Hao J C, Salem N, Peng X R, Kelly R B, Bennett M K
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA.
J Neurosci. 1997 Mar 1;17(5):1596-603. doi: 10.1523/JNEUROSCI.17-05-01596.1997.
The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.
包含囊泡相关膜蛋白2（VAMP - 2）以及质膜蛋白 syntaxin 1A和25 kDa的突触体相关蛋白（SNAP - 25）的多聚体蛋白复合物的组装，被认为反映了突触囊泡靶向、引发或融合的生化关联。利用多种蛋白质 - 蛋白质相互作用分析方法以及一系列缺失和点突变，我们研究了VAMP - 2与syntaxin 1A或SNAP - 25形成二元复合物以及与syntaxin 1A和SNAP - 25形成三元复合物所需的结构域。VAMP - 2中央保守结构域内的缺失消除了与syntaxin 1A或syntaxin 1A和SNAP - 25两者的结合。尽管所有缺失突变体都能够形成三元复合物，但其中只有一些复合物在十二烷基硫酸钠中对变性具有抗性。这些结果表明，协同相互作用导致形成至少两种生化性质不同的三元复合物类别。先前显示对VAMP - 2的细胞内运输有影响的两个点突变（M46A，内吞作用减少且分选至突触囊泡；N49A，分选至突触囊泡增强）位于syntaxin 1A和SNAP - 25结合所需的结构域内。M46A突变降低了syntaxin 1A和SNAP - 25的结合，而N49A突变增强了这种结合，这表明两个VAMP - 2点突变体的膜运输表型与其与syntaxin 1A或SNAP - 25形成复合物的能力之间存在相关性。
