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.

Regulation of synaptic vesicle acidification at the neuronal synapse

“…The combined use of an optimized purification protocol and the single vesicle assay allows for a highly sensitive, qualitative, and quantitative investigation of the acidification process. This study revealed that the clathrin coat on the endocytosed vesicle blocks the vATPase activity, while the uncoating of the same vesicle releases this inhibition . Therefore, vesicle coating acts as a checkpoint between the retrieval step and acidification/neurotransmitter refilling.…”

“…In an attempt to address this, Farsi et al (2018) optimized a protocol (adapted from Reference ) to isolate CCVs from mouse brains, and used this sample in a single vesicle assay designed to measure both components of electrochemical gradient, ΔpH and Δψ. The combined use of an optimized purification protocol and the single vesicle assay allows for a highly sensitive, qualitative, and quantitative investigation of the acidification process.…”

“…Therefore, vesicle coating acts as a checkpoint between the retrieval step and acidification/neurotransmitter refilling. Using this information, together with the structures of clathrin coat and of vATPase, a model was proposed in which clathrin coat formation around the vesicle inhibits vATPase activity by displacing the V 1 H subunit to accommodate vATPase complex into the hexagons of CCV lattices . Imaging of single vesicles derived from endocytic mutants will be crucial to understand the role of endocytic factors in the vATPase function and to clarify whether the clathrin alone is sufficient to block the pump, or other accessory factors are involved in this process, as well as when exactly the regulation by the coat is released in the endocytic pathway.…”

“…68 Furthermore, using a short acid pulse application following a series of action potential in hippocampal neurons, the reacidification kinetics was suggested to be~4-5 s. 69 Curiously, the time taken for endocytic retrieval was suggested to be~4 s. 70 In any case, while some reports suggested that endocytosed vesicles do not acidify while the clathrin coat is present around them, 71,72 others showed that acidification occurs in these vesicles. 41,73 In an attempt to address this, Farsi et al (2018) optimized a protocol (adapted from Reference 39) to isolate CCVs from mouse brains, 46 and used this sample in a single vesicle assay designed to measure both components of electrochemical gradient, ΔpH and Δψ. The combined use of an optimized purification protocol and the single vesicle assay allows for a highly sensitive, qualitative, and quantitative investigation of the acidification process.…”

“…This study revealed that the clathrin coat on the endocytosed vesicle blocks the vATPase activity, while the uncoating of the same vesicle releases this inhibition. 46 Therefore, vesicle coating acts as a checkpoint between the retrieval step and acidification/neurotransmitter refilling. Using this information, together with the structures of clathrin coat and of vATPase, a model was proposed in which clathrin coat formation around the vesicle inhibits vATPase activity by displacing the V 1 H subunit to accommodate vATPase complex into the hexagons of CCV lattices.…”