Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals Roles for Hsc70 and Auxilin

We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxilin prevented binding to Hsc70 in vitro and injecting this mutant protein inhibited CCV uncoating in vivo, demonstrating that the interaction of auxilin with Hsc70 is critical for CCV uncoating. These studies establish that auxilin and Hsc70 participate in synaptic vesicle recycling in neurons and that an interaction between these proteins is required for CCV uncoating.

[1]  R. Scheller,et al.  The molecular machinery for secretion is conserved from yeast to neurons. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[2]  P. De Camilli,et al.  The synaptic vesicle cycle: a single vesicle budding step involving clathrin and dynamin , 1996, The Journal of cell biology.

[3]  E. Ungewickell,et al.  Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation. , 2000, European journal of cell biology.

[4]  S. Bohlander,et al.  Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic. , 1999, Molecular biology of the cell.

[5]  P. Hanson,et al.  Neurotransmitter release — four years of SNARE complexes , 1997, Current Opinion in Neurobiology.

[6]  G. Augustine,et al.  A Role for the Clathrin Assembly Domain of AP180 in Synaptic Vesicle Endocytosis , 1999, The Journal of Neuroscience.

[7]  S. Schmid,et al.  Dominant-Interfering Hsc70 Mutants Disrupt Multiple Stages of the Clathrin-Coated Vesicle Cycle in Vivo , 2001, The Journal of cell biology.

[8]  G. Payne,et al.  Clathrin Coats— Threads Laid Bare , 1998, Cell.

[9]  E. Eisenberg,et al.  A protein cofactor is required for uncoating of clathrin baskets by uncoating ATPase. , 1993, The Journal of biological chemistry.

[10]  Walter E. Gall,et al.  The auxilin-like phosphoprotein Swa2p is required for clathrin function in yeast , 2000, Current Biology.

[11]  P. Maycox,et al.  Clathrin-coated vesicles in nervous tissue are involved primarily in synaptic vesicle recycling , 1992, The Journal of cell biology.

[12]  M. Douglas,et al.  A Conserved HPD Sequence of the J-domain Is Necessary for YDJ1 Stimulation of Hsp70 ATPase Activity at a Site Distinct from Substrate Binding (*) , 1996, The Journal of Biological Chemistry.

[13]  P. Bronk,et al.  Drosophila Hsc70-4 Is Critical for Neurotransmitter Exocytosis In Vivo , 2001, Neuron.

[14]  H. Nojima,et al.  GAK: a cyclin G associated kinase contains a tensin/auxilin‐like domain 1 , 1997, FEBS letters.

[15]  E. Eisenberg,et al.  Expression of auxilin or AP180 inhibits endocytosis by mislocalizing clathrin: evidence for formation of nascent pits containing AP1 or AP2 but not clathrin. , 2001, Journal of cell science.

[16]  S. Schmid,et al.  ATP- and cytosol-dependent release of adaptor proteins from clathrin-coated vesicles: A dual role for Hsc70. , 1998, Molecular biology of the cell.

[17]  T. A. Ryan,et al.  Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system , 2000, Nature Cell Biology.

[18]  S. Landry,et al.  Role of the J-domain in the cooperation of Hsp40 with Hsp70. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[19]  M. Mayer,et al.  Hsp70 chaperone systems: diversity of cellular functions and mechanism of action. , 1998, Biological chemistry.

[20]  R. Burgoyne,et al.  Mutational analysis of cysteine-string protein function in insulin exocytosis. , 1999, Journal of cell science.

[21]  J. Rothman,et al.  An enzyme that removes clathrin coats: purification of an uncoating ATPase , 1984, The Journal of cell biology.

[22]  T. Sasaki,et al.  Rabphilin-3A: A Multifunctional Regulator of Synaptic Vesicle Traffic , 1998, The Journal of general physiology.

[23]  E. Eisenberg,et al.  Effect of Nucleotide on the Binding of Peptides to 70-kDa Heat Shock Protein (*) , 1995, The Journal of Biological Chemistry.

[24]  E. Eisenberg,et al.  ATPase activity associated with the uncoating of clathrin baskets by Hsp70. , 1994, The Journal of biological chemistry.

[25]  Y. Endo,et al.  Structure, expression, and chromosomal localization of human GAK. , 1997, Genomics.

[26]  G. Prestwich,et al.  Regulation of AP-3 Function by Inositides , 1997, The Journal of Biological Chemistry.

[27]  E. Eisenberg,et al.  Role of auxilin in uncoating clathrin-coated vesicles , 1995, Nature.

[28]  E. Margoliash,et al.  A peptide binding protein having a role in antigen presentation is a member of the HSP70 heat shock family , 1989, The Journal of experimental medicine.

[29]  E. Margoliash,et al.  Identification of a peptide binding protein that plays a role in antigen presentation. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[30]  H. Jäckle,et al.  Role of Drosophila alpha-adaptin in presynaptic vesicle recycling. , 1997, Cell.

[31]  C. M. Nuno,et al.  Contractions of the squid stellate ganglion. , 1990, The Journal of experimental biology.

[32]  R. Burgoyne,et al.  The Molecular Chaperone Function of the Secretory Vesicle Cysteine String Proteins* , 1997, The Journal of Biological Chemistry.

[33]  G. Payne,et al.  A yeast DNA J protein required for uncoating of clathrin-coated vesicles in vivo , 2000, Nature Cell Biology.

[34]  J. Rothman,et al.  Mechanisms of intracellular protein transport , 1994, Nature.

[35]  P. Camilli,et al.  Accessory factors in clathrin-dependent synaptic vesicle endocytosis , 2000, Nature Reviews Neuroscience.

[36]  E. Ungewickell,et al.  Mechanism of clathrin basket dissociation: separate functions of protein domains of the DnaJ homologue auxilin , 1996, The Journal of cell biology.

[37]  D. McCormick,et al.  Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling , 1999, Cell.

[38]  T. Reese,et al.  EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION , 1973, The Journal of cell biology.

[39]  E. Eisenberg,et al.  Role of Cyclin G-associated Kinase in Uncoating Clathrin-coated Vesicles from Non-neuronal Cells* , 2000, The Journal of Biological Chemistry.

[40]  E. Eisenberg,et al.  Dissociation of clathrin from coated vesicles by the uncoating ATPase. , 1990, The Journal of biological chemistry.

[41]  H. Bellen,et al.  Synaptic Vesicle Size and Number Are Regulated by a Clathrin Adaptor Protein Required for Endocytosis , 1998, Neuron.

[42]  C. Gross,et al.  Interaction of the Hsp70 molecular chaperone, DnaK, with its cochaperone DnaJ. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[43]  G. Augustine,et al.  A functional role for GTP-binding proteins in synaptic vesicle cycling. , 1993, Science.

[44]  E. Ungewickell,et al.  Auxilin, a newly identified clathrin-associated protein in coated vesicles from bovine brain , 1990, The Journal of cell biology.

[45]  C. Georgopoulos,et al.  The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication. , 1994, The Journal of biological chemistry.

[46]  G. Augustine,et al.  A Conserved Clathrin Assembly Motif Essential for Synaptic Vesicle Endocytosis , 2000, The Journal of Neuroscience.

[47]  W. Kelley,et al.  The J-domain family and the recruitment of chaperone power. , 1998, Trends in biochemical sciences.

[48]  P. Evans,et al.  A Structural Explanation for the Binding of Multiple Ligands by the α-Adaptin Appendage Domain , 1999, Cell.

[49]  W. Betz,et al.  Nerve Activity but Not Intracellular Calcium Determines the Time Course of Endocytosis at the Frog Neuromuscular Junction , 1996, Neuron.

[50]  E. Eisenberg,et al.  Caenorhabditis elegans auxilin: a J-domain protein essential for clathrin-mediated endocytosis in vivo , 2001, Nature Cell Biology.

[51]  S. Lemmon.  Clathrin uncoating: Auxilin comes to life , 2001, Current Biology.

[52]  P. Camilli,et al.  Synaptic vesicle endocytosis , 1997, Current Opinion in Neurobiology.

[53]  E. Eisenberg,et al.  Interaction of Auxilin with the Molecular Chaperone, Hsc70* , 1997, The Journal of Biological Chemistry.