Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Berlin Institute for Medical Systems Biology, Max Delbrück Center for Molecular Medicine, Berlin, Germany.
Elife. 2018 Apr 13;7:e32569. doi: 10.7554/eLife.32569.
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.
新形成的突触小泡 (SVs) 被液泡三磷酸腺苷酶 (vATPases) 迅速酸化，产生质子电化学梯度，从而驱动神经递质的装载。网格蛋白介导的内吞作用是形成新的 SVs 所必需的，但尚不清楚内吞的囊泡何时在突触酸化和再填充。在这里，我们从老鼠大脑中分离出网格蛋白包被的囊泡 (CCVs)，以便在单个囊泡水平上直接测量它们的酸化。我们观察到，与 SVs 相比，ATP 诱导的 CCVs 酸化显著降低。值得注意的是，当从 CCVs 上去除涂层时，无涂层的囊泡恢复了 ATP 依赖性酸化，这表明 CCVs 含有功能性 vATPase，但它的功能被网格蛋白涂层抑制。考虑到 vATPase 和网格蛋白涂层的已知结构，我们提出了一个模型，其中涂层的形成包围了 vATPase 并抑制了它的活性。这种抑制对于 SV 再填充的适当时间可能是基础。
