Desnos C, Clift-O'Grady L, Kelly R B
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0534, USA.
J Cell Biol. 1995 Sep;130(5):1041-9. doi: 10.1083/jcb.130.5.1041.
Synaptic vesicles are synthesized at a rapid rate in nerve terminals to compensate for their rapid loss during neurotransmitter release. Their biogenesis involves endocytosis of synaptic vesicle membrane proteins from the plasma membrane and requires two steps, the segregation of synaptic vesicle membrane proteins from other cellular proteins, and the packaging of those unique proteins into vesicles of the correct size. By labeling an epitope-tagged variant of a synaptic vesicle protein, VAMP (synaptobrevin), at the cell surface of the neuroendocrine cell line PC12, synaptic vesicle biogenesis could be followed with considerable precision, quantitatively and kinetically. Epitope-tagged VAMP was recovered in synaptic vesicles within a few minutes of leaving the cell surface. More efficient targeting was obtained by using the VAMP mutant, del 61-70. Synaptic vesicles did not form at 15 degrees C although endocytosis still occurred. Synaptic vesicles could be generated in vitro from a homogenate of cells labeled at 15 degrees C. The newly formed vesicles are identical to those formed in vivo in their sedimentation characteristics, the presence of the synaptic vesicle protein synaptophysin, and the absence of detectable transferrin receptor. Brain, but not fibroblast cytosol, allows vesicles of the correct size to form. Vesicle formation is time and temperature-dependent, requires ATP, is calcium independent, and is inhibited by GTP-gamma S. Thus, two key steps in synaptic vesicle biogenesis have been reconstituted in vitro, allowing direct analysis of the proteins involved.
突触小泡在神经末梢以快速的速率合成，以补偿其在神经递质释放过程中的快速损耗。它们的生物发生涉及突触小泡膜蛋白从质膜的内吞作用，并且需要两个步骤，即突触小泡膜蛋白与其他细胞蛋白的分离，以及将那些独特的蛋白包装到正确大小的小泡中。通过在神经内分泌细胞系PC12的细胞表面标记突触小泡蛋白VAMP（突触融合蛋白）的表位标签变体，可以在数量和动力学方面相当精确地追踪突触小泡的生物发生。表位标签化的VAMP在离开细胞表面几分钟内就在突触小泡中被回收。通过使用VAMP突变体del 61-70可获得更有效的靶向。尽管内吞作用仍在发生，但在15℃时突触小泡并未形成。突触小泡可以由在15℃标记的细胞匀浆在体外产生。新形成的小泡在沉降特性、突触小泡蛋白突触素的存在以及未检测到转铁蛋白受体方面与体内形成的小泡相同。脑匀浆而非成纤维细胞胞质溶胶能使正确大小的小泡形成。小泡形成是时间和温度依赖性的，需要ATP，不依赖钙，并且被GTP-γS抑制。因此，突触小泡生物发生中的两个关键步骤已在体外重建，从而可以直接分析其中涉及的蛋白质。
