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.
- Ira Milosevic
- Carsten Mim
- Ira Milosevic
- Zohreh Farsi
- Sindhuja Gowrisankaran
- Eileen M Lafer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Animal experiments were conducted according to the European Guidelines for animal welfare (2010/63/EU) with approval by the Lower Saxony Landesamt fur Verbraucherschutz und Lebensmittelsicherheit (LAVES), registration number 14/1701.
- Margaret S. Robinson, Reviewing Editor, University of Cambridge, United Kingdom
- Received: October 6, 2017
- Accepted: April 7, 2018
- Accepted Manuscript published: April 13, 2018 (version 1)
© 2018, Farsi et al.
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