Journal: Elife / Year: 2018 Title: Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity. Authors: Zohreh Farsi / Sindhuja Gowrisankaran / Matija Krunic / Burkhard Rammner / Andrew Woehler / Eileen M Lafer / Carsten Mim / Reinhard Jahn / Ira Milosevic / Abstract: Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. ...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.
History
Deposition
Mar 14, 2018
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Header (metadata) release
Apr 25, 2018
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Map release
Apr 25, 2018
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Update
Apr 25, 2018
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Current status
Apr 25, 2018
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Model: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV
Details
Clathrin coated vesicles isolated from mouse brains.
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Image recording
Film or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 2 / Number real images: 2836 / Average exposure time: 2.5 sec. / Average electron dose: 23.8 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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