Dataset Information

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

ABSTRACT: Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recapture SVs after exocytosis. A ratio of vesicle-N-ethylmaleimide-sensitive factor attachment protein receptors to target-N-ethylmaleimide-sensitive factor attachment protein receptors, similar to that previously detected on SVs, supports a single-step model for SV sorting during CCV-mediated recycling of SVs. The uncovering of eight previously undescribed proteins, four of which have to date been linked to clathrin-mediated trafficking, further attests to the value of the current organelle-based proteomics strategy.

SUBMITTER: Blondeau F

PROVIDER: S-EPMC374330 | biostudies-literature | 2004 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Blondeau Francois F Ritter Brigitte B Allaire Patrick D PD Wasiak Sylwia S Girard Martine M Hussain Natasha K NK Angers Annie A Legendre-Guillemin Valerie V Roy Line L Boismenu Daniel D Kearney Robert E RE Bell Alexander W AW Bergeron John J M JJ McPherson Peter S PS

Proceedings of the National Academy of Sciences of the United States of America 20040308 11

Tandem MS has identified 209 proteins of clathrin-coated vesicles (CCVs) isolated from rat brain. An overwhelming abundance of peptides were assigned to the clathrin coat with a 1:1 stoichiometry observed for clathrin heavy and light chains and a 2:1 stoichiometry of clathrin heavy chain with clathrin adaptor protein heterotetramers. Thirty-two proteins representing many of the known components of synaptic vesicles (SVs) were identified, supporting that a main function for brain CCVs is to recap ...[more]

PMID: 15007177

Similar Datasets

Project description:Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles-LVs). Many proteins contribute to SV recycling, e.g., endophilin, synaptojanin, dynamin and clathrin, while the site of action of these proteins (at the plasma membrane (PM) vs. at the endosomal membrane) is only partially understood. Here, we investigated the roles of endophilin A (UNC-57), endophilin-related protein (ERP-1, homologous to human endophilin B1) and of clathrin, in SV recycling at the cholinergic neuromuscular junction (NMJ) of C. elegans. erp-1 mutants exhibited reduced transmission and a progressive reduction in optogenetically evoked muscle contraction, indicative of impaired SV recycling. This was confirmed by electrophysiology, where particularly endophilin A (UNC-57), but also endophilin B (ERP-1) mutants exhibited reduced transmission. By optogenetic and electrophysiological analysis, phenotypes in the unc-57; erp-1 double mutant are largely dominated by the unc-57 mutation, arguing for partially redundant functions of endophilins A and B, but also hinting at a back-up mechanism for neuronal endocytosis. By electron microscopy (EM), we observed that unc-57 and erp-1; unc-57 double mutants showed increased numbers of synaptic endosomes of large size, assigning a role for both proteins at the endosome, because endosomal disintegration into new SVs, but not formation of endosomes were hampered. Accordingly, only low amounts of SVs were present. Also erp-1 mutants show reduced SV numbers (but no increase in LVs), thus ERP-1 contributes to SV formation. We analyzed temperature-sensitive mutants of clathrin heavy chain (chc-1), as well as erp-1; chc-1 and unc-57; chc-1 double mutants. SV recycling phenotypes were obvious from optogenetic stimulation experiments. By EM, chc-1 mutants showed formation of numerous and large endosomes, arguing that clathrin, as shown for mammalian synapses, acts at the endosome in formation of new SVs. Without endophilins, clathrin formed endosomes at the PM, while endophilins A and B compensated for the loss of clathrin at the PM, under conditions of high SV turnover.

Dataset Information

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Publications

Tandem MS analysis of brain clathrin-coated vesicles reveals their critical involvement in synaptic vesicle recycling.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets