Coated Vesicles in Plant Cells Matthew J

Seminars in Cell & Developmental Biology 18 (2007) 471–478

Review Coated vesicles in plant cells Matthew J. Paul, Lorenzo Frigerio ∗ Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom Available online 10 July 2007

Abstract Coated vesicles represent vital transport intermediates in all eukaryotic cells. While the basic mechanisms of membrane exchange are conserved through the kingdoms, the unique topology of the plant endomembrane system is mirrored by several differences in the genesis, function and regulation of coated vesicles. Efforts to unravel the complex network of proteins underlying the behaviour of these vesicles have recently beneﬁted from the application in planta of several molecular tools used in mammalian systems, as well as from advances in imaging technology and the ongoing analysis of the Arabidopsis genome. In this review, we provide an overview of the roles of coated vesicles in plant cells and highlight salient new developments in the ﬁeld. © 2007 Elsevier Ltd. All rights reserved.

Keywords: Plant secretory pathway; Protein trafﬁcking; Endocytosis; Endoplasmic reticulum; Golgi; Vacuole; Coated vesicles; Clathrin; COPI; COPII; Retromer

Contents

1. Vesicular trafﬁcking in plant cells ...... 471 2. Small GTP-binding proteins and associated factors ...... 473 3. Plant vesicle coat families ...... 473 3.1. Clathrin ...... 473 3.2. COPI and COPII ...... 474 3.3. Retromer...... 475 4. Conclusions and perspectives ...... 475 Acknowledgements ...... 476 References ...... 476

1. Vesicular trafficking in plant cells The endoplasmic reticulum, a dynamic mesh-like structure traversing the cortical cytoplasm of plant cells, represents the The plant endomembrane system encompasses a series of entry compartment for nascent polypeptides destined for secre- compartments which provide specialised surfaces and segre- tion [1–3]. From the ER, cargo molecules following the secretory gated areas for the production and storage of biomolecules. An pathway reach a stack of the fragmented, mobile Golgi appara- overview of the plant secretory pathway and the coated vesicles tus [4] without passing through an intermediate compartment described in this review is shown in Fig. 1. such as the mammalian vesiculo-tubular compartment (VTC [5]). Compelling evidence now exists to support an extremely close functional and physical relationship between plant ER and Abbreviations: CCV, clathrin-coated vesicles; CHC, clathrin heavy chain; Golgi compartments ([6,7], reviewed in Ref. [8]). Nevertheless, CLC, clathrin light chain; ER, endoplasmic reticulum; ERES, endoplasmic retic- the formation of coatomer (COPI and COPII) transport inter- ulum export sites; GAP, GTPase activating protein; GEF, guanosine nucleotide mediates remains essential for the transfer of cargo and for the exchange factor; LV, lytic vacuole; MVB, multivesicular body; PVC, prevacuo- maintenance of membrane flux [6,9]. lar compartment; PSV, protein storage vacuole; VSR, vacuolar sorting receptor; Anterograde transport routes from the Golgi stacks and ret- VTC, vesiculo-tubular compartment ∗ Corresponding author. Fax: +44 24 765 23701. rograde (endocytic) routes from the cell surface converge at a E-mail address: [email protected] (L. Frigerio). poorly defined group of compartments, interchangeably termed

Fig. 1. A model for the distribution of coated vesicles and receptor proteins throughout the plant endomembrane system. Coloured circles represent soluble cargo molecules within the secretory/endocytic pathway and black arrows denote presumptive membrane fusion events. Cargoes entering the pathway at the level of the ER proceed towards the Golgi apparatus via COPII-coated vesicles, possibly following interaction with receptor proteins of the p24 family [84–86] or after passive diffusion into an ERES [8]. It is also possible that anterograde transport by cisternal maturation may occur for certain cargoes. ER resident proteins (orange circles) are recycled from downstream compartments by the plant ERD2 receptor homologue, while a distinct subpopulation of COPI coats (COPIb; [12]) mediates intra-Golgi retention and recycling events. Cargoes progress from the Golgi towards vacuoles either in uncoated, ‘dense’ vesicles [20,21] or clathrin-coated vesicles [12,22] depending on the nature of the cargo and/or the presence of a cognate cargo receptor. Clathrin-coated vesicles are also involved in endocytosis, e.g. that of the auxin transporter PIN2 [11], and these coats may be distinguished by the presence of distinct sets of adaptor proteins [55,59]. Finally, retromer coats have been implicated in the retrieval of cargo receptors from the PVC [23,26]. Abbreviations: ER, endoplasmic reticulum, ERES, ER export site, CCV, clathrin-coated vesicle, DV, dense vesicle, PVC, prevacuolar compartment, MVB, multivesicular body, VSR, vacuolar sorting receptor.

prevacuolar compartments (PVC) or endosomes [10]. Very tion of a full range of key plant orthologues for proteins with recently, clathrin-coated vesicles (CCV) have been convincingly established roles in clathrin-mediated transport in mammals shown to be implicated in endocytosis in the plant secretory and yeasts. pathway [11]. This is consistent with their observed distribution Plant cells also contain functional orthologues of the retromer within the cell [12–14]. In mammalian and yeast cells, CCV are coat complex which was first identified and characterised in also involved in post-Golgi trafficking routes to a terminal intra- yeast [23,24]. In yeast cells, retromer is required for the recy- cellular compartment [15,16]. In plant cells, the precise role of cling of cargo receptors from the pre-vacuolar compartment to clathrin in anterograde transport is still a matter of some debate the trans-Golgi [25]. Consistent with an equivalent role in plant as there exist at least two separate routes to functionally distinct cells, a component of the plant retromer complex is required for vacuolar compartments [17–19]. In addition, some plants can the correct sorting of vacuolar cargo in developing Arabidopsis handle post-Golgi sorting of storage proteins through electron- seed [26]. opaque, ‘dense’ vesicles (DV) with no detectable protein coat The similarity between vesiculation events in plants and those [20,21]. However, a role for clathrin in vacuolar transport in of mammalian and yeast cells has provided invaluable clues plant cells is illustrated by the recent in situ detection of clathrin into the function of the plant endomembrane system. However, coats in restricted regions around the trans-Golgi cisternae of the unique topology, compartments and cargoes present in the the Golgi apparatus [12,22]. The concept of multiple roles plant secretory pathway necessitate the close examination of for clathrin coats in plant cells is supported by the identifica- each aspect of vesicular transport in these cells. Here, we review M.J. Paul, L. Frigerio / Seminars in Cell & Developmental Biology 18 (2007) 471–478 473 the latest findings concerning the roles of various vesicle coats 3. Plant vesicle coat families in the selective recruitment of cargo at each step of this pathway and highlight plant-specific adaptations to the vesiculation 3.1. Clathrin machinery. The clathrin coat was the first proteinaceous vesicle coat to be 2. Small GTP-binding proteins and associated observed [38] and structurally characterised [39]. It remains the factors most fully understood coat complex in both plants and animals today. Three structural clathrin heavy chain molecules (CHC, In common with mammals and yeasts, plant vesiculation Mr ∼180 kDa) come together through the interactions between events are governed by members of the Ras superfamily of domains near the C-termini of the heavy chains to form a triske- small GTP binding proteins [27]. The Arf subfamily of small lion, the basic structural unit of the clathrin coat. Purified CHC GTPases, which includes the Sar, ARF and ARF-like ARL triskelia demonstrate self-assembly at a physiological pH [40] to GTPases, are involved in the formation and budding of vesi- form cages which have been the focus of detailed structural stud- cles throughout the plant endomembrane system [28,29]. ARF ies [41,42]. CHC is encoded by two genes in both mammals and GTPases are associated with clathrin coats as well as COPI Arabidopsis and displays a remarkable degree of conservation coated vesicles, whereas COPII coat nucleation is triggered by between the two organisms (∼75% similarity, [13]). Each heavy a GTPase of the Sar subfamily. Uniquely, the retromer coat chain is accompanied by a regulatory clathrin light chain (CLC, does not appear to contain a GTPase in any organism, includ- Mr ∼30 kDa) which is less recognisable against its mammalian ing plants [23,24]. Twelve AtARF (Arabidopsis thaliana ARF) counterpart. Out of a series of potential CLC genes in plants, sequences have been identified through a BLAST search of the Arabidopsis CLC1 has now been experimentally validated by the Arabidopsis genome against consensus mammalian and yeast demonstration of its ability to bind human CHC via a conserved ARF sequences [30]. The number of ARF homologues in Ara- central coiled-coil domain [43]. Two further proteins with simi- bidopsis exceeds that in the genomes of humans or fission larity to mammalian CLC were identified in this study (AtCLC2 yeast (6 and 3, respectively [31]), a diversity which may reflect and AtCLC3), and all three contained the N-terminal acidic both functional redundancy and the complexity of the plant motif implicated in preventing the spurious assembly of clathrin endomembrane system. In addition to this diversity, AtARF heavy chain triskelia into lattices [43]. Intriguingly, this discov- homologues may play several distinct roles within the plant cell. ery raises the possibility of an unparalleled degree of functional AtARF1 has been shown to play a role in a vacuolar sorting path- specialisation amongst Arabidopsis CLC sequences. This is sup- way in plants [29] while chimeric AtARF1-fluorescent protein ported by gene expression data indicating that the transcription fusions have been localised to unknown compartments derived of AtCLC1 may be relatively reduced in pollen granules, while from the Golgi apparatus [32] as well as putative endocytic that of AtCLC2 is raised in seeds (AtGenExpress developmental structures [33]. dataset [44]). In common with all Ras superfamily GTP-binding proteins, Recently, inhibition of clathrin triskelion formation by over- AtARFs adopt an active effector-binding conformation when expression of the ‘hub’ domain of clathrin heavy chain [40,45] in associated with GTP and, upon hydrolysis, an inactive confor- Arabidopsis protoplasts was used to prove that clathrin-mediated mation when bound to GDP. ARF GTPase activating proteins endocytosis is the principal endocytic mechanism in these cells (ARF GAPs) were initially characterised by their ability to [11]. It is not yet clear whether the same strategy can be success- stimulate the intrinsic GTPase activity of Arf, while guano- fully applied to study the role of clathrin in anterograde transport sine nucleotide exchange factors (GEFs) catalyse the reverse routes. reaction. However, it has recently become apparent that these Clathrin-mediated vesiculation occurs as a response to an proteins also frequently possess other biological activities medi- accumulation of peripheral and intrinsic membrane factors on ated through various protein–protein and protein–lipid domains the donor membrane. Clathrin adaptors are peripheral compo- [34]. The Arabidopsis genome contains a large number of ARF- nents of this network with the ability to interact with clathrin at GAPs and GEFs [30], raising the possibility that these regulators the N-terminal domain of CHC as well as with other components act in response to diverse cellular signals. Arabidopsis AGD7, of the network through well-characterised motifs and modules. a member of the ArfGAP1 family, has recently been shown to Clathrin adaptors range from 300 to 3000 amino acids in size activate AtARF1 in a phosphatidic acid-dependent manner [35]. and, in general, there exists no similarity between them. Recruit- Furthermore, the authors also show that the overexpression of ment of clathrin by these peripheral membrane components AGD7 led to the disruption of protein transport between the ER permits the system to avoid the induction of vesicles by recy- and the Golgi. Another report indicates that the uptake of auxin cling integral membrane proteins such as cargo receptors when at the plasma membrane may be impaired by the overexpres- away from their main point of function. Clathrin adaptors are sion of OsAGAP (Oryza sativa AGAP) in rice [36]. In view recruited from the cytoplasm by an activated small GTP-binding of these results, the overexpression of the ARF GAPs appears protein from the ARF family [46] often in conjunction with a to provide an informative tool to identify the multiple func- short-lived phospholipid species [47,48]. The prototype clathrin tions of individual ARF GTPases. Equally, different GEFs may adaptors are the heterotetrameric assembly proteins (APs). Four also mediate alternative functions for GTPases within the plant AP complexes each consisting of two large subunits (␤(1–4) cell [37]. and either of ␣, ␥, ␦, ␧ dependent on the complex), one medium 474 M.J. Paul, L. Frigerio / Seminars in Cell & Developmental Biology 18 (2007) 471–478 subunit (␮1–4) and one small subunit (␴1–4) have been identi- domain-bearing AtAP180, [65] and the ENTH-based adaptors fied to date in mammalian cells. Structural orthologues for each AtEPSINR1 and AtEPSIN2R [66,67]. AtAP180 was initially component of the four AP complexes have been identified in the identified as a binding partner for the large subunit of the plant Arabidopsis genome [49]. Several of these components have AP-2 complex, At-␣C-Ad, which is implicated in receptor- been functionally characterised in plant cells: a ␥-adaptin, three mediated endocytosis. In rats, AP180 is a neuronal adaptor of ␤-adaptins [50], and a ␮-adaptin [51] all from Arabidopsis, along ∼90 kDa with the ability to induce the formation of coated vesi- with two Arabidopsis ␴-adaptins and a ␴1 from a Chinese med- cles with a remarkably narrow size distribution [68]. AtAP180 ical tree (Camptotheca acuminata) [52,53] and a ␴2 from maize was found to retain this assembly activity based on a critical [54]. Each AP complex is associated with specific membranes DLL clathrin-binding motif. Despite being unable to promote through interactions with specific membrane phosphoinositides the assembly of clathrin, mutant AtAP180 lacking the classical (PtdInsP) mediated by one of the large subunits (␣, ␥, ␦ or ␧). DLL clathrin-box could still bind the heavy chain. It was con- In mammals and yeasts, AP1 is associated with intracellular cluded that additional, undescribed clathrin binding motifs must membranes and primarily the Golgi apparatus, AP2 with the be present within the adaptor and that the assembly activity was plasma membrane, AP3 with lysosomes while AP4 is present likely to be mediated by a structural array of such motifs [65]. on endosomes [55] (reviewed in Ref. [56]). Plant cells share the A similar arrangement of clathrin-binding motif has been pro- enzymes necessary for the synthesis of the PtdInsP2 series of posed for AtEPSINR1, which has also been shown to bind a large phosphoinositide isomers [57]. The recruitment of clathrin adap- AtAP-2 subunit [67]. However, a possible role in endocytosis tors from the cytosol may also occur in response to the build-up for this adaptor is contrasted with strong evidence implicating of cargo molecules themselves. In this case, a transmembrane AtEPSINR1 in the vacuolar trafficking of soluble cargo pro- receptor is required to transduce the sorting signal of the lumi- teins at the TGN: AtEPSINR1 has been shown to interact with nal cargo protein or extracellular ligand to the cytoplasmic side the vacuolar sorting receptor AtVSR1 and the TGN v-SNARE of the membrane. Several classes of peptide motifs within the VTI11 with which it was observed to colocalise. Moreover, an cytosolic tails of these receptor proteins are recognised by ␮AP AtEPSINR1 T-DNA insertional mutant line missorted 100% of subunits, whereas other classes interact via additional adaptor the lytic vacuolar cargo sporamin:GFP to the apoplast. Con- proteins (reviewed in Ref. [58]). Two families of cargo receptors sistent with a role at distinct membranes, AtEPSINR1 binds a with anterograde trafficking roles and several receptors involved putative AP-1 ␥-subunit with greater affinity than the ␣-subunit in events analogous to mammalian receptor-mediated endocy- of AP-2 [67]. tosis have been identified in plants. Of these, the tyrosine-based Recently published data on a second Arabidopsis epsin homo- sorting signals present in the VSR family of cargo receptors logue, AtEPSINR2, indicate that this clathrin adaptor interacts [51] and a leucine-rich receptor kinase [59] have been shown to with a plant AP subunit with the strongest degree of similarity to recruit ␮-adaptin, and thus potentially traffic via clathrin-coated the ␦-subunit of AP-3, thereby providing a link between clathrin vesicles. and AP3 in plant cells [66] that has not been observed in studies Several families of monomeric clathrin adaptors bear- of yeast or mammalian AP3 [69,70]. AtEPSINR2 was found to ing characteristic conserved N-terminal domains have been localise with the TGN and, through interactions mediated by described in animal and yeast cells. Three Arabidopsis proteins the adaptor ENTH domain, with a potentially novel post-Golgi contain a consensus phosphoinositide-binding epsin NH3- compartment enriched in Ptd(3)InsP and the ␦-adaptin related terminal homology (ENTH) domain and seven contain the protein. Intriguingly, the functional identity of this compartment related AP180 NH3-terminal homology (ANTH) domain. In may be compromised by the phosphoinositide-3-kinase inhibitor non-plant systems, each monomeric adaptor is associated with wortmannin, a fungal metabolite which has been previously specific, diverse cargo including the glutamate receptor (HIP1) shown to interfere with vacuolar trafficking [71,72]. Recent [60], ubiquitinated receptors (epsins) [61] and v-SNAREs reports have also indicated that Ptd(3)InsP is transiently enriched (synaptobrevin [62], Vti1b and Vps27/Hrs [63]). Recently, a in certain vesicular membranes during cytokinesis [73]. It seems detailed sequence analysis of the Arabidopsis E/ANTH domain likely that new roles for clathrin coats within the plant endomem- proteins was performed in order to establish potential roles for brane system will be forthcoming from further functional studies these adaptors in plants [64]. Outside of the characteristic N- of the adaptor protein complement. terminal domain, clathrin binding motifs were identified in each homologue. Importantly however, no Arabidopsis E/ANTH 3.2. COPI and COPII domain protein contains the established actin or ubiquitin binding motifs which are intrinsic to the reported functions of many Vesicles associated with polymeric coatomer (COP) coat non-plant adaptors of this type. Furthermore, structural changes complexes are associated with trafficking events at the ER and in the ␣-helices of the plant E/ANTH domains indicate an altered Golgi apparatus. A strong body of evidence supports a role phosphoinositide specificity when compared with the domain for COPII coats in the movement of cargo from the ER to the archetypes [64]. These observations suggest that these plant Golgi apparatus [74]. Plant orthologues for several components adaptors may drive substantially different vesiculation events of the COPII coat have been isolated (Sar1 and Sec12 [75]; compared with their animal and yeast counterparts. Sec24 [76]; Sec13 [77]). A further component, Sec16, has been Functional data have very recently been published for implicated in the formation and function of mobile ER export three monomeric clathrin adaptors in Arabidopsis, the ANTH- sites (ERES) in yeasts and mammalian cells [78,79]. Although M.J. Paul, L. Frigerio / Seminars in Cell & Developmental Biology 18 (2007) 471–478 475 a plant Sec16 orthologue has not yet been identified, similar and Vps5p). Vps35p interacts with the cargo receptor Vps10p ERES structures are apparent in the plant ER [6,80]. Recent evi- and the complex is anchored to the membrane via Vps26p dence suggests that plant COPII vesicles are formed at ERES de and to the small subunit via Vps29p [97]. The two small sub- novo in response to the accumulation of cargo signals present unit components of the yeast retromer complex can dimerise in membrane proteins [80,81]. This model would appear to dis- via BAR protein-protein interaction domains [98] and interact tinguish the formation of plant COP-coated vesicles from the with Ptd(3)InsP-enriched membranes via Phox homology (PX) constitutive formation of CCV at the plasma membrane [11].It domains [99]. The Arabidopsis genome contains orthologues for has been established that COPII vesicles are also involved in the each of the large retromer subunits (3 for VPS35p, 2 for VPS26p trafficking of soluble proteins from the ER [6,9], although these and 1 for VPS29p) and three for the small subunit Vps5p [23]. cargoes alone do not induce the formation of ERES in plants. A The plant vacuolar sorting receptor VSR1 [100], which plays passive ‘bulk-flow’ model has therefore been proposed for the an analogous role to yeast Vps10p, has been immunoprecipi- trafficking of these soluble cargoes [9,82]. tated with antisera cognate for AtVPS35. Fluorescent reporter A family of type I transmembrane protein factors, the p24 pro- constructs based on these two proteins have been shown to teins, was identified as potential cargo receptors for ER export co-localise under the microscope [23]. These observations sug- and as structural components of coatomer coated vesicles in gest that plant retromer coats, like their yeast and mammalian mammals, yeast and now plants [83–86]. A recent investigation counterparts, are involved in the retrieval of cargo receptors into plant p24 proteins has revealed a synergy between two dis- from downstream compartments. The interaction of VSR1 with tinct sorting motifs in the cytosolic tail of these receptors which both the retromer at the PVC and the clathrin-recruiting AP- mediate binding in vitro to both Sec23 and subunits of the COPI 2␮ subunit at the TGN also suggests that specific vesiculation coat complex [85,86]. The precise role of the COPI complex events occur in response to membrane-intrinsic factors and not remains unclear; although it has been associated with vesicular cargo receptors themselves. Recently, an allele of AtVPS29 transport within the Golgi apparatus and from the Golgi appa- (maigo1-1) was identified in a screen for Arabidopsis mutants ratus to the ER [87]. Plants contain a full complement of COPI that accumulate abnormal levels of the precursors of the major components (ARF1, ␦-, ␧-COP [88], ␣-, ␤-, ␤-, ␥-COP [89], seed storage proteins [26]. This striking phenotype is likely due Sec21p [76]). The binding of plant p24 proteins to both COPII to AtVPS29 being the only retromer component without further and COPI coated vesicles provides a possible means for receptor potential homologues in Arabidopsis. This result is important as recycling in planta and is consistent with the situation in mam- it indicates that correct, retromer-mediated recycling of VSR1 malian cells [90]. This model of bidirectional cargo transport between the Golgi and the PVC is necessary to guarantee the between the ER and the Golgi via coated vesicles has been sup- proper sorting of all vacuole-bound cargo. It is not yet clear, ported by recently published EM observations of COPI-coated however, whether VSR1 participates directly in this sorting event vesicles associated with the Golgi apparatus of Scherffelia dubia [17]. and Arabidopsis [12]. The authors identify a subpopulation of The presence of additional VPS35 and VPS26 genes in plants retrograde COPI vesicles (COPIa) which appear to be concen- is unusual and, assuming they are functional, may point to mul- trated in the space between the ER and the Golgi along with tiple cargo specificities for large retromer subunits in plants. In COPII-coated vesicles. contrast, membrane-selective small retromer subunits are drawn In yeast cells, the total ablation of the p24 family leads to from a family of phosphoinositide-binding proteins with roles in the accumulation of the secreted enzymes invertase and Gas1p membrane trafficking, the sorting nexins (SNX). In mammalian [91,92], as well as interfering with the activity of the H/KDEL cells, SNX1, 2, 5 and 6 have been described as components of receptor ERD2 which functions to retrieve ER-resident proteins retromer coats, while no Vps17p orthologue exists [101,102]. from downstream compartments [93]. COPI-coated vesicles are The three Vps5p/SNX orthologues identified to date in plant also likely to be involved in the recycling of the H/KDEL cells may therefore interact with each other or with further cur- receptor in plants. Stripped membranes from tobacco plants rently unidentified factors to form a complete small subunit with overexpressing an ER-retained reporter can be induced to form a unique membrane specificity. Given that no small Arf fam- COPI vesicles containing detectable levels of the reporter and ily GTPase has been implicated in retromer coat formation in the soluble ER chaperone calreticulin [88]. However, no report mammalian or yeast systems, these interactions may be of piv- highlighting the requirement for p24 proteins in the trafficking otal importance to the function of these coat complexes in plant of the plant ERD2 receptor or of specific plant cargoes has yet cells. been published. 4. Conclusions and perspectives 3.3. Retromer In this brief review we have attempted to describe the cur- In yeast cells, the vacuolar sorting receptor Vps10p is recy- rently known features of plant coated vesicles. 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