Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes
Reconstitution of synaptic vesicle biogenesis from PC12 cell membranes
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DOI：
10.1006/meth.1998.0662
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发表时间：
1998-10-01
期刊：
影响因子：
--
通讯作者：
Kelly, RB
中科院分区：
文献类型：
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作者：
Clift-O'Grady, L;Desnos, C;Kelly, RB
神经内分泌PC 12细胞含有小微泡，其物理和化学性质与突触囊泡非常相似。突触囊泡的两个决定性特征是它们均匀的大小和它们独特的蛋白质组成。由于突触囊泡是由质膜的内吞作用产生的，因此神经末梢和PC 12细胞必须包含分子机制，以将突触囊泡从其他膜蛋白中分选出来，并从前体隔室中夹出正确直径的囊泡。开发了无细胞重建系统，其在ATP和脑胞质溶胶的存在下从PC 12膜前体产生囊泡，并且是温度依赖性的。在15摄氏度时，表面标记的突触囊泡蛋白在供体隔室中积累，而标记的突触囊泡不能被检测到。在15摄氏度下突触囊泡形成的阻断使得能够使用单克隆抗体KT 3（表位标记的突触囊泡蛋白VAMP-TAg的特异性标记物）来标记突触囊泡生物发生途径中的前体。从膜标记在体内在15摄氏度，在体外产生的囊泡在37摄氏度的甘油速度梯度上的神经内分泌突触囊泡的沉降特性，并排除转铁蛋白受体。因此，可以在这种无细胞系统中研究囊泡形成和分选。(C)北京：科学出版社.
Neuroendocrine PC12 cells contain small microvesicles that closely resemble synaptic vesicles in their physical and chemical properties. Two defining characteristics of synaptic vesicles are their homogeneous size and their unique protein composition. Since synaptic vesicles arise by endocytosis from the plasma membrane, nerve terminals and PC12 cells must contain the molecular machinery to sort synaptic vesicles from other membrane proteins and pinch off vesicles of the correct diameter from a precursor compartment. A cell-free reconstitution system was developed that generates vesicles from PC12 membrane precursors in the presence of ATP and brain cytosol and is temperature dependent. At 15 degrees C, surface-labeled synaptic vesicle proteins accumulate in a donor compartment, while labeled synaptic vesicles cannot be detected. The block of synaptic vesicle formation at 15 degrees C enables the use of the monoclonal antibody, KT3, a specific marker for the epitope-tagged synaptic vesicle protein, VAMP-TAg, to label precursors in the synaptic vesicle biogenesis pathway. From membranes labeled in vivo at 15 degrees C, vesicles generated in vitro at 37 degrees C had the sedimentation characteristics of neuroendocrine synaptic vesicles on glycerol velocity gradients, and excluded the transferrin receptor. Therefore, vesiculation and sorting can be studied in this cell-free system. (C) 1998 Academic Press.