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Synaptic Vesicle Recycling: The Ferrari of endocytosis?
STEPHEN A. MORRIS AND SANDRA L. SCHMID
SYNAPTIC VESICLE RECYCLING
The Ferrari of endocytosis? After neurotransmission, neurons internalize and recycle the membrane components of synaptic vesicles remarkably quickly; they may have a 'rebuilt, turbo-charged' endocytic engine to achieve these speeds. Specialized regions of the cell surface known as clathrincoated pits mediate efficient endocytosis of membrane receptors and their ligands. For receptors free to diffuse along the cell surface, the rate of endocytosis depends on the number of coated pits, the ability of a given receptor to be recruited and concentrated into a coated pit, and the time taken for coated pit assembly and coated vesicle budding. For the transferrin receptor, considered a paradigm of efficiency, the half-time of internalization is 3-5 minutes in fibroblastic cells. By contrast, following neurotransmission, synaptic vesicle membranes are internalized within seconds, and the recycled synaptic vesicles can be reloaded with neurotransmitter within 1-2 minutes. Does this greatly enhanced speed necessarily suggest an alternative vehicle, or can rapid endocytosis at the synapse be accomplished by 'revving-up' the clathrincoated vesicle engine? Coated vesicle formation in fibroblastic cells is initiated by the recruitment of assembly, or adaptor, protein (AP2) complexes from the cytosol to saturable, high-affinity binding sites on the plasma membrane [1]. Clathrin is then recruited from the cytosol to the membrane-bound AP2 complexes, in a reaction that is presumably mediated by the AP2 molecules, which have been shown to be capable of self-aggregating and enhancing clathrin assembly in vitro [2]. The interaction of AP2 complexes with 'internalization signals' in the cytoplasmic domains of receptors is required for the receptors' concentration into coated pits [3,4]. Coated pits invaginate and eventually bud to form coated vesicles carrying receptor-ligand complexes into the cell. Reconstitution of these latter events in vitro requires ATP and GTP hydrolysis [5]. Recently, dynamin has been identified as one of the GTPases that regulate late stages in coated-vesicle formation [6-8]. How might endocytosis mediated by clathrin-coated vesicles be accelerated in neurons? The number of coated pits and the efficiency of their assembly could be enhanced simply by overexpressing coated pit constituents. In fact, all of the known components of the coated vesicle engine, including AP complexes, clathrin, dynamin and the uncoating ATPase (hsc70) are expressed at 10-50-fold higher levels in neuronal cells than in nonneuronal cells. In addition, neuron-specific, perhaps performance-enhancing, isoforms exist for most of the known components of the coated vesicle machine. Separate genes encode neuron-specific isoforms of the o subunit of AP2 complexes [4] and of dynamin [9]; and differential RNA splicing leaves neuron-specific inserts in
the adaptin subunit of AP2 complexes [4] and in both clathrin light chains [10]. 'Optional' equipment has also been added to the neuron's coated vesicle engine. For example, AP3 is a neuronspecific assembly protein which is approximately fourtimes more efficient at stimulating clathrin assembly than are other clathrin-binding proteins [11]. Auxilin is another neuron-specific clathrin-binding protein [12], the function of which is not yet known. Importantly, expression patterns in the rat brain of the neuron-specific forms of the clathrin light chain, the AP2 subunits and auxilin (S.A.M., E. Ungewickell and E Brodsky, unpublished data), and of dynamin [13], correlate with those of proteins known to be involved in synaptic vesicle function. These data argue that neurons use more of the basic components, as well as specifically designed components, to enhance the performance of the endocytic coatedvesicle engine (see Fig. 1). A recent report [14] suggests that synaptic vesicle membrane components may actively participate in their'own rapid uptake. AP2 molecules bind with extremely high affinity (half-maximal binding at -4 x 10-10 M) to carbonate-stripped synaptic vesicles plated on microtitre wells. Binding apparently occurs through interactions with the cytoplasmic domain of synaptotagmin, a synaptic vesicle integral membrane protein believed to be involved in Ca2+-regulation of synaptic vesicle exocytosis [15]. These results led to the provocative suggestion that synaptic vesicles might contribute to their own rapid recycling by carrying with them an AP2 receptor that triggers coat assembly and endocytosis [14]. This mechanism might allow for direct recycling of individual functional synaptic vesicles, perhaps without an obligatory endosomal intermediate (Fig. 1). If, as suggested, synaptotagmin is the only protein on synaptic vesicles with which coat components interact, then efficient recycling of the entire complement of synaptic vesicle components would require that they directly or indirectly interact with synaptotagmin. Such complexes have been shown to exist [16,17], and coated vesicles isolated from the synapse contain all the membrane constituents of synaptic vesicles in their appropriate molar ratios [18]. While intriguing, there are several caveats to this report. First, the affinities for AP2 interaction with glutathioneS transferase (GST)-fusion proteins encoding the cytoplasmic domain of synaptotagmin in solution, as assessed by competition studies, were considerably lower (-,M)
© Current Biology 1995, Vol 5 No 2
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Current Biology 1995, Vol 5 No 2 Fig. 1. 'Revving-up' the endocytic coated engine The recycling of synaptic vesicle membrane components occurs within minutes, a rate more than 10-times faster than membrane recycling in nonneuronal cells. To accomplish these speeds, abundant, neuron-specific components of the clathrin-coated-vesicle machinery are used for endocytosis. Synaptic vesicles could also be regenerated directly from coated vesicles by using a short-cut that bypasses the recycling endosomal compartment.
than those measured using immobilized proteins (
fuse, or a reduced ability to regenerate synaptic vesicles through endocytosis. A recent, more detailed, phenotypic analysis of hippocampal neurons from synaptotagmindeficient gene 'knock-out' mice [23] has established synaptotagmin's role in Ca2+-dependent synaptic-vesicle exocytosis; however, an additional role for synaptotagmin in endocytosis is not ruled out by these results. Impaired synaptic function as a result of defective endocytosis is the most striking phenotype of shibire mutant fruitflies. In these flies, a mutation in the shibire gene, which encodes a dynamin homologue, causes rapid paralysis after a shift to the non-permissive temperature. Within minutes, synaptic vesicles are depleted from the neuromuscular junctions of affected flies, and both coated and uncoated budding structures accumulate at the synaptic membrane [24]. The rapidity of this response argues strongly that dynamin-dependent endocytosis is directly required for synaptic vesicle recycling. Mammals, but not Drosophila, carry a neuron-specific isoform of dynamin, referred to as dynamin-1, that is 79% identical to a ubiquitously expressed isoform, dynamin-2. The greatest sequence divergence between the two dynamin isoforms occurs in their -100 residue carboxyterminal proline-rich domains (PRDs) [9]. Dynamin GTPase activity can be regulated through interactions with this domain [25]. In dynamin-1, but not dynamin2, the PRD is also the site for regulation of dynamin GTPase activity by phosphorylation/dephosphorylation
DISPATCH
[9]. Phosphorylation of dynamin by protein kinase C stimulates its intrinsic GTPase activity, yet dynamin is rapidly dephosphorylated by calcineurin following depolarization, just when synaptic vesicle recycling is required. This has led to the suggestion that dephosphorylated dynamin is locked in an 'activated', GTP-bound conformation for endocytosis [9]. However, it is also possible that phosphorylated dynamin might be poised for rapid endocytosis in resting neurons, but unable to interact through its phosphorylated PRD with essential downstream partners. Rapid dephosphorylation of the PRD might serve to release dynamin for interaction with its partners to trigger endocytosis. Phosphorylation of proline-rich domains on other proteins is known to block their ability to interact with functional partners [26]. Interestingly, upon depolarization, dynamin dephosphorylation occurs in a reciprocal manner to phosphorylation of synapsin-1, a synaptic vesicle protein required for exocytosis. Phosphorylation within the carboxy-terminal PRD of synapsin-1 is believed to regulate its interaction with the actin cytoskeleton [27]. A third, 145 kD, PRDcontaining protein at the synapse was recently identified as another dephosphin and shown to closely co-localize with dynamin, suggesting that it too might contribute to rapid endocytosis following depolarization and synapticvesicle release 13]. Thus phosphorylation/dephosphorylation reactions might serve to rapidly engage both the exocytic and endocytic machinery. In summary, neuron-specific mechanisms appear to affect all of the steps regulating the rate of endocytosis in nonneuronal cells. More coat-protein components could allow for increases in coated pit numbers. High-affinity interactions with coat constituents may enhance the endocytosis of synaptic vesicle components, and these interactions may themselves trigger coated-pit assembly. Finally, neuron-specific, perhaps 'turbo-charged', components or adaptations of the clathrin-coated vesicle machinery could accelerate the process of coated vesicle formation. Whether these modifications are sufficient to account for rapid synaptic vesicle recycling, or whether an alternative vehicle must be employed, remains a matter of dispute. An understanding of the role of the neuron-specific features of the endocytic apparatus, and of the degree to which these function to 'rev-up' the clathrin-coatedvesicle engine, will require new model systems in which endocytosis can be studied in the context of neurotransmission. After all, you cannot measure the fastest lap time for a Ferrari unless you watch it on a racetrack. Acknovnledxemients: S.A. Morris is supported by a Senior Postdoctoral Fellowship from the American Cancer Society, California Division. S.L. Schmid is an Established Investigator of the American Heart Association. We thank our colleagues for helpful discussions. References 1. Mahaffey DT, Peeler JS, Brodsky FM, Anderson RGW: Clathrincoated pits contain an integral membrane protein that binds the AP2 subunit with high affinity. Biol Chem 1990, 265:16514-16520.
2. Keen JH: Clathrin and associated assembly and disassembly proteins. Annu Rev Cell Biol 1990, 16:49-68. 3. Trowbridge IS, Collawn F and, Hopkins CR: Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol 1993, 9:129-161. 4. Robinson MS: Adaptins. Trends Cell Biol 1992, 2:293-297. 5. Schmid SL: Coated-vesicle formation in vitro: conflicting results using different assays. Trends Cell Biol 1993, 3:145-148. 6. van der Bliek AM, Redelmeier TE, Damke H, Tisdale EJ,Meyerowitz EM, Schmid SL: Mutations in human dynamin block an intermediate stage in coated vesicle formation. Cell Biol 1993, 122:553-563. 7. Herskovits S, Burgess CC, Obar RA, Vallee RB: Effects of mutant rat dynamin on endocytosis. I Cell Biol 1993, 122:565-578. 8. Damke H, Baba T, Warnock DE, Schmid SL: Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 1994, 127:915-934. 9. Robinson PJ, Liu J-P, Powell, KA, Fykse EM, Sdhof TC: Phosphorylation of dynamin I and synaptic-vesicle recycling. Trends Neurosci 1994, 17:348-353. 10. Brodsky FM, Hill BL, Acton SL, Nathke I, Wong DH, Ponnambalam S, Parham P: Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics. Trends Biochem Sci 1991, 16:208-213. 11. Lindner R, Ungewickell E: Clathrin-associated proteins from bovine brain coated vesicles: an analysis of their number and assemblypromoting activity. J Biol Chem 1992, 267:16567-16573. 12. Ahle S, Ungewickell E: Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain. J Cell Biol 1990, 111:19-29. 13. McPherson PS, Takei K, Schmid SL, De Camilli P: p145, a major grb2-binding protein in brain, is colocalized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation. Biol Chem 1994, 269:30132-30139. 14. Zhang JZ, Davletov BA, Sudhof TC, Anderson RGW: Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 1994, 78:751-760. 15. DeBello WM, Betz H, Augustine GJ: Synaptotagmin and neurotransmitter release. Cell 1993, 74:947-950. 16. Bennett MK, Calakos N, Kreiner T, Scheller RH: Synaptic vesicle membrane proteins interact to form a multimeric complex. J Cell Bio 1992, 116:761 775. 17. Calakos N, Scheller RH: Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle. J Biol Chem 1994, 269:24534-24537. 18. Maycox PR, Link E, Reetz A, Morris SA, Jahn R: Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling. J Cell Biol 1992, 118:1379-1388. 19. Sorkin A, Carpenter G: Interaction of activated EGF receptors with coated pit adaptins. Science 1993, 261:612-615. 20. West MA, Bretsher MS, Watts C: Distinct endocytic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J Cell Biol 1989, 109:2731-2739. 21. Nonet ML, Grundahl K, Meyer BJ, Rand JB: Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell 1993, 73:1291-1305. 22. Broadie K, Bellen H, DiAntonio A, Littleton T, Schwarz TL: Absence of synaptotagmin disrupts excitation-secretion coupling during synaptic transmission. Proc Natl Acad Sci USA 1994, 91:10727-10731. 23. Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Sudhof TC: Synaptotagmin : A major Ca2+ sensor for transmitter release at a central synapse. Cell 1994, 79:717-727. 24. Koenig JH, Ikeda K: Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J Neurosci 1989, 11:3844-3860. 25. Herskovits S, Shpetner HS, Burgess CC, Vallee RB: Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain. Proc Natl Acad Sci USA 1993, 90: 11468-11472. 26. Williamson MP: The structure and function of proline-rich regions in proteins. Biochem J 1994, 297:249-260. 27. Greengard P Valtorta F, Czernik A, Benfenati F: Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 1993, 259:780-785.
Stephen A. Morris, School of Pharmacy, University of California, 513 Parnassus Ave., San Francisco, California 94143, USA. Sandra L. Schmid, Department of Cell Biology, The Scripps Research Institute, 10666 N. Torrey Pines Road, LaJolla, California 92037, USA.
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SYNAPTIC VESICLE RECYCLING
The Ferrari of endocytosis? After neurotransmission, neurons internalize and recycle the membrane components of synaptic vesicles remarkably quickly; they may have a 'rebuilt, turbo-charged' endocytic engine to achieve these speeds. Specialized regions of the cell surface known as clathrincoated pits mediate efficient endocytosis of membrane receptors and their ligands. For receptors free to diffuse along the cell surface, the rate of endocytosis depends on the number of coated pits, the ability of a given receptor to be recruited and concentrated into a coated pit, and the time taken for coated pit assembly and coated vesicle budding. For the transferrin receptor, considered a paradigm of efficiency, the half-time of internalization is 3-5 minutes in fibroblastic cells. By contrast, following neurotransmission, synaptic vesicle membranes are internalized within seconds, and the recycled synaptic vesicles can be reloaded with neurotransmitter within 1-2 minutes. Does this greatly enhanced speed necessarily suggest an alternative vehicle, or can rapid endocytosis at the synapse be accomplished by 'revving-up' the clathrincoated vesicle engine? Coated vesicle formation in fibroblastic cells is initiated by the recruitment of assembly, or adaptor, protein (AP2) complexes from the cytosol to saturable, high-affinity binding sites on the plasma membrane [1]. Clathrin is then recruited from the cytosol to the membrane-bound AP2 complexes, in a reaction that is presumably mediated by the AP2 molecules, which have been shown to be capable of self-aggregating and enhancing clathrin assembly in vitro [2]. The interaction of AP2 complexes with 'internalization signals' in the cytoplasmic domains of receptors is required for the receptors' concentration into coated pits [3,4]. Coated pits invaginate and eventually bud to form coated vesicles carrying receptor-ligand complexes into the cell. Reconstitution of these latter events in vitro requires ATP and GTP hydrolysis [5]. Recently, dynamin has been identified as one of the GTPases that regulate late stages in coated-vesicle formation [6-8]. How might endocytosis mediated by clathrin-coated vesicles be accelerated in neurons? The number of coated pits and the efficiency of their assembly could be enhanced simply by overexpressing coated pit constituents. In fact, all of the known components of the coated vesicle engine, including AP complexes, clathrin, dynamin and the uncoating ATPase (hsc70) are expressed at 10-50-fold higher levels in neuronal cells than in nonneuronal cells. In addition, neuron-specific, perhaps performance-enhancing, isoforms exist for most of the known components of the coated vesicle machine. Separate genes encode neuron-specific isoforms of the o subunit of AP2 complexes [4] and of dynamin [9]; and differential RNA splicing leaves neuron-specific inserts in
the adaptin subunit of AP2 complexes [4] and in both clathrin light chains [10]. 'Optional' equipment has also been added to the neuron's coated vesicle engine. For example, AP3 is a neuronspecific assembly protein which is approximately fourtimes more efficient at stimulating clathrin assembly than are other clathrin-binding proteins [11]. Auxilin is another neuron-specific clathrin-binding protein [12], the function of which is not yet known. Importantly, expression patterns in the rat brain of the neuron-specific forms of the clathrin light chain, the AP2 subunits and auxilin (S.A.M., E. Ungewickell and E Brodsky, unpublished data), and of dynamin [13], correlate with those of proteins known to be involved in synaptic vesicle function. These data argue that neurons use more of the basic components, as well as specifically designed components, to enhance the performance of the endocytic coatedvesicle engine (see Fig. 1). A recent report [14] suggests that synaptic vesicle membrane components may actively participate in their'own rapid uptake. AP2 molecules bind with extremely high affinity (half-maximal binding at -4 x 10-10 M) to carbonate-stripped synaptic vesicles plated on microtitre wells. Binding apparently occurs through interactions with the cytoplasmic domain of synaptotagmin, a synaptic vesicle integral membrane protein believed to be involved in Ca2+-regulation of synaptic vesicle exocytosis [15]. These results led to the provocative suggestion that synaptic vesicles might contribute to their own rapid recycling by carrying with them an AP2 receptor that triggers coat assembly and endocytosis [14]. This mechanism might allow for direct recycling of individual functional synaptic vesicles, perhaps without an obligatory endosomal intermediate (Fig. 1). If, as suggested, synaptotagmin is the only protein on synaptic vesicles with which coat components interact, then efficient recycling of the entire complement of synaptic vesicle components would require that they directly or indirectly interact with synaptotagmin. Such complexes have been shown to exist [16,17], and coated vesicles isolated from the synapse contain all the membrane constituents of synaptic vesicles in their appropriate molar ratios [18]. While intriguing, there are several caveats to this report. First, the affinities for AP2 interaction with glutathioneS transferase (GST)-fusion proteins encoding the cytoplasmic domain of synaptotagmin in solution, as assessed by competition studies, were considerably lower (-,M)
© Current Biology 1995, Vol 5 No 2
113
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Current Biology 1995, Vol 5 No 2 Fig. 1. 'Revving-up' the endocytic coated engine The recycling of synaptic vesicle membrane components occurs within minutes, a rate more than 10-times faster than membrane recycling in nonneuronal cells. To accomplish these speeds, abundant, neuron-specific components of the clathrin-coated-vesicle machinery are used for endocytosis. Synaptic vesicles could also be regenerated directly from coated vesicles by using a short-cut that bypasses the recycling endosomal compartment.
than those measured using immobilized proteins (
fuse, or a reduced ability to regenerate synaptic vesicles through endocytosis. A recent, more detailed, phenotypic analysis of hippocampal neurons from synaptotagmindeficient gene 'knock-out' mice [23] has established synaptotagmin's role in Ca2+-dependent synaptic-vesicle exocytosis; however, an additional role for synaptotagmin in endocytosis is not ruled out by these results. Impaired synaptic function as a result of defective endocytosis is the most striking phenotype of shibire mutant fruitflies. In these flies, a mutation in the shibire gene, which encodes a dynamin homologue, causes rapid paralysis after a shift to the non-permissive temperature. Within minutes, synaptic vesicles are depleted from the neuromuscular junctions of affected flies, and both coated and uncoated budding structures accumulate at the synaptic membrane [24]. The rapidity of this response argues strongly that dynamin-dependent endocytosis is directly required for synaptic vesicle recycling. Mammals, but not Drosophila, carry a neuron-specific isoform of dynamin, referred to as dynamin-1, that is 79% identical to a ubiquitously expressed isoform, dynamin-2. The greatest sequence divergence between the two dynamin isoforms occurs in their -100 residue carboxyterminal proline-rich domains (PRDs) [9]. Dynamin GTPase activity can be regulated through interactions with this domain [25]. In dynamin-1, but not dynamin2, the PRD is also the site for regulation of dynamin GTPase activity by phosphorylation/dephosphorylation
DISPATCH
[9]. Phosphorylation of dynamin by protein kinase C stimulates its intrinsic GTPase activity, yet dynamin is rapidly dephosphorylated by calcineurin following depolarization, just when synaptic vesicle recycling is required. This has led to the suggestion that dephosphorylated dynamin is locked in an 'activated', GTP-bound conformation for endocytosis [9]. However, it is also possible that phosphorylated dynamin might be poised for rapid endocytosis in resting neurons, but unable to interact through its phosphorylated PRD with essential downstream partners. Rapid dephosphorylation of the PRD might serve to release dynamin for interaction with its partners to trigger endocytosis. Phosphorylation of proline-rich domains on other proteins is known to block their ability to interact with functional partners [26]. Interestingly, upon depolarization, dynamin dephosphorylation occurs in a reciprocal manner to phosphorylation of synapsin-1, a synaptic vesicle protein required for exocytosis. Phosphorylation within the carboxy-terminal PRD of synapsin-1 is believed to regulate its interaction with the actin cytoskeleton [27]. A third, 145 kD, PRDcontaining protein at the synapse was recently identified as another dephosphin and shown to closely co-localize with dynamin, suggesting that it too might contribute to rapid endocytosis following depolarization and synapticvesicle release 13]. Thus phosphorylation/dephosphorylation reactions might serve to rapidly engage both the exocytic and endocytic machinery. In summary, neuron-specific mechanisms appear to affect all of the steps regulating the rate of endocytosis in nonneuronal cells. More coat-protein components could allow for increases in coated pit numbers. High-affinity interactions with coat constituents may enhance the endocytosis of synaptic vesicle components, and these interactions may themselves trigger coated-pit assembly. Finally, neuron-specific, perhaps 'turbo-charged', components or adaptations of the clathrin-coated vesicle machinery could accelerate the process of coated vesicle formation. Whether these modifications are sufficient to account for rapid synaptic vesicle recycling, or whether an alternative vehicle must be employed, remains a matter of dispute. An understanding of the role of the neuron-specific features of the endocytic apparatus, and of the degree to which these function to 'rev-up' the clathrin-coatedvesicle engine, will require new model systems in which endocytosis can be studied in the context of neurotransmission. After all, you cannot measure the fastest lap time for a Ferrari unless you watch it on a racetrack. Acknovnledxemients: S.A. Morris is supported by a Senior Postdoctoral Fellowship from the American Cancer Society, California Division. S.L. Schmid is an Established Investigator of the American Heart Association. We thank our colleagues for helpful discussions. References 1. Mahaffey DT, Peeler JS, Brodsky FM, Anderson RGW: Clathrincoated pits contain an integral membrane protein that binds the AP2 subunit with high affinity. Biol Chem 1990, 265:16514-16520.
2. Keen JH: Clathrin and associated assembly and disassembly proteins. Annu Rev Cell Biol 1990, 16:49-68. 3. Trowbridge IS, Collawn F and, Hopkins CR: Signal-dependent membrane protein trafficking in the endocytic pathway. Annu Rev Cell Biol 1993, 9:129-161. 4. Robinson MS: Adaptins. Trends Cell Biol 1992, 2:293-297. 5. Schmid SL: Coated-vesicle formation in vitro: conflicting results using different assays. Trends Cell Biol 1993, 3:145-148. 6. van der Bliek AM, Redelmeier TE, Damke H, Tisdale EJ,Meyerowitz EM, Schmid SL: Mutations in human dynamin block an intermediate stage in coated vesicle formation. Cell Biol 1993, 122:553-563. 7. Herskovits S, Burgess CC, Obar RA, Vallee RB: Effects of mutant rat dynamin on endocytosis. I Cell Biol 1993, 122:565-578. 8. Damke H, Baba T, Warnock DE, Schmid SL: Induction of mutant dynamin specifically blocks endocytic coated vesicle formation. J Cell Biol 1994, 127:915-934. 9. Robinson PJ, Liu J-P, Powell, KA, Fykse EM, Sdhof TC: Phosphorylation of dynamin I and synaptic-vesicle recycling. Trends Neurosci 1994, 17:348-353. 10. Brodsky FM, Hill BL, Acton SL, Nathke I, Wong DH, Ponnambalam S, Parham P: Clathrin light chains: arrays of protein motifs that regulate coated-vesicle dynamics. Trends Biochem Sci 1991, 16:208-213. 11. Lindner R, Ungewickell E: Clathrin-associated proteins from bovine brain coated vesicles: an analysis of their number and assemblypromoting activity. J Biol Chem 1992, 267:16567-16573. 12. Ahle S, Ungewickell E: Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain. J Cell Biol 1990, 111:19-29. 13. McPherson PS, Takei K, Schmid SL, De Camilli P: p145, a major grb2-binding protein in brain, is colocalized with dynamin in nerve terminals where it undergoes activity-dependent dephosphorylation. Biol Chem 1994, 269:30132-30139. 14. Zhang JZ, Davletov BA, Sudhof TC, Anderson RGW: Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell 1994, 78:751-760. 15. DeBello WM, Betz H, Augustine GJ: Synaptotagmin and neurotransmitter release. Cell 1993, 74:947-950. 16. Bennett MK, Calakos N, Kreiner T, Scheller RH: Synaptic vesicle membrane proteins interact to form a multimeric complex. J Cell Bio 1992, 116:761 775. 17. Calakos N, Scheller RH: Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle. J Biol Chem 1994, 269:24534-24537. 18. Maycox PR, Link E, Reetz A, Morris SA, Jahn R: Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling. J Cell Biol 1992, 118:1379-1388. 19. Sorkin A, Carpenter G: Interaction of activated EGF receptors with coated pit adaptins. Science 1993, 261:612-615. 20. West MA, Bretsher MS, Watts C: Distinct endocytic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J Cell Biol 1989, 109:2731-2739. 21. Nonet ML, Grundahl K, Meyer BJ, Rand JB: Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin. Cell 1993, 73:1291-1305. 22. Broadie K, Bellen H, DiAntonio A, Littleton T, Schwarz TL: Absence of synaptotagmin disrupts excitation-secretion coupling during synaptic transmission. Proc Natl Acad Sci USA 1994, 91:10727-10731. 23. Geppert M, Goda Y, Hammer RE, Li C, Rosahl TW, Stevens CF, Sudhof TC: Synaptotagmin : A major Ca2+ sensor for transmitter release at a central synapse. Cell 1994, 79:717-727. 24. Koenig JH, Ikeda K: Disappearance and reformation of synaptic vesicle membrane upon transmitter release observed under reversible blockage of membrane retrieval. J Neurosci 1989, 11:3844-3860. 25. Herskovits S, Shpetner HS, Burgess CC, Vallee RB: Microtubules and Src homology 3 domains stimulate the dynamin GTPase via its C-terminal domain. Proc Natl Acad Sci USA 1993, 90: 11468-11472. 26. Williamson MP: The structure and function of proline-rich regions in proteins. Biochem J 1994, 297:249-260. 27. Greengard P Valtorta F, Czernik A, Benfenati F: Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 1993, 259:780-785.
Stephen A. Morris, School of Pharmacy, University of California, 513 Parnassus Ave., San Francisco, California 94143, USA. Sandra L. Schmid, Department of Cell Biology, The Scripps Research Institute, 10666 N. Torrey Pines Road, LaJolla, California 92037, USA.
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