; At1g10630; At5g14670 plant class A ARF7 – ARF8 At5g17060; At3g03120 * plant class B ARF9 At2g15310 * ARF-GEFs Gea/GNOM/GBF GNOM, GNL1, GNL2 At1g13980; At5g39500; At5g19610 * Sec7p/BIG class AtBIG1-5 At3g43300; At1g01960; At3g60860; * At4g35380; At4g38200 ARF-GAPs ARF-GAP1 class 3 members At2g35210; At4g17890; At5g46750 * Age2p class 2 members At3g17660; At5g54310 * Age2p-like class 3 members At3g07940; At4g05330; At4g21160 * Gcs1p class 2 members At2g37550; At3g53710 * Rab GTPases 12 subfamilies 57 members See reference (44) Rab effectors exocyst 8 of 8 subunits At1g47550, At3g10380, At1g71820 * (At1g21170/At1g76850), At5g03540 (At1g10385/At5g49830), At5g12370 (At3g56640/At4g02350) VFT 3 of 3 subunits (At1g71270/At1g71300), At1g50500, * At4g19490 HOPS 6 of 6 subunits At2g05170, At2g38020 (VCL1), * At1g12470, At1g08190, At4g36630, At3g54860 Sec34/35p 5 of 8 subunits See reference (52) SNAREs SYP (syntaxins) 24 members See reference (57) VAMP 14 members SNAP25 hom. 3 members NPSN 3 members VTI, Gos1, 3, 2, 2, 2 members, Bet1, Membrin respectively Sec1 family Sec1 group KEULE, Sec1a, Sec1b See reference (57) Vps45 group VPS45 Vps33 group VPS33 Sly1 group Sly1

Traffic 2002; 3: 605–613 607 Jürgens and Geldner sion with, the correct target membrane. Screening of the Ara- of an N-terminal dimerisation domain and are related to yeast bidopsis genome for related sequences to key players of in- Gea1/2p and Sec7p large ARF-GEFs, respectively (22,26). tracellular trafficking in nonplant organisms gives some idea Unlike mammals with only one Gea1/2p class (GBF) and two of conserved elements of the machinery between yeast, Sec7p class (BIG1/2) ARF-GEFs, the Arabidopsis genome plants and animals. For the sake of brevity, we will limit our encodes 3 Gea1/2p and 5 Sec7p-like exchange factors. The discussion to two processes: vesicle budding from the donor sequence divergence between family members (20–60% membrane and vesicle interaction with the target membrane. identity) suggests at least partially non-redundant functions. Only the Gea1/2p class ARF-GEF GNOM has been functionally characterised. Although gnom embryos have severe pat- Vesicle Budding terning defects, the mutant cells are viable and can be grown in culture (27–29). GNOM might act in signal-dependent re- Vesicle budding requires small GTPases of the ARF family, cycling of plasma membrane proteins from endosomal com- their guanine-nucleotide exchange factors (ARF-GEFs) and partments to the cell surface (29) (Figure 1 and see below). GTPase-activating proteins (ARF-GAPs) for coat recruitment Neither yeast nor mammalian ARF-GEFs of the Gea/GBF/ and cargo selection. ARFs not only act to recruit COPI and GNOM class appear to act in endosomal recycling, which in clathrin coats to membranes but also play a role in the control mammals rather involves mammal-specific ARF-GEFs, such of membrane lipid composition, actin remodeling and related as EFA6, ARNO or ARF-GEP100 (22,23). Thus, plants seem events (18). Structurally related ARF-like proteins (ARLs) are to have evolved new members of an existing ARF-GEF class members of the same subgroup of the ras superfamily, but to regulate recycling events, whereas ARF-GEFs with newly have different functions. For example, Arabidopsis ARL2 is assembled domain structures have evolved for the same pro- involved in microtubule formation (16). The six mammalian cess in animals. ARFs have been grouped into three classes based on their structure and function. Class I consists of three members Yeast ARF1-GAP mediates interaction between ER-Golgi v- (ARF1–3), class II is represented by ARF4 and ARF5, SNAREs and COPI coat proteins (30). Three other yeast ARF- whereas ARF6 constitutes the most divergent class III (19). GAPs act at the TGN, whereas mammals also have structur- By contrast, there are only three ARFs in yeast. Class I ARF1 ally divergent ARF-GAPs functioning in the periphery of the and ARF2 are functionally redundant (20). Yeast ARF3 is di- cell (18,31). In Arabidopsis, ten genes encode ARF-GAPs of vergent from both yeast class I ARFs and human ARF6 (21). which three groups are related to the yeast ARF-GAPs acting In Arabidopsis, six of nine putative ARF genes encode class at the TGN, Age2p, Gcs1p and Glo3p, respectively, while the I ARF proteins with 98–100% amino acid identity (Table1). members of the fourth group show similarities to human Four of them can be grouped into two pairs derived from ARF-GAP1 (31,32) (Table1). segmental genome duplication events. Additionally, there are three more divergent ARFs, including one pair of duplicates, Transport vesicles can be distinguished by their coats which with about 60% amino acid identity to human ARF1. The are formed by a limited set of proteins, including COPI, COPII third divergent ARF seems to represent a subclass of its own and clathrin. Coat proteins are captured by specific adaptor (Table1). The less conserved Arabidopsis ARFs are also di- proteins and aid in cargo recruitment and vesicle budding vergent from human ARF6 or yeast ARF3, arguing for an in- (33). Whereas COPI- and COPII-coated vesicles traffic be- dependent evolution of divergent ARF classes from class I tween ER and cis-Golgi or between Golgi cisternae, clathrin- ancestors in the three kingdoms. coated vesicles bud from the TGN or the plasma membrane and are thus involved in post-Golgi trafficking. The ARF GDP/GTP exchange factors (ARF-GEFs) also under- went diversification during the separate evolution to multicel- Clathrin-coated vesicles have been detected at the TGN and lularity. Yeast have four such regulators of ARFs: the largely plasma membrane, and at the cell plate in dividing cells of redundant couple Gea1p/Gea2p, and Sec7p and Syt1p (18). Arabidopsis (34–36). Arabidopsis has two genes encoding ARF-GEFs are defined by the catalytic Sec7 domain. The slightly divergent putative clathrin heavy chains, and clathrin mammalian cell displays a much greater diversity of Sec7 has been immunolocalised to budding vesicles (35). In ad- domain proteins than yeast. There are to date five different dition, one of three putative clathrin light chains was shown classes of exchange factors described, accounting for about to interact with light chain-free mammalian clathrin hubs in 10 different ARF-GEFs three of which show a completely vitro (37). new arrangement of domains that are not present in yeast ARF-GEFs (22,23). These new ARF-GEF classes appear to Clathrin is recruited by adaptor (AP) complexes that consist have evolved for specialised transport functions in a multicel- of four subunits: two large subunits (b and one of a, g, d or lular context, such as matrix adhesion or receptor down-regu- e), the medium subunit m and the small subunit s. Like mam- lation (24,25). mals, and in contrast to Drosophila or yeast, Arabidopsis has four adaptor complexes, AP-1 to AP-4 (38). AP-2 mediates Plant ARF-GEFs appear to show a different pattern of diversi- clathrin recruitment in endocytosis from the plasma mem- fication. Arabidopsis has only two classes of exchange fac- brane, whereas AP-1, AP-3 and AP-4 are associated with the tors which can be distinguished by the presence or absence trans-Golgi network and/or endosomes in mammals.

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Whereas AP-1 and AP-2 function with clathrin, AP-4 is most member of the rab5 subfamily, which lacks the strictly con- likely part of a nonclathrin coat. Interestingly, the Arabidopsis served C-terminal isoprenylation (47). Instead, N-myristoyl- genome encodes three putative g subunits of AP-1 as op- ation and palmitoylation of an N-terminal extension is necess- posed to two in mammals and only in one each in Drosophila ary for proper localisation of Ara6 to endosomal compart- and yeast, and three b subunits that cannot be clearly as- ments (Figure 1). Since only a subset of early endosomes signed to either AP-1 or AP-2 (38,39). The reason for this appears to be Ara6-positive, plants may have functionally dis- potential diversity of AP-1 and, possibly, AP-2 complexes re- tinct early endosome populations. mains to be determined as no functional data are available in Arabidopsis. Rab effectors are specific to the target membrane and often consist of protein complexes. For example, the exocyst com- Clathrin adaptors recruit cargo proteins via interaction with plex of eight proteins conserved between yeast and mam- their cytosolic tails, e.g. m2-adaptin of AP-2 recognises a tyro- mals mediates tethering of Sec4p/rab3 vesicles to the sine-based endocytic sorting motif (40). In plants, the only plasma membrane (48). In yeast, the exocyst targets vesicles evidence for cargo recruitment is the in vitro interaction of to a specific site of the bud plasma membrane via the Sec3p the TGN-localised vacuolar cargo receptor AtELP with the protein, although t-SNAREs appear more broadly distributed mammalian TGN-specific AP-1 clathrin-adaptor complex in the target membrane (49). The mammalian exocyst targets (41,42). vesicles to the baso-lateral surface of epithelial cells (50). A Ypt6p effector complex named VFT (for Vps 53) mediates tethering of putatively endosome-derived vesicles to the Interaction of Vesicles with the Target trans-Golgi network (51). A Ypt1p effector complex named Membrane Sec34/35 complex appears to tether vesicles to the cis-Golgi stack (52). Interestingly, several components of these three The fusion of transport vesicles with their target membrane complexes show similarities that suggest a common evol- is preceded by tethering and docking, both of which contrib- utionary origin (53). The Arabidopsis genome encodes ute to the target specificity of vesicle fusion. Initially, rab-GTP homologues of all eight exocyst, three VFT and five of eight on the vesicle membrane interacts with effectors that tether Sec34/35 complex proteins (Table 1). However, no functional the vesicle to the target membrane (43). This is followed by analyses have been performed. Vesicles trafficking to the the formation of trans-SNARE complexes that dock vesicles vacuole are tethered to the target membrane via the Ypt7p to target membranes, facilitating subsequent fusion. effector complex named HOPS, which is also conserved between yeast and mammals (54). Again, homologues of all Rab proteins are thought to determine the fusion com- six components of the HOPS complex are encoded in the petence of membranes and to specify target membranes by Arabidopsis genome (Table1). In this case, mutations in the acting as molecular scaffolds and regulators of membrane VACUOLELESS1 (VCL1) gene encoding the Vps16p homolo- composition. As many as 57 rabs have been identified in gue were shown to be embryonic lethal and to lead to de- Arabidopsis, nearly matching the number of rabs in mam- fects in vacuole biogenesis (55). mals and by far exceeding those of yeast, Drosophila or Ca- enorhabditis elegans (44). Rab functions are conserved Vesicle docking is brought about by the pairing of comple- across eukaryotes, such that their subcellular localisation can mentary SNAREs on opposite membranes (56). SNARE be inferred from known localisations of members of the same complexes consist of pathway-specific synaptobrevins (v- subfamily in other species. The large number of Arabidopsis SNAREs) and syntaxins (t-SNAREs), whereas more pro- rab proteins results from diversification within 12 subfamilies. miscuous t-SNARE light chains or SNAP25s contribute the The rab11 subfamily, for example, contains 26 members, remaining two coiled-coil domains of the four-helical bundle which may reflect differential tissue expression or functional in yeast and mammals. Again, the Arabidopsis genome en- redundancy of late-endosomal rabs. Alternatively, this diversi- codes a larger number of syntaxins (called SYP, syntaxins of fication might suggest a complex subdomain structure of re- plant) than does the human genome (57). Especially, there cycling endosomes in plants [for review, see (43)]. The rab11 are nine SYP1 proteins related to the plasma-membrane syn- homologue PRA2 has been reported to localise to the ER and taxins Sso1/2p and syntaxin1. They include KNOLLE, a cyto- to regulate brassinosteroid biosynthesis in response to light, kinesis-specific syntaxin only found in plants, and the plasma an unprecedented case of a rab function possibly unrelated membrane-located Syr-1 required for protein secretion (58– to membrane trafficking (45). However, the same protein 60) (Figure 1). It will be interesting to determine the tissue was recently localised to the Golgi and endosomes rather specificity and the subcellular location of the remaining than the ER (46) (Figure 1). Regardless of this discrepancy, members of this group. Members of four other SYP subfamil- both reports agree on differences in localisation or function ies form at least five complexes involved in TGN/prevacuolar between PRA2 and its closest homologue PRA3, supporting compartment (PVC) trafficking (61). Members of the SYP2 the notion of functional diversity among rab11 subfamily and SYP4 syntaxin family are closely related by sequence, members in plants. but SYP41 and SYP42 have been shown to localise to different regions of the TGN. In addition, functional analysis by Another example of a plant-specific rab variant is Ara6, a reverse genetics suggests unique requirements of each, as

Traffic 2002; 3: 605–613 609 Jürgens and Geldner evidenced by pollen lethality (62). As in yeast and animals, ferent subdomains of the plasma membrane. Other proteins the SNAP25-homologue SNAP33 appears to be more pro- localised to specific subdomains include PIN2, which ac- miscuous, interacting with KNOLLE at the cell plate and also cumulates at the apical end of root epidermal cells (74), and localising to the plasma membrane (63) (Figure 1). PIN3, which is predominantly observed at lateral plasma membranes of root cortical cells (75). Arabidopsis encodes 16 putative synaptobrevins, of which 11 form a subfamily related to the endosomal/lysosomal How these differential localisations are achieved by targeted VAMP7 (57). By contrast, there are no close homologues secretion in plant cells is not known. In mammalian polarised to the plasma membrane synaptobrevins Snc1/2p and epithelial cells, syntaxins are differentially localised to either VAMP1/2. On the other hand, the Arabidopsis genome en- apical or basolateral plasma membrane domains (76), and codes 3 ‘novel plant SNAREs’ (NPSNs) that lack close homo- this was shown to be important for correct sorting of marker logues in nonplant organisms. NPSN11 interacts with the proteins (77). It is important to note that epithelial cells have cytokinesis-specific syntaxin KNOLLE and also localises to mechanical diffusion barriers that prevent correctly targeted the cell plate during cytokinesis (64). It remains to be deter- proteins from diffusing laterally into the neighbouring plasma- mined whether NPSNs can substitute for conventional syn- membrane domain. Such barriers are not apparent in the de- aptobrevins. picted root meristem cell (Figure 1), suggesting that different mechanisms prevent the mixing of apical, lateral and basal Arabidopsis encodes six putative members of the Sec1 fam- markers. One possible mechanism would be a self-organis- ily which in nonplant organisms interact with SNAREs in dif- ing scaffolding machinery. Alternatively or in addition, segre- ferent ways. Whereas nSec1 stabilises the closed confor- gated proteins might be continually resorted by recycling, as mation of syntaxin1A (65), Sly1 interaction with the N-ter- will be discussed below. However, where does sorting hap- minus of the cis-Golgi SNARE Sed5 may contribute to the pen in the first place? specificity of SNARE complex formation (66,67). In contrast to yeast, the Arabidopsis VPS45 is located at the TGN and The TGN has been recognised as a major sorting site in the interacts with the TGN syntaxins of the SYP4 family (68) (Fig- secretory pathway where proteins destined for the lysosome/ ure 1). The only Arabidopsis Sec1 protein that has been func- vacuole are segregated away from exocytic cargo. This is also tionally characterised is KEULE which interacts with the cyto- the case in plants since the vacuolar sorting receptor AtELP kinesis-specific syntaxin KNOLLE and is required for vesicle has been localised to the TGN (41,42) (Figure 1). It has also fusion during cell-plate formation (69,70). been shown recently through live imaging that transport vesicles from the TGN directly fuse with the plasma membrane, In conclusion, the sequence analysis of the Arabidopsis ge- without passing through any intermediate compartment (78). nome suggests conservation of key regulators of vesicle traf- If the TGN acts as a last sorting station before outer space, ficking. However, in the case of protein families, there has differential secretion towards plasma membrane subdomains been differential expansion of subfamilies in plant evolution, would require budding of several distinct vesicle populations which points to possibly divergent roles of individual mem- from the TGN. However, there is evidence from both mam- bers. To clarify this issue, functional analysis needs to be malian and yeast cells for ‘indirect’ trafficking to the plasma done by reverse genetics in Arabidopsis. membrane, with vesicles first being delivered to the endosomal system (79–82). The endosome is an important sorting centre in the cell where cargo destined for degradation is Post-Golgi Trafficking Pathways in Plants: separated from proteins to be recycled back to the surface. Where Does Sorting Take Place? Thus, it is a rather straightforward idea that newly synthesised proteins travel from the TGN to the endosome Protein secretion to the cell surface delivers integral plasma where they join proteins from the endocytic pathway, and membrane proteins, such as cellulose synthases, the t- both sets of proteins are sorted together for their respective SNAREs SYR1 and SNAP33 or Hπ-ATPase, and extracellular destinations at this point. The direct pathway from the TGN proteins located in the cell wall, such as lipid transfer protein, to the plasma membrane could be taken by cargo such as endoxyloglucan transferase or endoglucanase, and secreted cell-wall material or secreted proteins that would not undergo signaling peptides, such as CLV3 or SCR. In all these cases, recycling through endosomes. In a growing plant cell, this the entire plasma membrane is the target of vesicle traffick- would probably still constitute the major part of vesicle flow ing. However, there are other proteins that accumulate in spe- to the plasma membrane. However, things are likely to be cific subdomains of the plasma membrane. This is illustrated more complicated, since sorting of some apical or basolateral by the apical localisation of the auxin uptake carrier AUX1 markers does clearly also take place in the TGN. This is even (71), the basal localisation of the putative auxin efflux carrier the case in nonpolarised cells, where different vesicle popula- PIN1 (28,72) and the lateral localisation of COBRA, a GPI- tions are not destined to distinct plasma membrane compart- anchored protein necessary for correct differential cell ments (78). Currently, data from mammals and yeast only elongation (73) (Figure 1). These three proteins can be found indicate the existence of two pathways from the TGN to the in the same cells of the Arabidopsis seedling root, sug- plasma membrane, but we are far from understanding why gesting that targeted secretion can distinguish between dif- one protein takes the direct route and another travels via the

610 Traffic 2002; 3: 605–613 Late Secretory Pathway in Plants endosome, and what contributions the two pathways make References to overall secretion. 1. Dupree P. The Golgi bypassed. Trends Cell Biol 1999;9:130. In plants, post-Golgi trafficking to the plasma membrane has 2. Sanderfoot AA, Raikhel NV. The specificity of vesicle trafficking: coat not been analysed and it is thus not clear whether there are proteins and SNAREs. Plant Cell 1999;11:629–641. multiple pathways. However, BFA treatment leads to rapid 3. Dixit R, Nasrallah JB. Recognizing self in the self-incompatibility re- internalisation of plasma membrane proteins, which is most sponse. Plant Physiol 2001;125:105–108. likely due to a block of re-secretion to the plasma membrane 4. Rojo E, Sharma VK, Kovaleva V, Raikhel NV, Fletcher JC. CLV3 is localized to the extracellular space, where it activates the Arabidopsis CLA- (29). BFA-induced internal accumulation appears to be plant VATA stem cell signaling pathway. Plant Cell 2002;14:669–677. specific, since in animals endocytosis and recycling of 5. Hepler PK, Vidali L, Cheung AY. Polarized cell growth in higher plants. plasma membrane markers are resistant to BFA (83,84). Annu Rev Cell Dev Biol 2001;17:159–187. Thus, BFA is a tool to study post-Golgi secretion in plants. 6. Higashiyama T, Yabe S, Sasaki N, Nishimura Y, Miyagishima S, Kuro- Several plasma membrane proteins accumulate intracellularly iwa H, Kuroiwa T. Pollen tube attraction by the synergid cell. Science in response to BFA treatment (28,29,63,85). It has been 2001;293:1480–1483. shown that PIN1 accumulates in so-called BFA compart- 7. Cosgrove DJ. Assembly and enlargement of the primary cell wall in ments that consist of aggregates of membrane vesicles and plants. Annu Rev Cell Dev Biol 1997;13:171–201. are distinct from adjacent Golgi stacks (29). The BFA com- 8. Hülskamp M. How plants split hairs. Curr Biol 2000;10:R308–R310. partments involved in PIN1 recycling most likely represent 9. Mathur J, Hülskamp M. Cell growth: how to grow and where to grow. Curr Biol 2001;11:R402–R404. endosomes. If this is the case, proper targeting of PIN1 and 10. Ma F, Peterson CA. Development of cell wall modifications in the en- possibly other plasma membrane proteins may be effected dodermis and exodermis of Allium cepa roots. Can J Bot by sorting in endosomes rather than at the TGN. By compari- 2001;79:621–634. son, newly synthesised PIN1 accounts only for a minor pro- 11. Staehelin LA, Moore I. The plant Golgi apparatus. structure, functional portion of transported PIN1 protein in the cell. Thus, regard- organization and trafficking mechanisms. Annu Rev Plant Physiol Plant less of whether this minor fraction is sorted at the TGN or Mol Biol 1995;46:261–288. passed on to the endosome for sorting, the determining step 12. Boevink P, Oparka K, Cruz SS, Martin B, Betteridge A, Hawes C. Stacks for the polar localisation of PIN1 should be sorting at the on tracks: the plant Golgi apparatus traffics on an actin/ER network. endosome. How sorting and targeting to the correct plasma Plant J 1998;15:441–447. membrane subdomain is brought about is entirely unknown. 13. Nebenführ A, Gallagher LA, Dunahay TG, Frohlick JA, Mazurkiewicz For example, none of the several putative plasma membrane AM, Meehl JB, Staehelin LA. Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol SNAREs of Arabidopsis has been shown to be asymmetri- 1999;121:1127–1142. cally localised in the plasma membrane, in contrast to ani- 14. Vitale A, Raikhel NV. What do proteins need to reach different vacu- mals (76). In the case of the COBRA protein, which is local- oles? Trends Plant Sci 1999;4:149–155. ised to the lateral subdomain of the plasma membrane, its 15. Lloyd C, Hussey P. Microtubule-associated proteins in plants – why GPI anchor may be instructive for targeting since, in animals, we need a MAP. Nat Rev Mol Cell Biol 2001;2:40–47. segregation into lipid rafts has been postulated to target GPI- 16. Steinborn K, Maulbetsch C, Priester B, Trautmann S, Pacher T, Geiges anchored proteins to the apical end of epithelial cells (86). B, Küttner F, Lepiniec L, Stierhof YD, Schwarz H, Jürgens G, Mayer U. The Arabidopsis PILZ group genes encode tubulin-folding cofactor orthologs required for cell division but not cell growth. Genes Dev 2002;16:959–971. Future Perspectives 17. Bassham DC, Raikhel NV. Unique features of the plant vacuolar sorting machinery. Curr Opin Cell Biol 2000;12:491–495. 18. Donaldson JG, Jackson CL. Regulators and effectors of the ARF Both the analysis of the Arabidopsis genome and the still GTPases. Curr Opin Cell Biol 2000;12:475–482. limited number of functional studies point to a similar com- 19. Moss J, Vaughan M. Structure and function of ARF proteins: acti- plexity of the secretory system in plants as in animals. More vators of cholera toxin and critical components of intracellular vesicu- functional studies are required to analyse mechanisms of lar transport processes. J Biol Chem 1995;279:12327–12330. plant protein secretion, and the necessary tools are now be- 20. Stearns T, Kahn RA, Botstein D, Hoyt MA. ADP ribosylation factor is coming available. Specifically, reverse-genetics analysis of an essential protein in Saccharomyces cerevisiae and is encoded by Arabidopsis genes has become commonplace with the avail- two genes. Mol Cell Biol 1990;10:6690–6699. ability of large collections of insertion lines, and facile trans- 21. Lee FJ, Stevens LA, Kao YL, Moss J, Vaughan M. Characterization of formation makes it easy to express specific variants of regula- a glucose-repressible ADP-ribosylation factor 3 (ARF3) from Sac- tory components of vesicle trafficking. In addition, the paucity charomyces cerevisiae. J Biol Chem 1994;269:20931–22093. of suitable subcellular markers for identifying membrane 22. Jackson CL, Casanova JE. Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors. Trends Cell Biol 2000;10:60–67. compartments will be overcome by the widespread use of 23. Someya A, Sata M, Takeda K, Pacheco-Rodriguez G, Ferrans VJ, Moss GFP fusions. We can thus expect a dramatic increase in J, Vaughan M. ARF-GEP (100), a guanine nucleotide-exchange pro- knowledge about protein secretion in plants, which will tein for ADP-ribosylation factor 6. Proc Natl Acad Sci USA eventually enable us to differentiate between a common eu- 2001;98:2413–2418. karyotic heritage and plant-specific ways of sorting out 24. Geiger C, Nagel W, Boehm T, van Kooyk Y, Figdor CG, Kremmer E, things. Hogg N, Zeitlmann L, Dierks H, Weber KS, Kolanus W. Cytohesin-1

Trafﬁc 2002; 3: 605–613 611 Jürgens and Geldner

regulates beta-2 integrin-mediated adhesion through both ARF-GEF nals are integrated via a dark-induced small G protein in etiolated function and interaction with LFA-1. EMBO J 2000;19:2525–2536. seedling growth. Cell 2001;105:625–636. 25. Mukherjee S, Gurevich VV, Jones JC, Casanova JE, Frank SR, Maizels 46. Inaba T, Nagano Y, Nagasaki T, Sasaki Y. Distinct localization of two ET, Bader MF, Kahn RA, Palczewski K, Aktories K, Hunzicker-Dunn M. closely related Ypt3/Rab11 proteins on the trafficking pathway in The ADP ribosylation factor nucleotide exchange factor ARNO pro- higher plants. J Biol Chem 2002;277:9183–9188. motes beta-arrestin release necessary for luteinizing hormone/chori- 47. Ueda T, Yamaguchi M, Uchimiya H, Nakano A. Ara6, a plant-unique ogonadotropin receptor desensitization. Proc Natl Acad Sci USA novel type Rab GTPase, functions in the endocytic pathway of Arabi- 2000;97:5901–5906. dopsis thaliana. EMBO J 2001;20:4730–4741. 26. Grebe M, Gadea J, Steinmann T, Kientz M, Rahfeld JU, Salchert K, 48. Guo W, Sacher M, Barrowman J, Ferro-Novick S, Novick P. Protein Koncz C, Jürgens G. A conserved domain of the Arabidopsis GNOM complexes in transport vesicle targeting. Trends Cell Biol protein mediates subunit interaction and cyclophilin 5 binding. Plant 2000;10:251–255. Cell 2000;12:343–356. 49. Finger FP, Hughes TE, Novick P. Sec3p is a spatial landmark for polar- 27. Mayer U, Büttner G, Jürgens G. Apical-basal pattern formation in the ized secretion in budding yeast. Cell 1998;92:559–571. Arabidopsis embryo: studies on the role of the gnom gene. Develop- 50. Grindstaff KK, Yeaman C, Anandasabapathy N, Hsu SC, Rodriguez- ment 1993;117:149–162. Boulan E, Scheller RH, Nelson WJ. Sec6/8 complex is recruited to 28. Steinmann T, Geldner N, Grebe M, Mangold S, Jackson CL, Paris S, cell-cell contacts and specifies transport vesicle delivery to the basal- Galweiler L, Palme K, Jürgens G. Coordinated polar localization of lateral membrane in epithelial cells. Cell 1998;93:731–740. auxin efflux carrier PIN1 by GNOM ARF GEF. Science 1999;286:316– 51. Conibear E, Stevens TH. Vps52p, Vps53p, and Vps54p form a novel 318. multisubunit complex required for protein sorting at the yeast late 29. Geldner N, Friml J, Stierhof YD, Jürgens G, Palme K. Auxin transport Golgi. Mol Biol Cell 2000;11:305–323. inhibitors block PIN1 cycling and vesicle trafficking. Nature 52. Whyte JR, Munro S. The Sec34/35 Golgi transport complex is related 2001;413:425–428. to the exocyst, defining a family of complexes involved in multiple 30. Rein U, Andag U, Duden R, Schmitt HD, Spang A. ARF-GAP-me- steps of membrane traffic. Dev Cell 2001;1:527–537. diated interaction between the ER–Golgi v-SNAREs and the COPI 53. Short B, Barr FA. Membrane traffic: exocyst III – makes a family. Curr coat. J Cell Biol 2002;157:395–404. Biol 2002;12:R18–R20. 31. Donaldson JG. Filling in the GAPs in the ADP-ribosylation factor story. 54. Sato TK, Rehling P, Peterson MR, Emr SD. Class C Vps protein com- Proc Natl Acad Sci USA 2000;97:3792–3794. plex regulates vacuolar SNARE pairing and is required for vesicle 32. Jensen RB, Lykke-Andersen K, Frandsen GI, Nielsen HB, Haseloff J, docking/fusion. Mol Cell 2000;6:661–671. Jespersen HM, Mundy J, Skriver K. Promiscuous and specific phos- 55. Rojo E, Gillmor CS, Kovaleva V, Somerville CR, Raikhel NV. VACU- pholipid binding by domains in ZAC, a membrane-associated Arabi- OLELESS1 is an essential gene required for vacuole formation and dopsis protein with an ARF GAP zinc finger and a C2 domain. Plant morphogenesis in Arabidopsis. Dev Cell 2001;1:303–310. Mol Biol 2000;44:799–814. 56. Chen YA, Scheller RH. SNARE-mediated membrane fusion. Nat Rev 33. Kirchhausen T. Three ways to make a vesicle. Nat Rev Mol Cell Biol Mol Cell Biol 2001;2:98–106. 2000;1:187–198. 57. Sanderfoot AA, Assaad FF, Raikhel NV. The Arabidopsis genome. An 34. Otegui MS, Mastronarde DN, Kang BH, Bednarek SY, Staehelin LA. abundance of soluble N-ethylmaleimide-sensitive factor adaptor pro- Three-dimensional analysis of syncytial-type cell plates during endo- tein receptors. Plant Physiol 2000;124:1558–1569. sperm cellularization visualized by high resolution electron tomo- 58. Lauber MH, Waizenegger I, Steinmann T, Schwarz H, Mayer U, graphy. Plant Cell 2001;13:2033–2051. Hwang I, Lukowitz W, Jürgens G. The Arabidopsis KNOLLE protein is 35. Lam BC, Sage TL, Bianchi F, Blumwald E. Role of SH3 domain–con- a cytokinesis-specific syntaxin. J Cell Biol 1997;139:1485–1493. taining proteins in clathrin–mediated vesicle trafficking in Arabidopsis. 59. Leyman B, Geelen D, Blatt MR. Localization and control of expression Plant Cell 2001;13: 2499–2512. of Nt-Syr1, a tobacco SNARE protein. Plant J 2000;24:369–381. 36. Staehelin LA, Hepler PK. Cytokinesis in higher plants. Cell 60. Geelen D, Leyman B, Batoko H, Di Sansabastiano GP, Moore I, Blatt 1996;84:821–824. MR. The abscisic acid-related SNARE homolog NtSyr1 contributes to 37. Scheele U, Holstein SE. Functional evidence for the identification of secretion and growth: evidence from competition with its cytosolic an Arabidopsis clathrin light chain polypeptide. FEBS Lett domain. Plant Cell 2002;14:387–406. 2002;514:355–360. 61. Sanderfoot AA, Kovaleva V, Bassham DC, Raikhel NV. Interactions be- 38. Boehm M, Bonifacino JS. Adaptins: the final recount. Mol Biol Cell tween syntaxins identify at least five SNARE complexes within the 2001;12:2907–2920. Golgi/prevacuolar system of the Arabidopsis cell. Mol Biol Cell 39. Holstein S. Clathrin and plant endocytosis. Traffic 2002; 3:614–620. 2001;12:3733–3743. 40. Kirchhausen T. Clathrin adaptors really adapt. Cell 2002;109:413–416. 62. Sanderfoot AA, Pilgrim M, Adam L, Raikhel NV. Disruption of individual 41. Sanderfoot AA, Ahmed SU, Marty-Mazars D, Rapoport I, Kirchhausen members of Arabidopsis syntaxin gene families indicates each has T, Marty F, Raikhel NV. A putative vacuolar cargo receptor partially essential functions. Plant Cell 2001;13:659–666. colocalizes with AtPEP12p on a prevacuolar compartment in Arabi- 63. Heese M, Gansel X, Sticher L, Wick P, Grebe M, Granier F, Jürgens G. dopsis roots. Proc Natl Acad Sci USA 1998;95:9920–9925. Functional characterization of the KNOLLE-interacting t-SNARE AtS- 42. Ahmed SU, Rojo E, Kovaleva V, Venkataraman S, Dombrowski JE, NAP33 and its role in plant cytokinesis. J Cell Biol 2001;155:239–250. Matsuoka K, Raikhel NV. The plant vacuolar sorting receptor AtELP is 64. Zheng H, Bednarek SY, Sanderfoot AA, Alonso J, Ecker JR, Raikhel involved in transport of NH (2) -terminal propeptide-containing vacuo- NV. NPSN11 is a cell plate-associated SNARE protein that interacts lar proteins in Arabidopsis thaliana. J Cell Biol 2000;149:1335–1344. with the syntaxin KNOLLE. Plant Physiol 2002;129:530–539. 43. Zerial M, McBride H. Rab proteins as membrane organizers. Nat Rev 65. Misura KM, Scheller RH, Weis WI. Three-dimensional structure of the Mol Cell Biol 2001;2:107–119. neuronal-Sec1-syntaxin 1a complex. Nature 2000;404:355–362. 44. Pereira-Leal JB, Seabra MC. Evolution of the Rab family of small GTP- 66. Yamaguchi T, Dulubova I, Min SW, Chen X, Rizo J, Südhof TC. Sly1 binding proteins. J Mol Biol 2001;313:889–901. binds to Golgi and ER syntaxins via a conserved N-terminal peptide 45. Kang JG, Yun J, Kim DH, Chung KS, Fujioka S, Kim JI, Dae HW, Yoshi- motif. Dev Cell 2002;2:295–305. da S, Takatsuto S, Song PS, Park CM. Light and brassinosteroid sig- 67. Peng R, Gallwitz D. Sly1 protein bound to Golgi syntaxin Sed5p allows

612 Trafﬁc 2002; 3: 605–613 Late Secretory Pathway in Plants

assembly and contributes to specificity of SNARE fusion complexes. 77. Low SH, Roche PA, Anderson HA, van Ijzendoorn SC, Zhang M, Mos- J Cell Biol 2002;156:645–655. tov KE, Weimbs T. Targeting of SNAP-23 and SNAP-25 in polarized 68. Bassham DC, Sanderfoot AA, Kovaleva V, Zheng H, Raikhel NV. epithelial cells. J Biol Chem 1998;273:3422–3430. AtVPS45 complex formation at the trans-Golgi network. Mol Biol Cell 78. Keller P, Toomre D, Diaz E, White J, Simons K. Multicolour imaging of 2000;11:2251–2265. post-Golgi sorting and trafficking in live cells. Nat Cell Biol 69. Waizenegger I, Lukowitz W, Assaad F, Schwarz H, Jürgens G, Mayer 2001;3:140–149. U. The Arabidopsis KNOLLE and KEULE genes interact to promote 79. Futter CE, Connolly CN, Cutler DF, Hopkins CR. Newly synthesized vesicle fusion during cytokinesis. Curr Biol 2000;10:1371–1374. transferrin receptors can be detected in the endosome before they 70. Assaad FF, Huet Y, Mayer U, Jürgens G. The cytokinesis gene KEULE appear on the cell surface. J Biol Chem 1995;270:10999–11003. encodes a Sec1 protein that binds the syntaxin KNOLLE. J Cell Biol 80. Leitinger B, Hille-Rehfeld A, Spiess M. Biosynthetic transport of the 2001;152:531–543. asialoglycoprotein receptor H1 to the cell surface occurs via endo- 71. Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Bennett somes. Proc Natl Acad Sci USA 1995;92:1010 9–10113. M. Localization of the auxin permease AUX1 suggests two function- 81. Laird V, Spiess M. A novel assay to demonstrate an intersection of ally distinct hormone transport pathways operate in the Arabidopsis the exocytic and endocytic pathways at early endosomes. Exp Cell root apex. Genes Dev 2001;15:2648–2653. Res 2000;260:340–345. 72. Gälweiler L, Guan C, Müller A, Wisman E, Mendgen K, Yephremov A, 82. Harsay E, Schekman R. A subset of yeast vacuolar protein sorting Palme K. Regulation of polar auxin transport by AtPIN1 in Arabidopsis mutants is blocked in one branch of the exocytic pathway. J Cell Biol vascular tissue. Science 1998;282:2226–2230. 2002;156:271–285. 73. Schindelman G, Morikami A, Jung J, Baskin TI, Carpita NC, Derbyshire 83. Lippincott-Schwartz J, Yuan L, Tipper C, Amherdt M, Orci L, Klausner P, McCann MC, Benfey PN. COBRA encodes a putative GPI-anchored RD. Brefeldin A’s effects on endosomes, lysosomes, and the TGN protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis. Genes Dev 2001;15:1115–1127. suggest a general mechanism for regulating organelle structure and 74. Müller A, Guan C, Gälweiler L, Tanzler P, Huijser P, Marchant A, Parry membrane traffic. Cell 1991;67:601–616. G, Bennett M, Wisman E, Palme K. AtPIN2 defines a locus of Arabi- 84. Hunziker W, Whitney JA, Mellman I. Selective inhibition of transcytosis dopsis for root gravitropism control. EMBO J 1998;17:6903–6911. by brefeldin A in MDCK cells. Cell 1991;67:617–627. 75. Friml J, Wisniewska J, Benkova E, Mendgen K, Palme K. Lateral relo- 85. Grebe M, Friml J, Swarup R, Ljung K, Sandberg G, Terlou M, Palme cation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis. K, Bennett MJ, Scheres B. Cell polarity signaling in Arabidopsis in- Nature 2002;415:806–809. volves a BFA-sensitive auxin influx pathway. Curr Biol 2002;12:329– 76. Low SH, Chapin SJ, Weimbs T, Komuves LG, Bennett MK, Mostov KE. 334. Differential localization of syntaxin isoforms in polarized Madin-Darby 86. Keller P, Simons K. Post-Golgi biosynthetic trafficking. J Cell Sci canine kidney cells. Mol Biol Cell 1996;7:2007–2018. 1997;110:3001–3009.

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