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.
Highlights
Neuronal synapses are capable of regenerating SVs locally with high efficiency and fidelity in order to meet the demands of neuronal activity
Detailed analysis of clathrin-coated vesicles (CCVs) samples by mass spectrometry revealed that the large majority of CCVs seems to be derived from the synapses, yet some CCV come from neuronal cell body/intracellular membranes
Since, according to our knowledge, there is no evidence of a specific protein-protein interaction that blocks the vATPase activity at the newly formed vesicles at the synapse in the presence of clathrin or adaptor proteins, we propose that the vATPase may be sterically hindered by the three-dimensional clathrin scaffold that includes both clathrin triskelia and adaptor proteins (e.g. AP2, AP180, etc., adaptor proteins are essential to recruit clathrin triskelia to the membrane in order to initiate and organize the formation of clathrin coats)
Summary
Neuronal synapses are capable of regenerating SVs locally with high efficiency and fidelity in order to meet the demands of neuronal activity. Clathrin-mediated endocytosis is a classic example of vesicle formation mediated by a coat assembly, and it occurs at the synapse (Saheki and De Camilli, 2012; Milosevic, 2018a). It has been intensely studied for over four decades, yet numerous details, including when the endocytosed vesicle initiates acidification, remain unclear. Acidification of endocytic compartments is primarily mediated by vATPases. VATPases are well studied at the synapse, where they traffic with other SV proteins through the SV cycle and generate a proton electrochemical gradient (DmH+) across the vesicular membrane, fueling the reloading of SVs with neurotransmitters. To weigh in on this debate, and to investigate whether vATPase is active on CCVs, we have performed a full characterization of the DmH+ at the single CCV level
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