Protoplasma (2000) 210:123-132

PROTOPLASMA 9 Springer-Verlag2000 Printed in Austria

Membrane transport in the endocytic pathway: animal versus plant cells M. J. Marcote 1, E Gu 2, J. Gruenberg 2, and E Aniento 3'* Instituto de Biologfa Molecular y Celular de Plantas, Universidad Politdcnica de Valencia, Consejo Superior de Investigaciones Cientificas, Valencia, 2Department of Biochemistry, Sciences II, University of Geneva, Geneva, 3Department de Bioquimica i Biologfa Molecular, Facultat de Farmacia, Universitat de Valencia, Valencia Received January 27, 1999 Accepted March 23, 1999

Summary. The endocytic pathway is a well established process in animal cells,but it is not well understood in plant cells.At the morphological level, all the compartments involved in endocytosis in animal cells seem to have counterparts in plant cells, and the organization of the pathway appears to share some striking similarities. Several Rab homologues have been found in plant cells, including homologues of Rab5, Rab7, and Rabll, markers of endocytic compartments in animal cells. Coat proteins are also present in plant cells, including clathrin, adaptins, and ADP ribosylation factor proteins. However, endocytic compartments in plant cells also exhibit specific features both in organization and function. The molecular composition of these compartments remains to be established, and future work will be necessary to identify the key regulators of endocytic trafficking in plant cells.

Keyworfls: Endocytosis; Membrane transport; Vesicle formation; Coat proteins; Rab proteins.

Abbreviations: EE early endosome; LE late endosome; ECV-MVB, endosomal carrier vesicle-multivesicularbody; PCR partially coated reticulum; MPR mannose 6-phosphate receptor; TGN trans-Golgi network.

Introduction Endocytosis is a well established process in animal cells. The organelles involved have been extensively characterized not only at the morphological level but also in terms of molecular composition (see below and Fig. 1 A). In contrast, much less is k n o w n about this process in plant cells. Unlike animal cells, plant cells develop a hydrostatic pressure termed turgor, which has constituted one of the main arguments against the existence of endocytosis in plant cells. However, the

evidence supporting the existence of the process in plant cells is already compelling (for reviews, see Low and Chandra 1994; D. Robinson 1996; D. Robinson et al. 1992, 1998b). A t the morphological level, plant cells appear to contain organelles presumably involved in endocytosis (see below), but their functional role remains unclear. Endocytosis in plants could be a means to balance exocytosis and to regulate m e m brane recycling and repair. By analogy with animal cells, plant cells could also undergo receptor-mediated endocytosis. Receptor-mediated endocytosis could be used for the uptake of certain nutrients too polar to traverse the plasma m e m b r a n e , for the downregulation of extracellular signals (such as those induced by plant growth factors or elicitors) or to r e m o v e toxins. Indeed, some elicitors and toxins have been shown to bind with high affinity to the plasma m e m b r a n e (Low and Chandra 1994). Certainly, further characterization of the pathway is required in order to understand the functional role of the process. In this review, we will focus on the general organization of the pathway, both in animal and in plant cells, and on the molecules that are involved in endosomal m e m b r a n e transport, m a n y of which have been extensively studied in animal cells and are just starting to be found in plant cells.

Organization of the endocytic pathway A n i m a l cells

* Correspondence and reprints: Departamcnt dc Bioqufmica i Biologfa Molecular, Facultat de Farmacia, Universitat de Valencia, Avenida Vicent Andr4s Estell6s s/n, E-46t00 Burjassot, Spain.

Figure 1 A outlines the general organization of the endocytic pathway in animal cells. Cell surface pro-

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A ANIMAL CELLS

vesicular portions with a diameter of ca. 0.3-0.4 gm which contain internal membranes, much like multivesicular bodies (see below). They constitute a major sorting station in the endocytic pathway and therefore have been also called sorting endosomes (Gruenberg and Maxfield 1995). EEs are mildly acidic, with a lumenal p H of ca. 6.2, maintained by an ATPdriven proton pump (the vacuolar ATPase), which allows ligand-receptor dissociation. After uncoupling from their ligands, most of the receptors, as well as other m e m b r a n e proteins and approximately 70-80% of the early-endosomal content follow the recycling pathway. In contrast, a specific subset of proteins, in particular down-regulated receptors, are transported to late endosomes (LEs) and lysosomes to be degraded. A common view is that recycling back to the plasma m e m b r a n e may occur directly from the sorting endosome (fast cycle) or indirectly via the so-called recycling endosome (Fig. 1A) (Hopkins et al. 1994). The recycling endosome consists of narrow tubules with a diameter of approximately 50 nm which often appear in the perinuclear region of the cell. In some cell types, its lumenal p H is ca. 0.3-0.4 units more alkaline than that of the sorting endosome. There appear to be also differences in the protein composition of EEs and recycling endosomes. Recycling endosomes are typically enriched in transferrin receptor (Yamashiro et al. 1984), cellubrevin (McMahon et al. 1993), and Rab11 (Ullrich et al. 1996). EEs contain the small GTPases Rab5 and Rab4 (Chavrier etal. 1990, Novick and Zerial 1997), a n n e x i n I I (Emans et al. 1993), and E E A 1 (early endosomal antigen 1, see below), but it is not clear to what extent some of these proteins may also be present in recycling endosomes. Endosomes also communicate with the biosynthetic pathway by vesicular transport. Newly synthesized lysosomal enzymes bound to the mannose-6phosphate receptors (MPR) are delivered from the trans-Golgi network (TGN) to endosomes, presumably EEs, and then routed towards lysosomes, whereas the receptors are recycled from LEs back to the TGN. Very recently, evidence has been presented of a trafficking pathway from EEs and recycling endosomes back to the Golgi apparatus, distinct from the recycling pathway followed by the MPR, and sorting probably involves adaptor protein type 1 (AP-1) clathrin coats (Mallard et al. 1998). Finally, observations in yeast and mammalian cells support the notion that the AP3 adaptor complex plays a role in transport between

RE ~


(Rab11)

EE

~

(RabS,Rab4 Anll,EEA1)

G

LE (Rab7,Rab9)

Lys Q

B PLANT CELLS

fl

~

M

l

/

MVB

Fig. 1A, B. Membrane transport in the endocytic pathway. A Animal cells. During transport to lysosomes,endocytosed materials appear sequentially in early endosomes (EE), then in late endosomes (LE), and eventually in lysosomes (Lys). Endosomal carrier vesicles-multivesicular bodies (ECV/MVB) function as transport intermediates between early and late endosomes. These vesicles form from early endosomes,in a process dependent on COPI proteins and on the luminal pH, and are transported along microtubules towards the perinuclear region, where they fuse with late endosomes. Recyclingback to the cell surface may occur either directly (fast cycle) or indirectly via the recycling endosomes (RE). Molecules that have been localized to different endocytic compartments are also shown.B Plant cells.Markers endocytosedin plant cellsfirst appear in the partially coated reticulum (PCR) and in the Golgi region. Markers that followthe degradative pathway then appear in multivesicular bodies (MVB) and in the central vacuole (V). Recycling occurs presumably via the Golgi apparatus (G). Transport of proteins from the endoplasmic reticulum or Golgi apparatus to the vacuole may occur via MVB to either protein storage vacuoles or lytic vacuoles and finally into the central vacuole (see text, for details). Arrows indicate routes of membrane transport. PM Plasma membrane

teins (including receptors) and lipids, as well as solutes are internalized via clathrin-coated pits and vesicles into early endosomes (EE), at the cell periphery. EEs are formed of networks of tubules, cisternae, and

M. J. Marcote et al.: M e m b r a n e transport in the endocytic pathway

TGN and endosomes and lysosomes (Cowles et al. 1997, Simpson et al. 1997). Transport between EEs and LEs could imply either a maturation-type process or a vesicular-transport step (Gruenberg an Maxfield 1995). After leaving EEs, molecules destined for degradation are found within spherical multivesicular vesicles with a diameter of ca. 0.4 gm, before they appear in LEs and lysosomes. We have proposed that these vesicles are transport intermediates between EEs and LEs (Gruenberg et al. 1989;Aniento et al. 1993,1996), and therefore we have called them endosomal carrier vesicles-multivesicular bodies (ECV-MVBs) to emphasize both their function as transport intermediates and their multivesicular appearance (Gruenberg et al. 1989). Indeed, EEs and LEs exhibit a high tendency to undergo homotypic fusion in vitro, but, in contrast, there is no direct fusion between EEs and LEs (Gorvel et al. 1991, Aniento et al. 1993). Transport between EEs and LEs may occur via the formation of ECV-MVBs from EEs at the cell periphery. ECV-MVB formation requires some, but not all, components of the COPI coat complex (Aniento et al. 1996, Whitney et al. 1996, Gu et al. 1997, Daro et al. 1997). Once formed, ECV-MVBs move towards LEs in the perinuclear region in a process dependent on microtubules and on the minusend-directed motor cytoplasmic dynein in nonpolarized cells (Aniento et al. 1993). Then ECV-MVBs dock onto and fuse with LEs (Aniento et al. 1993, 1996), and this step requires NSF (N-ethylmaleimide-sensitive factor) and o~-SNAP (soluble NSF attachment protein) (L. Robinson et al. 1997) as well as perhaps the small GTPase Rab7 (Feng et al. 1995). LEs (Fig. 1 A), located in the perinuclear region, contain a complex system of internal membranes in their lumen and the small GTPases Rab7 and Rab9. Both LEs and lysosomes are highly enriched in distinctive, highly glycosylated and conserved lysosomal membrane glycoproteins (Lgps, Lamps, etc.), which are generally depleted from EEs and the plasma membrane (Kornfeld and Mellman 1989). It has been shown recently that the internal membranes contain large amounts of a unique lipid, lysobisphosphatidic acid, and thus form specialized lipid domains within endosomes (Kobayashi et al. 1998a, b). Indeed, some proteins are selectively enriched within these internal membranes, including members of the tetraspan family (Escola et al. 1998), MPR while in transit through endosomes (Griffiths et al. 1988, Kobayashi et al. 1998b), and the down-regulated EGF receptor (Futter et al. 1996). In

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contrast, Lgp120/Lampl is exclusively found on the limiting membrane (Griffiths et al. 1988, Aniento et al. 1993, Kobayashi et al. 1998b). Studies using the MPR as a marker have shown that these internal proteinlipid domains are involved in protein sorting and/or trafficking in the endocytic pathway (Kobayashi et al. 1998a, b). The relationships between LEs and lysosomes seem to be quite dynamic and thus not easily defined. It has been proposed that dense-core lysosomes can fuse with LEs and are re-formed from the resultant hybrid organelles (Bright et al. 1997). Plant cells

Electron microscopy studies have identified a number of organelles involved in endocytosis in plants (Fig. 1B). As in animal cells, endocytosed markers first appear in clathrin-coated pits and vesicles, which closely resemble their counterparts in animal cells. Coated pits and coated vesicles are particularly numerous in cells actively depositing new cell wall material and at the cell plate region of dividing plant cells. Indeed, a positive correlation has been found between active growth involving cell wall deposition and the frequency of coated pits (for reviews, see Low and Chandra 1994; D. Robinson et al. 1992, 1998b). Then, markers appear in the so-called partially coated reticulum (PCR) and in the Golgi region (Pesacreta and Lucas 1985, Hillmer et al. 1988, Tanchak et al. 1988, Fowke et al. 1991, Staehlin and Moore 1995).The PCR is a system of interconnected tubular membranes bearing coated regions (hence its name) and often surrounded by coated vesicles. The PCR occasionally appears in a ring configuration and it is often characterized by a rounded membrane dilation containing small internal vesicles. Markers within the Golgi complex are located principally at the periphery of medial and trans-Golgi cisternae and also in clathrincoated and apparently smooth vesicles in the vicinity of the Golgi cisternae, and in the trans-Golgi membranes and associated vesicles (Galway et at. 1993, Staehelin and Moore 1995). Probably, the PCR plays a role similar to that of the sorting endosome in animal cells, sorting material destined to be recycled, which would then be transferred to the Golgi, from material destined to be degraded, which would continue the degradative pathway. Structurally, the TGN and the PCR are two nearly indistinguishable organelles at the morphological level, which has led to considerable

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controversy (Hillmer et al. 1988, Griffing et al. 1991, Staehelin and Moore 1995). Both are branched, tubular-membrane systems with clathrin- and nonclathrin-coated terminal buds, and many of the ramifying PCR networks are closely associated with the trans side of the Golgi stacks. It has been therefore proposed that TGN and PCR could represent two functional domains of the same organelle. The next station in the degradative pathway are structures containing internal membranes, which have been referred to as multivesicular bodies (MVBs). However, all endosomes of the degradation pathway contain internal membranes in animal cells (see above) and the same is likely to be true in plant cells. It is thus possible that structures with a multivesicular appearance observed in plant cells encompass both ECV-MVBs and LEs of animal cells. MVBs are single-membrane-bounded organelles, approximately 250-500 nm in diameter, containing numerous small membranous internal vesicles. They are distributed throughout the cytoplasm but are preferentially located in the perinuclear region of the cells (Tanchak et al. 1984, Hitlmer et al. 1986, Fowke et al. 1991, D. Robinson and Hinz 1997). The final destination of endocytosed markers that follow the degradative pathway is the central vacuole. Mature plant cells are characterized by a large central vacuole and numerous small vacuoles in the peripheral cytoplasm. Vacuoles have a variety of functions in plant cells, including storage (proteins, amino acids, sugars), waste disposal (crystals, organic acids), maintenance of cell turgor, and cell elongation. In addition, there is considerable evidence that plant vacuoles contain a range of hydrolytic enzymes and thus represent the lytic compartment or lysosome of plants. Vacuoles within individual cells are specialized to accommodate this range of functions. Indeed, there is evidence indicating that plant cells contain at least two different types of vacuoles (Paris et al. 1996, Di Sansebastiano et al. 1998; Swanson et al. 1998), characterized by the presence of specific tonoplast-intrinsic proteins (TIPs). Protein storage vacuoles, containing seed-type storage proteins, are marked by the presence of cz-TIR while lytic or degradative vacuoles are marked by the presence of 7-TIR Both types of vacuoles are connected with the biosynthetic pathway by two separate pathways (Jiang and Rogers 1998). Most probably, transfer to the vacuole involves an intermediate compartment, presumably MVBs, and double immunogold labeling experiments suggest that MVBs fuse with protein

storage vacuoles (D. Robinson et al. 1998a). It is not clear whether there are prevacuolar intermediate compartments for the pathway to each type of vacuole, but both appear to converge ultimately on the central vacuole (Schroeder et al. 1993). Recently, a third type of functionally distinct vacuole has been described, which is specifically marked by 8-TIP and which is used to store pigments and vegetative storage proteins, proteins synthesized in response to developmental and environmental cues (Jauh et al. 1998). The organization of the endocytic pathway in yeast cells is very similar to animal or plant cells. During transport to the vacuole, endocytosed markers appear first in a peripheral compartment with a tubularvesicular structure, corresponding to the EEs, and then in large (ca. 200 nm in diameter) oval structures containing internal membranes, much like MVBs and often found near the vacuole (Prescianotto-Baschong and Riezman 1998, Wendland et al. 1998). The final destination of endocytosed markers is the central vacuole, but in contrast to plant cells, yeast cells do not appear to contain different types of vacuoles.

Proteins implicated in endosomai membrane transport

Rab proteins In mammalian cells, small GTPases of the Rab family localize to specific subcellular compartments and play key regulatory roles in membrane trafficking (for reviews, see Novick and Zerial 1997, Schimm611er et al. 1998). Rabs cycle between an active, GTP-bound form and an inactive, GDP-bound form. Studies of endosome fusion in vitro have shown that GTP hydrolysis converts Rab5 into its GDP-bound state after fusion has occurred. A cytosolic protein, termed GDI (GDP-dissociation inhibitor), retrieves prenylated GDP-bound Rab proteins and presumably delivers the Rabs to Rab-specific, nucleotide exchange factors, for reloading of the active, GTP-bound form (Horiuchi et al. 1997). Some Rab proteins are well known to play a role in membrane docking and fusion. These appear to initiate vesicle docking and facilitate SNARE complex formation but are not core elements of such complexes (Schimm011er et al. 1998). Recently, a role for Rabs in vesicle formation has also been proposed (Schimm611er et al. 1998,Woodman 1998). Rab5 regulates the transport from the plasma membrane to EEs as well as the homotypic fusion between EEs

M. J. Marcote et al.: Membrane transport in the endocytic pathway

(Gorvel et al. 1991, Bucci et al. 1992). In contrast, Rab4 has been implicated in recycling from EEs back to the plasma membrane (fast cycle) (Van der Sluijs et al. 1992). Rabaptin-5 acts as an cffector for Rab5 function: it is essential for the Rab5-dependent endosome fusion and is recruited by Rab5 on the membrane in a GTP-dependent manner (Stenmark et al. 1995, Horiuchi et al. 1997, Gournier et al. 1998). Rabaptin-5 also interacts with the GTP-bound form of Rab4. This may indicate that rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic (Vitale et al. 1998). Recent studies have shown that the GTP-bound form of Rab5 can also interact with EEA1 (see below). Rab9 is required for transport from LEs to the TGN (Lombardi et al. 1993). Rab7 appears to be implicated at later stages of endosomal protein transport (Feng et al. 1995, M6resse et al. 1995), although the precise step(s) in which it is implicated has not been unequivocally determined. The recent discovery of putative Rab effectors, which are not related to each other and seem to play different roles, suggests that Rab proteins may be involved in the regulation of more than one cellular process (Martinez and Goud 1998). In plant cells, numerous Rab homologues have been reported (Borg et al. 1997, Haizel et al. 1995, Ma 1994). 29 members of this protein family homologous to Rabl, Rab2, Rab5, Rab7, Rab8, and R a b l l have been recently identified in developing root nodules of Lotus japonicus (Borg et al. 1997). Lj-rab5A and -rab5B are 60% and 50%, respectively, identical to mammalian Rab5. Comparison of Rab5 functional domains with the homologous plant Rab5 sequences shows that they are closely related. Of these, Lj-rab5A may be the functional counterpart of mammalian Rab5a protein (Borg et al. 1997). Lj-rab7A to -rab7D constitute a subclass of proteins that are highly homologous (more than 70% identical) to mammalian Rab7. Alignment of the mammalian Rab 7 protein with the plant homologue demonstrates the high degree of homology between these proteins, specially in the domains important for binding and hydrolysis of GTP and in functional domains known from other Ypt/Rab proteins (Borg et al. 1997). Additional homologues to Rab5 and Rab7 have been isolated from other plants by other investigators (Borg et al. 1997, Ma 1994). In particular, a homologue of Rab7 in soybean has been shown to be involved in the biogenesis of the peribacteroid membrane, a subcellular compartment formed de ~lovo during root nodule symbiosis (Cheon et al.

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1993). Finally a large subgroup of Ypt/Rab proteins related to mammalian Rablls are also present in plant cells (Borg et al. 1997). Since homologues for GDI (Beyser and Fabry 1996) and GAP(GTPase-activating protein)-like activity (Anai et al. 1994) have been also identified in plants, it is a safe assumption that the Rab cycle functions in the same way as it does in mammalian and yeast cells, and that membrane dynamics in plants is also under the control of Rab proteins. Whether each of these Rabs has the same subcellular distribution as in mammalian cells and plays (a) similar role(s) in plant cells remains to be established, and certainly an effort should be directed towards this direction to study vesicle transport in plant cells.

NSF, SNAPs, and SNAREs According to the "SNARE hypothesis" (Rothman and Warren 1994), a family of compartmentally specific and cytoplasmically oriented integral membrane proteins provides the core mechanism that specifically pairs membranes. Vesicle- and target-associated SNAREs (v- and t-SNAREs) bind each other in a pairwise, cognate fashion, and must reside in opposite membranes for fusion to occur. SNAREs appear to be the minimal machinery required for membrane fusion (Weber et al. 1998). NSF, a member of the triple A ATPase family, and SNAPs were proposed to assemble into a fusion complex together with SNAREs at most docking and fusion steps. Evidence indicates that another triple A ATPase, p97/cdc48, is also involved at early steps of the biosynthetic pathway. However, the role of each component of this complex at precise steps of docking and fusion is controversial and unclear (for a review, see L. Robinson and Martin 1998). NSF and SNAPs are mainly cytosolic proteins, but they were also found to be associated with membranes (L. Robinson et al. 1997). In the endocytic pathway several SNARE proteins have been described, including cellubrevin (Daro etal. 1996), which localizes primarily to recycling endosomes, and endobrevin, which was observed in perinuclear early endosomes (Wong etal. 1998). Several members of the syntaxin family have been implicated in endocytosis in yeast, including PEP12p, required for the ligand-induced internalization of the s-factor receptor (Holthuis et al. 1998a), Vamp3p, required for homotypic fusion of vacuoles (Nichols et al. 1997), and Tlglp and Tlg2p, t-SNAREs located in the TGN-endosome system of yeast (Seron et al. 1998, Holthuis et al.

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1998b). NSF and SNAPs have been shown to be required for all homotypic and heterotypic docking and fusion events during transport from early to late endosomes (Colombo et al. 1996, L. Robinson et al. 1997, Rodriguez et al. 1994). In plant cells, an NSF homologue has been reported in red pepper fruits (Capsicum annuum), which catalyzes ATP-dependent fusion and/or translocation of an integral protein of chromoplast vesicle donors to chromoplast vesicle acceptors (Huegeney et al. 1995). Additional homologues have been found in rice and barley (Mogelsvang and Simpson 1998). To our knowledge, SNAP homologues have not yet been identified in plants. Concerning SNAREs, three syntaxin homologues in plants have been identified so far. One is a PEP12p homologue in Arabidopsis thaliana, a tSNARE localized to a prevacuolar compartment (Conceicao da Silva et al. 1997, Sanderfoot et al. 1998). Another one is the KNOLLE protein, also from A. thaliana, which appears to be involved in homotypic fusion of Golgi-derived vesicles during cell plate formation (Lukowitz et al. 1996). The third one is AtVamp3p, which is implicated in vacuole biogenesis in A. thaliana (Sato et al. 1997). Future studies will be necessary to identify the other members of this protein family in plants.

which type of vesicles would use this novel coat. In mammalian cells, biogenesis of ECV-MVB depends on the acidic lumenal pH of EEs, and some, but not all, components of the COPI coat (Aniento et al. 1996, Clague et al. 1994). Both mechanisms are functionally related, since COP association to endosomes, but not to biosynthetic membranes, is itself pH dependent. These observations suggest that the acidification properties of EEs may control the formation of vesicles destined for LEs by controlling coat recruitment onto the membranes. This mechanism is consistent with the observation that ECVs can be more acidic than EEs. It is not clear how information on the lumenal pH may then be transferred to the cytoplasmic face of the membrane. The simplest view is that pH differences may be sensed by the conformational change of a membrane protein. We have speculated that the conformation of an endosomal transmembrane protein, possibly a COP receptor, is pH sensitive and thus serves as a sensor of the endosomal pH. Endosomal and biosynthetic COPs thus exhibit, at least in part, different properties. In addition, two subunits of the biosynthetic COPI coat, ? and S, are not present on endosomes (Whitney et al. 1995, Aniento et al. 1996), suggesting that the composition of the endosomal coat is simpler or that the endosomal homologues of ? and ~ have not been identified. A functional dissection of the role of COP I in ECV/MVB formation has shown that the 1~, [~', and X-COP subunits can bind to endosomes in a pH-dependent and GTP-dependent manner, while the a and t-COP subunits are required to confer functionality to the process of vesicle formation, probably facilitating coat assembly (Gu et al. 1997). Recruitment of COP I subunits on EEs appears to be mediated also by the ARF1 protein, as on biosynthetic membranes, and ARF1 binding is itself pH dependent (E Gu and J. Gruenberg pers. commun.). In plant cells, coats are clearly visible at the electron microscopical level, including typical clathrin-coated buds and vesicles at the plasma membrane and others as yet unidentified coats (Emons and Traas 1986, Fowke et al. 1991, D. Robinson et al. 1998b). The PCR is the paradigm of coated regions (Pesacreta and Lucas 1985, Hillmer et al. 1988, Tanchak et al. 1988, Fowke et al. 1991), which may probably be involved in sorting events in the endocytic pathway in plant cells. Coated regions can also be observed in MVBs (Hillmer et al. 1986, D. Robinson and Hinz 1997). However, the nature of these coated regions remains to be established. Clathrin and adaptins have been

Coat proteins and A R F proteins Two types of clathrin-coated vesicles deliver cargo to the endocytic pathway by assembling on distinct compartments through interactions with different heterotetrameric adaptor protein complexes, AP-1 and AP-2 on TGN- and plasma membrane-derived vesicles, respectively (M. Robinson 1994). A different type of clathrin-coated vesicles seems to bud from EEs (Stoorvogel et al. 1996, Whitney et al. 1995). These vesicles contain transferrin receptor, suggesting a role in recycling, but they are devoid of a- or ?-adaptin, present in AP-1 and AP-2, suggesting that they may contain a novel type of adaptor complex (Stoorvogel et al. 1996). A third adaptor complex, AP-3, has a similar four-subunit composition as AP-1 and AP-2 and is presumably involved in transport between Golgi-TGN and endosome-lysosomes. Whether this complex also associates with clathrin remains controversial (Simpson et al. 1997, Dell'Angelica et al. 1998). AP-3 is recruited on endosomal membranes in an A R F I ( A D P ribosylation factor)-dependent manner (Ooi et al. 1998). It remains to be established, however,

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identified in plants (Coleman et al. 1987, 1991; Holstein et al. 1994; Beevers 1996; D. Robinson 1996; D. Robinson et al. 1998b). The same is true for proteins of the A R F family (Memon et al. 1993, Regard et al. 1993). Evidence for other coat proteins is not yet available, although it is quite likely that they will come up pretty soon.

is present in plant cells, and if so, whether its distribution and role are similar to those in mammalian cells.

Lipid and protein-lipid domains Recent studies have revealed the role of specialized lipids and/or protein-lipid domains in endocytic membrane traffic (Kobayashi et al. 1998b). As discussed above, late endosomes contain lysobisphosphatidic acid-rich internal membranes, which are also enriched in some membrane proteins (Kobayashi et al. 1998a). In addition, it is becoming clear that phosphoinositides (PIs) and PI kinases are also involved in the endocytic pathway (for reviews, see Corvera and Czech 1998, De Camilli et al. 1996). Inhibitors of PI3-kinase, like wortmannin, affect several steps in the endocytic pathway, including transport to LEs and lysosomes, traffic of MPR from endosomes to TGN and transferrin receptor recycling. Wortmannin also causes changes in the morphology of endocytic compartments. EEA1, a 170 kDa hydrophilic, peripheral membrane protein found as an antigen in a patient with a mild form of Lupus erythematosus, localizes to early endosomes and is a direct target of PtdIns(3)R Indeed, EEA1 binds directly to Ptdlns(3)P through the Zn2+-containing RING finger in its C terminus. EEA1 is emerging as a key regulator of endosome traffic. It interacts strongly with Rab5 in a yeast two-hybrid screen and probably interacts directly with the GTP-bound form of Rab5. Consistent with this idea, homotypic fusion of EEs depends on both active Rab5 and PI3-kinase (for reviews, see Corvera and Czech 1998, Mills et al. 1998). Recently, the yeast FAB1 gene was identified to be a PI3,5-kinase involved in the maintenance of vacuolar size and membrane homeostasis (Gary et al. 1998). The function of such kinase is not known, but one of its products, the novel polyphosphoinositide PtdIns (3,5)P2 has been shown to be implicated in the response to osmotic stress in yeast and is present both in mammalian and in plant cells (Dove et al. 1997). Plant cells contain PI3-kinase (Welters et al. 1994) and PI-4-P 5-kinase. The latter has been shown to be induced by water stress and abcisic acid in A. thaliana (Mikami etal. 1998). Future studies witl also be required to establish whether lysobisphosphatidic acid

Conclusions and perspectives It is clear that a wealth of information is becoming available on the endocytic pathway in animal cells. We are starting to have a precise picture of the compartments in the pathway, their morphology, protein and lipid composition, and on the processes of vesicle formation and vesicle fusion. In addition, the set of molecules involved in endosomal membrane transport is starting to be unraveled. Plant cells appear to contain most, if not all, organelles presumably involved in the housekeeping functions of the endocytic pathway in mammalian cells, although the precise functions of the pathway should still be clarified in plants. Plant cells also appear to contain key components of the protein machinery involved in membrane transport in the endocytic pathway of animal cells. Effort should be made in the following years into understanding whether these molecules play similar roles in plant cells. Finally, plant cells also exhibit a characteristic membrane organization and functions which appear different from those observed in the endocytic pathway of mammalian cells; the identification and characterization of membrane components and factors will be a challenge for the future.

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