@article{4807d0e25a5f47f98cf2203e9248d052,
title = "Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATpase activity",
abstract = "Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.",
author = "Zohreh Farsi and Sindhuja Gowrisankaran and Matija Krunic and Burkhard Rammner and Andrew Woehler and Lafer, {Eileen M.} and Carsten Mim and Reinhard Jahn and Ira Milosevic",
note = "Funding Information: The cryo-EM data were collected at the Cryo-EM Swedish National Facility funded by the Knut and Alice Wallenberg Family, Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University and Ume{\aa} University. The structure has been deposited with the EMDB-ID: 4335. We thank R Fer-nandez-Busnadiego (MPI for Biochemisty, M{\"u}nchen), D Riedel (EM Facility, MPI for Biophysical Chemistry, G{\"o}ttingen) and W M{\"o}bius (EM Facility, MPI for Experimental Medicine, G{\"o}ttingen) for their expert help and advice regarding the EM data, and J Rubinstein (Hospital for Sick Children, Toronto) for the discussions on the vATPase structure. We thank MPIbpc and ENI animal facilities, B Barg-Kues and M Costa for genotyping, K-W Li (University of Amsterdam) and CK Frese (Proteomics Core Facility, University of Cologne, CECAD Research Center) for the help with the MS, and N Rai-mundo for the help with proteomics. This work was supported by Schram-Stiftung T287/25457 and Deutsche Forschungsgemeinschaft (Emmy Noether Young Investigator Award MI-1702/1 and SFB889/A8) to IM, Human Frontier Science Program (Young Investigator Grant RGY0074/16) to CM, Peter-und-Traudl-Engelhorn fellowship to ZF, SySy fellowship to SG, and the NIH Grant GM118933 to EML. Conceptualization IM and RJ; Investigation and/or Analysis ZF, SG, MK, AW, CM, EML and IM; Model BR, SG and IM; Writing ZF and IM, all coauthors contributed to the final ms. The authors declare no competing financial interests. Funding Information: The cryo-EM data were collected at the Cryo-EM Swedish National Facility funded by the Knut and Alice Wallenberg Family, Erling Persson and Kempe Foundations, SciLifeLab, Stockholm University and Ume{\aa} University. The structure has been deposited with the EMDB-ID: 4335. We thank R Fernandez-Busnadiego (MPI for Biochemisty, M{\"u}nchen), D Riedel (EM Facility, MPI for Biophysical Chemistry, G{\"o}ttingen) and W M{\"o}bius (EM Facility, MPI for Experimental Medicine, G{\"o}ttingen) for their expert help and advice regarding the regarding the EM data, and J Rubinstein (Hospital for Sick Children, Toronto) for the discussions on the vATPase structure. We thank MPIbpc and ENI animal facilities, B Barg-Kues and M Costa for genotyping, K-W Li (University of Amsterdam) and CK Frese (Proteomics Core Facility, University of Cologne, CECAD Research Center) for the help with the MS, and N Rai mundo for the help with proteomics. This work was supported by Schram-Stiftung T287/25457 and Deutsche Forschungsgemeinschaft (Emmy Noether Young Investigator Award MI-1702/1 and SFB889/A8) to IM, Human Frontier Science Program (Young Investigator Grant RGY0074/16) to CM, Peter-und-Traudl-Engelhorn fellowship to ZF, SySy fellowship to SG, and the NIH Grant GM118933 to EML. Conceptualization IM and RJ; Investigation and/or Analysis ZF, SG, MK, AW, CM, EML and IM; Model BR, SG and IM; Writing ZF and IM, all coauthors contributed to the final ms. The authors declare no competing financial interests. Engelhorn Stiftung Postdoc fellowship Zohreh Farsi Synaptic System PhD fellowship Sindhuja Gowrisankaran National Institutes of Health GM118933 Eileen M Lafer Human Frontier Science Program Young Investigator Grant RGY0074/16 Carsten Mim Deutsche Forschungsgemeinschaft Emmy Noether Young Investigator Award MI-1702/ 1 Ira Milosevic Schram Stiftung T287/25457 Ira Milosevic Deutsche Forschungsgemeinschaft SFB88/A8 Ira Milosevic. Publisher Copyright: {\textcopyright} Farsi et al.",
year = "2018",
month = apr,
day = "13",
doi = "10.7554/eLife.32569",
language = "English (US)",
volume = "7",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications",
}