Clathrin-mediated endocytosis: membrane factors pull the trigger

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1 Trends in Cell Biology Volume 11, Issue 9, 1 September 2001, Pages Clathrin-mediated endocytosis: membrane factors pull the trigger Kohji Takei, a and Volker Haucke, b a Dept of Neuroscience, Okayama University Graduate School of Medicine and Dentistry, Shikatacho, Okayamashi, Okayama , Japan b Zentrum Biochemie & Molekulare Zellbiologie, University of Goettingen, Humboldtallee 23, D Goettingen, Germany Abstract Clathrin-mediated endocytosis is a vesicular transport event involved in the internalization and recycling of receptors participating in signal transduction events and nutrient import as well as in the reformation of synaptic vesicles. Recent studies in vitro and in living cells have provided a number of new insights into the initial steps of clathrin-coated vesicle formation and the membrane factors involved in this process. The unexpected complexity of these interactions at the cytosol-membrane interface suggests that clathrin-coated vesicle assembly is a highly cooperative process occurring under tight regulatory control. In this review, we focus on the role of membrane proteins and lipids in the nucleation of clathrin-coated pits and provide a hypothetical model for the early steps in clathrin-mediated endocytosis. Clathrin-mediated endocytosis is a process by which virtually all eukaryotic cells internalize nutrients, antigens, growth factors, pathogens and recycling receptors [1 and 2]. Internalization can occur either constitutively or in response to certain stimuli, such as in the recycling of synaptic vesicles after exocytosis [3, 4 and 5]. The basic mechanisms underlying endocytosis have fascinated cell biologists for more than two decades, but insights into how clathrin-coated vesicle budding from the plasma membrane might be initiated at the molecular level [6] have been gained only recently. While a large number of cytosolic factors regulating endocytic vesicle formation have been identified over the years [4 and 7], the molecular events that determine the nucleation of clathrin-coated buds at endocytic `hot spots' are only now beginning to reveal their secrets. Here, we provide an overview of the components involved in the nucleation of coated pits at the plasma membrane and postulate a hypothetical model for how multiple partners might act synergistically to initiate this event. An overview of clathrin-mediated endocytosis Endocytosis of plasma membrane or synaptic vesicle components is effected by the progressive and sequential assembly of clathrin-coated vesicles that serve to concentrate cargo proteins and lipids into the emerging vesicle and provide a mechanical means to deform the membrane into a vesicular bud [6, 8 and 9]. This bud matures and eventually pinches off, giving rise to a free 1

2 clathrin-coated vesicle. Energy-dependent uncoating restores a vesicle, which can either undergo fusion with endosomes or, in the case of a synaptic vesicle, be refilled with neurotransmitter and re-enter the synaptic vesicle pool [4, 5 and 10] (Fig. 1). At first glance, the principal steps of clathrin-mediated endocytosis are similar to other vesicular transport reactions such as the budding of nascent secretory vesicles by COPI and COPII coat proteins, but these trafficking pathways differ considerably in their mechanistic details [6]. Fig. 1. Overview of the steps involved in clathrin-mediated endocytosis. Shown are the sequential steps involved in clathrin-mediated endocytosis and several key components that function in each step. Protein-protein or protein-lipid interactions that play putative roles in the coat nucleation and assembly step are depicted in more detail in Fig. 2. Abbreviation: PIP 2, phosphatidylinositol (4,5)-bisphosphate. 2

3 Clathrin coats were identified initially by their distinctive molecular appearance in electron micrographs that arises from the presence of polygonal clathrin lattices that form the outer shell and main scaffold of the coat. The clathrin coat is assembled on the cytoplasmic face of the plasma membrane by the recruitment of the adaptor complex AP-2, a heterotetramer comprising two large subunits, alpha and beta2 (each of ~100 kda), and two smaller chains, mu2 (50 kda) and sigma2 (20 kda). Adaptors serve to link clathrin to the membrane and to coordinate the structural assembly of the coat with the selection of cargo proteins and lipids (Fig. 1, step 1) [2, 6, 8 and 9]. Most notably, the medium chain (mu2) of AP-2 is capable of recognizing tyrosinebased endocytic sorting motifs of multiple cargo proteins [11 and 12], thereby concentrating them in the emerging bud. In many cases, the coat contains an additional monomeric protein, termed AP180 in neurons and CALM in other tissues, that appears to define the size of the coated vesicle [13 and 14] by assisting coat formation. The assembly of clathrin-coated buds is aided by an array of mostly cytosolic proteins, often referred to as `accessory proteins', that form a dynamic network of protein-protein interactions by associating with multiple partner proteins during as yet ill-defined stages of endocytosis [4, 5 and 7] (Box 1). These modular proteins could help in coat assembly and membrane fission and coordinate these events with changes in the actin cytoskeleton [15] and in lipid metabolism [16]. During the progressive assembly of clathrin-coated buds and their maturation, the membrane acquires increasing curvature until a deeply invaginated coated pit forms (Fig. 1, step 2). Fission of the coated pit requires the action of the dynamin GTPase [17] (Fig. 1, step 3), the best-studied accessory protein of endocytosis. While the exact molecular mechanism by which fission is effected is not yet fully understood, it seems clear that hydrolysis of GTP by oligomeric rings of dynamin around the neck of endocytic intermediates [17, 18, 19, 20 and 21] is required for vesicle scission. This could occur by direct constriction [22] (`pinchase'), by helical expansion of dynamin spirals at the neck [23] (`poppase') or with the aid of downstream effectors [24] such as the lysophosphatidic acid acyl transferase endophilin [25 and 26] (Box 1). Finally, free clathrincoated vesicles undergo rapid uncoating catalyzed by the ATP-hydrolyzing molecular chaperone hsc70 [27] and its DnaJ-like partner protein auxilin [28] (Fig. 1, step 4). Box 1. Components of the endocytic machinery Domain structure and established or putative functions of clathrin coat components, membrane factors and accessory proteins of clathrin-mediated endocytosis (Fig. I). Abbreviations: C2, protein kinase C homology 2; DPW, Asp-Pro-Trp; SH3, Src homology 3; EH, eps15 homology domain; ENTH, epsin amino-terminal homology; GED, GTPase-enhancing domain; J, DnaJ domain; LPA-ATase, lysophosphatidic acid acyl transferase; NPF, Asp-Pro-Phe; PIP 2, phosphatidylinositol (4,5)-bisphosphate; PH, pleckstrin homology domain; PRD, proline-rich domain; Sac1, suppressor of actin 1; YxxPhi, tyrosine-based endocytic motif. 3

4 Triskelia built from 170-kDa heavy and 35-kDa (regulatory) light chains polymerize into coats that form the outer shell of clathrin-coated buds and vesicles. Heterotetrameric complex of alpha ( kda), beta2 (105 kda), mu2 (50 kda) and sigma2 (20 kda) subunits that links the clathrin shell to the membrane through interactions of its mu2 and alpha subunits with membrane proteins and lipids. NOTE: Hip1R has an ENTH domain Accessory component (95 kda protein; runs at 180 kda on SDS- PAGE) of clathrin coats that might regulate vesicle size. Contains PIP 2- binding ENTH and clathrin-assembly domains. AP-2 binding protein of synaptic vesicles and the presynaptic plasmalemma that facilitates vesicle recycling by promoting coated pit nucleation. Multiple isoforms in brain but also in other tissues. Interacts with AP-2, AP-180, synaptotagmin and epsin and might help to initiate or stabilize clathrin-ap-2 coats at the membrane. Cargo proteins containing tyrosine-based endocytic motifs bind to mu2 and might assist coated pit nucleation by synergistically promoting AP- 2 recruitment to synaptotagmin. The affinity of these motifs for AP-2 is modulated by phosphoinositides. 100-kDa GTPase that polymerizes into oligomeric rings at the neck of invaginating buds and catalyzes vesicle fission upon GTP hydrolysis. Lysophosphatidic acid acyl transferase (40 kda) with putative roles in coated bud maturation and vesicle fission. Binds to dynamin and synaptojanin. Binding partner of clathrin, AP-2 and dynamin. Putative role in fission. Large modular protein ( kda isoforms) with multiple partners in endocytosis. Two related proteins of kda that bind to clathrin, AP-2, Eps15 and PIP 2. Inositol-phosphatase ( kda isoforms) that regulates PIP 2 metabolism and the stability of clathrin-ap-2 coats. The 170-kDa isoform contains NPF motifs. J-domain protein (100 kda) that assists hsc70 in uncoating. Binding partner ( kda) of synaptotagmin, AP-2 and proteins containing EH-domains, with a putative role in endocytosis and coat dynamics. 4

5 From the overview provided above, it is clear that clathrin-mediated endocytosis is initiated at the membrane-to-cytosol interface. How then do membrane factors trigger coated pit assembly? Membrane protein factors in clathrin-coated pit nucleation: synaptotagmin and AP-2 Various stages of coated pit formation have been dissected at synapses, where clathrin-coated vesicles are involved in synaptic vesicle formation. Endocytic recycling of synaptic vesicles not only is an unusually rapid form of clathrin-ap-2-mediated endocytosis but also is tightly coupled to the exocytic fusion of neurotransmitter-filled vesicles [29 and 30]. Morphological studies [4] have provided strong evidence that plasma membrane-associated factors exposed or generated during the exocytic limb of the vesicle cycle trigger the onset of clathrin-coated pit formation [31]. Although the putative membrane factor was not identified in this study, it seems likely that synaptic vesicle proteins form part of the trigger. Indeed, synaptic vesicle proteins represent the main cargo of synaptic clathrin-coated vesicles from brain [32]. A likely candidate molecule is the AP-2-binding protein synaptotagmin [33], a major component of synaptic vesicles and the presynaptic plasma membrane [34]. Synaptotagmin is abundant in brain, and isoforms have been detected in virtually all cells and tissues [34, 35 and 36] and at all membranes from which clathrin-ap-2-coated vesicles can arise, including lysosomes [37]. Synaptotagmin comprises an N-terminal transmembrane segment followed by the cytoplasmic domain comprising two Ca 2+ - and phospholipid-binding C2 domains. Although both C2 domains are structurally similar, only the second C2 domain (C2B) contains a unique cluster of lysine residues implicated in AP-2 binding [38]. Genetic experiments in mice [39 and 40], Drosophila [41, 42 and 43] and Caenorhabditis elegans [44] have shown that synaptotagmin is involved in both exocytosis and endocytosis. Mutants expressing truncated forms of synaptotagmin lacking the AP-2-binding C2B domains in C. elegans [44] or Drosophila [43] are severely impaired in neurotransmission and display a strongly reduced number of synaptic vesicles following stimulation, indicative of a defect in vesicle recycling. Likewise, microinjection of antibodies against the C2B domain of synaptotagmin in giant terminals of the squid inhibits synaptic vesicle endocytosis at an early stage in living synapses [45]. Since clathrin-mediated endocytosis represents the main pathway of synaptic vesicle formation [4 and 5], these data imply a function for synaptotagmin in endocytosis at the synapse. Biochemical experiments using lysed brain nerve terminals and isolated plasmalemmal sheets have corroborated the hypothesis that synaptotagmin acts as a docking site for AP-2 at the plasma membrane [46 and 47]. The association of synaptotagmin with AP-2 is unusual in that it involves a dual interaction with both the alpha and mu2 subunits of AP-2 [47], thus providing a means of flexibility and possibly regulation (Fig. 2). Other cargo proteins such as internalized plasma membrane receptors carrying tyrosine-based endocytic motifs strengthen the synaptotagmin-ap-2 interaction by inducing a conformational change within the AP-2 complex. This suggests a possible mechanism for how coated pit nucleation could be coupled to the selection of multiple cargo proteins [46]. Recent experiments in non-neuronal cells [47 and 48] have shown that synaptotagmin needs to oligomerize in order to effectively recruit AP-2 to the plasma membrane and facilitate endocytosis [48]. These combined data provide evidence for the hypothesis that synaptotagmin, along with other cargo proteins, might act as an important regulator of clathrin-coated pit nucleation. 5

6 Fig. 2. Possible interactions between membrane factors and AP-2 in coated pit nucleation. Hypothetical model for the cooperative recruitment of AP-2 to the presynaptic plasma membrane. Cooperative interactions of membrane-localized factors including synaptotagmin and phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) might convert `loosely' membrane-associated AP- 2 weakly bound to tyrosine-based motifs of cargo proteins into `tightly' membrane-bound nucleation sites for clathrin-coated pit assembly. Note the simultaneous binding of AP-2 to phosphatidylinositol (4,5)-bisphosphate, synaptotagmin and cargo proteins containing tyrosinebased endocytic motifs. Although not depicted here, cytosolic factors that bind to phosphatidylinositol (4,5)-bisphosphate, such as AP180/CALM and epsin, might also contribute to coated pit nucleation (see Box 1). C2A, first C2 domain of synaptotagmin C2B, second C2 domain of synaptotagmin; YxxPhi, tyrosine-based endocytic motif. Membrane lipids regulate clathrin-ap-2 coated pit formation While synaptotagmin could serve an important function in the spatial and temporal regulation of coated pit nucleation, several lines of evidence suggest that it does so in concert with membrane lipids and, in particular, phosphoinositides. Phosphoinositides interact specifically with several proteins implicated in endocytosis. The alpha subunit of the AP-2 adaptor binds to phosphoinositides, with high preference for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ] and PtdIns(3,4,5)P 3 at physiological concentrations [49]. The binding site within alpha-adaptin is contained within residues 21-80, with a positively charged lysine triad at the core of the PtdIns(4,5)P 2 -binding site. Expression of a mutated version of alpha-adaptin in which the crucial lysine residues have been replaced by alanines reduces the affinity of AP-2 for the plasma membrane [50]. In agreement with these findings, it has been reported that masking of PtdIns(4,5)P 2 with either neomycin, a PtdIns(4,5)P 2 -binding aminoglycoside, or the pleckstrin homology (PH) domain of 6

7 phospholipase C inhibits recruitment of AP-2 onto endosomes or the plasma membrane [51 and 52]. In addition, phosphoinositides might regulate AP-2 self-assembly [9] and its interaction with tyrosine-based endocytosis motifs [53] and synaptotagmin [46]. Other endocytic proteins such as dynamin, AP180 and epsin also associate with phosphoinositides. Binding of dynamin to PtdIns(4,5)P 2 or PtdIns(3,4,5)P 3 through its PH domain stimulates its GTPase activity and dynamin self-assembly in vitro [54 and 55]. Conversely, expression of a dynamin mutant incapable of associating with phosphoinositides has a dominant-negative effect on clathrinmediated endocytosis [56 and 57]. Thus, phosphoinositides might play a role in dynaminmediated vesicle fission. Epsin, another accessory protein of endocytosis (see Box 1), comprises an N-terminal homology (ENTH) domain that is conserved in several other endocytic proteins such as AP180/CALM and Hip1R. The highly related ENTH domains of epsin and AP180/CALM bind to PtdIns(4,5)P 2 but, surprisingly, utilize structurally distinct mechanisms involving clusters of positively charged arginine and lysine residues [58 and 59]. Mutation of the ENTH domain of epsin not only abolishes phosphoinositide binding but also blocks clathrinmediated endocytosis upon overexpression [59]. As both epsin and AP180/CALM can either directly or indirectly associate with AP-2 and clathrin, it is likely that binding of these proteins to phosphoinositides contributes to coated pit nucleation (see also Fig. 1). Accordingly, enzymes that catalyze the formation and turnover of phosphoinositides play a pivotal role in endocytosis. The generation of PtdIns(4,5)P 2, which is catalyzed by phosphatidylinositol 4-kinase (PI4K) and phosphatidylinositol 4-phosphate 5-kinase (PI4P5K), is stimulated by phosphatidic acid (PtdOH) [60]. PtdOH production in turn involves known endocytic proteins: endophilin is a cytosolic lysophosphatidic acid (LPA) acyltransferase [26] capable of synthesizing PtdOH by fatty acylation. Alternatively, PtdOH can be generated by phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine. The enzymatic activity of PLD is stimulated by Arf and PtdIns(4,5)P 2 (Ref. [51]), thus generating a positive-feedback loop, and can be inhibited by the endocytic proteins synaptojanin and amphiphysin [61 and 62]. There is also evidence for a regulatory function of 3' phosphoinositides in clathrin-mediated endocytosis. Class II phosphatidylinositol 3-kinase contains a phosphoinositide-binding C2 domain and occurs abundantly in clathrin-coated vesicles. Its enzymatic activity is stimulated by clathrin, suggesting a regulatory function for this enzyme in coated vesicle dynamics [63]. One possible role for phosphatidylinositol 3-phosphates is the modulation of cargo selection by increasing the affinity of mu2-adaptin for tyrosine-based endocytic motifs of accessory cargo proteins [53]. The important role of phosphoinositides in nucleating clathrin-ap-2 coated pit assembly in turn requires that the levels of PtdIns(4,5)P 2 and other inositol lipids be tightly regulated. Recent experiments have provided strong evidence for this proposal. Disruption of the inositol phosphatase synaptojanin 1 in mice rendered the animals inviable and resulted in the accumulation of clathrin-coated vesicles at the synapse, possibly owing to elevated levels of PtdIns(4,5)P 2 (Ref. [64]). These data support the notion that PtdIns(4,5)P 2 is an important regulator of clathrin-ap-2 coat stability in vivo. In fact, endocytic vesicle formation in vitro is accompanied by degradation of PtdIns(4,5)P 2 (K. Takei, unpublished), but when exactly the PtdIns(4,5)P 2 is being hydrolyzed in vivo is unknown at present. 7

8 Consistent with the observed interactions between phosphoinositide lipids and the endocytic machinery, clathrin-coated buds have been reconstituted in vitro by incubating protein-free liposomes made from brain lipids with cytosol or purified clathrin coat proteins [65] (Fig. 3). Although no strict lipid requirements were observed, the formation of clathrin-ap-2 coated buds differed qualitatively, depending on the lipid content [65]. Similar observations have been made for the assembly of clathrin-ap-1 or clathrin-ap-3-coated buds on synthetic liposomes [66 and 67]. Thus, it appears likely that clathrin-coated pit nucleation is regulated by compositional changes in the lipid bilayer. Fig. 3. Electron micrograph of clathrincoated buds formed on liposomes. Clathrincoated buds were generated on liposomes of total brain lipids by incubating with a bovine brain fraction highly enriched in clathrin coat proteins. The coated structures are indistinguishable from those observed in vivo. Bar: 220 nm (a), 100 nm (b). (Images reproduced, with permission, from Ref. [65].) A hypothetical model for clathrin-coated pit nucleation at the synapse As outlined above, accumulating evidence implicates both synaptotagmin and phosphoinositides in clathrin-ap-2 coated pit nucleation at the plasma membrane, in particular within the presynaptic nerve terminal. As both AP-2 [50] and synaptotagmin [34] can bind to PtdIns(4,5)P 2, it seems tempting to speculate that phosphoinositides cooperate with synaptotagmin in nucleating coat assembly. In the case of synaptic vesicle recycling, a regulatory form of rapid endocytosis, it has been proposed that the reversible phosphorylation of inositol lipids could link the exocytic and endocytic reactions and provide a device to confer directionality onto the synaptic vesicle cycle [68]. Recent data indicate that other membrane lipids such as phosphatidic acid [46 and 60] or cholesterol [16 and 69] could also contribute to clathrin-ap-2 coated pit nucleation, possibly by forming raft-like `hot spots' of endocytosis. It is noteworthy that synaptotagmin, like synaptophysin (another major synaptic vesicle membrane protein), can bind to cholesterol [69]. 8

9 Thus, in a hypothetical scenario, the synergistic action of synaptotagmin and phosphoinositides could act as a trigger to convert loosely membrane-associated AP-2 molecules, which can weakly associate with accessory cargo proteins, into tightly bound nucleation sites for clathrincoated pits (Fig. 2). Binding of phosphoinositides to AP-2 could not only assist its association with the membrane [50, 60 and 64] but also enhance the affinity of AP-2 for tyrosine-based endocytic motifs [53], which in turn might facilitate the association between AP-2 and synaptotagmin [46]. In this way, synaptotagmin, non-obligatory cargo and phosphoinositides could serve the role of `coincidence' detectors [6] in order to allow for the regulation of clathrincoated pit nucleation in time and space. The putative role of synaptotagmin and other cargo proteins in coated pit nucleation need not prevent there being a synergistic action for accessory cytoplasmic proteins during the early stages of coated pit formation. Phosphoinositide-binding proteins such as epsin and AP180/CALM, therefore, are likely contributors to coated pit nucleation as well [58 and 59]. By associating with clathrin-ap-2 [5, 6, 7, 8 and 9], these molecules could assist the transition from AP-2-containing nucleation sites to clathrin-coated pits. Thus, a multiplicity of cooperative interactions between synaptotagmin and other cargo proteins, accessory factors and phosphoinositides is likely to be involved in initiating coated pit assembly. The highly cooperative nature of clathrin-coated pit formation at the synapse is consistent with this speculative model. Vesicle uncoating: a reversal of coated pit assembly? Free clathrin-coated vesicles are rarely observed in living cells or at stimulated synapses, suggesting that these vesicles undergo rapid uncoating [4 and 5]. What exactly provides the trigger for coat removal and how this reaction is coupled to vesicle budding are unknown at present. The uncoating reaction itself involves disassembly of the polygonal clathrin lattice by heat-shock cognate protein hsc70 (`uncoating ATPase'), a member of the DnaK family of 70- kda heat-shock proteins. On its own, this molecular chaperone exhibits a relatively low ATPase activity that can be substantially stimulated by auxilin, an accessory protein comprising a DnaJ domain [28]. Auxilin is highly concentrated at nerve terminals and aids the targeting of hsc70 to clathrin coats by associating with AP-2 and clathrin [7]. Although hsc70 and auxilin also participate in removing AP-2 from the membrane, biochemical studies have indicated the presence of an additional, currently uncharacterized, 100-kDa protein in adaptor release [70]. Given the tight association of AP-2 with phosphoinositides and synaptotagmin at the membrane, it is not surprising that specialized factors are required to trigger AP-2 release. One such candidate molecule is the recently identified human homolog of Drosophila stoned B (hstnb/stonin 2). Like its counterpart in fly [71 and 72], hstnb/stonin 2 associates with synaptotagmin [73 and 74] through a domain homologous to mu2-adaptin and unexpectedly also associates with AP-2 [73] in this way. Overexpression of hstnb/stonin 2 in cells inhibits clathrin-mediated endocytosis by promoting the dissociation of AP-2 from the plasma membrane [74]. Moreover, the purified mu2 homology domain of hstnb/stonin 2 competes with AP-2 in binding to synaptotagmin and potently stimulates release of clathrin-ap-2 from coated vesicles in vitro [73]. These findings imply that hstnb/stonin 2 might negatively regulate coat stability by 9

10 promoting vesicle uncoating. Thus, removal of clathrin-ap-2 from the membrane might, at least in part, be a reversal of coated pit assembly. It is, however, possible that hstnb/stonin 2 serves additional functions during endocytosis such as cargo selection or modulation of coat assembly [74]. An additional factor that could contribute to uncoating is the inositol-phosphatase synaptojanin 1 [4, 5 and 7] (Box 1). As described above, mice deficient in synaptojanin 1 accumulate both PtdIns(4,5)P 2 and clathrin-coated vesicles at the synapse [64]. These data again suggest that uncoating could be triggered by `reversing' the conditions for coat assembly that is, hydrolysis of PtdIns(4,5)P 2. An intriguing possibility, not yet supported by experimental data, is that PtdIns(4,5)P 2 hydrolysis might already be occurring during membrane fission, thereby releasing accessory proteins such as epsin and dynamin from the emerging vesicle and coupling vesicle budding to the uncoating reaction. Outlook The past few years have witnessed enormous progress in our understanding of the mechanisms of endocytosis. The identification and characterization of a plethora of new components of the endocytic machinery and their initial functional analysis has provided us with a roadmap for future studies that will be aimed at a detailed mechanistic inspection of coated vesicle formation in real time at increasingly higher spatial resolution. With ever-better tools to hand, we shall soon be able to untangle the complex network of protein-protein and protein-lipid interactions during endocytosis and to determine the precise role of each component in coated pit nucleation. Having analyzed most of the basic components of the coat and its associated machinery, one of the most fascinating tasks will be to unravel how these factors are regulated under certain physiological conditions, how they interact in time and space and how they manage to sort a vast array of cargo proteins and lipids. Acknowledgements Work in the authors' laboratories was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan (to K.T.) and from the Deutsche Forschungsgemeinschaft (SFB523) and the Fonds der Chemischen Industrie (to V.H.). We thank members of our laboratories for discussions, Hiroshi Yamada for help with the illustrations and Michael Krauss for critical comments on the manuscript. References 1. I. Mellman, Endocytosis and molecular sorting. Annu. Rev. Cell Biol. 12 (1996), pp J. Hirst and M.S. Robinson, Clathrin and adaptors. Biochim. Biophys. Acta 1404 (1998), pp M.J. Hannah et al., Synaptic vesicle biogenesis. Annu. Rev. Cell Dev. Biol. 15 (1999), pp L. Brodin et al., Sequential steps in clathrin-mediated synaptic vesicle endocytosis. Curr. Opin. Neurobiol. 10 (2000), pp P. De Camilli et al.in: M.W. Cowan et al.synapses, The Johns Hopkins University Press (2001), pp

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