Synaptic vesicle membrane proteins interact to form a multimeric complex.
Synaptic vesicle membrane proteins interact to form a multimeric complex.
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DOI：
10.1083/jcb.116.3.761
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发表时间：
1992-02
期刊：
影响因子：
--
通讯作者：
Scheller RH
中科院分区：
文献类型：
--
作者：
Bennett MK;Calakos N;Kreiner T;Scheller RH
通过洗涤剂溶解，然后通过大小分级或免疫沉淀法分析大鼠脑突触囊泡的膜组分之间的潜在相互作用。通过Western印迹法监测六种突触囊泡膜蛋白以及质膜蛋白的行为。在CHAPS中突触囊泡膜的溶解导致了一个大的蛋白质复合物的恢复，包括SV 2，p65，p38，囊泡相关的膜蛋白，和空泡质子泵。在辛基葡糖苷溶解导致SV 2，p38，和rab 3A之间的相互作用的保存，而与Triton X-100的突触囊泡溶解导致两个主要的相互作用，一个涉及p65和SV 2，和其他涉及p38和囊泡相关的膜蛋白。用CHAPS溶解保存的多组分复合物在辛基葡萄糖苷溶解和随后针对CHAPS透析后部分重构。通过凝胶过滤色谱法减少CHAPS浓度导致多组分复合物的回收率增加。通过负染色EM从CHAPS溶解的囊泡中分离的大复合物的检查显示具有多个球状结构域的结构，其中一些用针对p65和SV 2的金缀合抗体特异性标记。本报告中定义的蛋白质相互作用可能是神经递质分泌、膜交通和神经末梢内囊泡空间组织的基础。
Potential interactions between membrane components of rat brain synaptic vesicles were analyzed by detergent solubilization followed by size fractionation or immunoprecipitation. The behavior of six synaptic vesicle membrane proteins as well as a plasma membrane protein was monitored by Western blotting. Solubilization of synaptic vesicle membranes in CHAPS resulted in the recovery of a large protein complex that included SV2, p65, p38, vesicle-associated membrane protein, and the vacuolar proton pump. Solubilization in octylglucoside resulted in the preservation of interactions between SV2, p38, and rab3A, while solubilization of synaptic vesicles with Triton X-100 resulted in two predominant interactions, one involving p65 and SV2, and the other involving p38 and vesicle-associated membrane protein. The multicomponent complex preserved with CHAPS solubilization was partially reconstituted following octylglucoside solubilization and subsequent dialysis against CHAPS. Reduction of the CHAPS concentration by gel filtration chromatography resulted in increased recovery of the multicomponent complex. Examination of the large complex isolated from CHAPS-solubilized vesicles by negative stain EM revealed structures with multiple globular domains, some of which were specifically labeled with gold-conjugated antibodies directed against p65 and SV2. The protein interactions defined in this report are likely to underlie aspects of neurotransmitter secretion, membrane traffic, and the spatial organization of vesicles within the nerve terminal.