Bhalla Akhil, Chicka Michael C, Tucker Ward C, Chapman Edwin R
Howard Hughes Medical Institute, University of Wisconsin, 1300 University Avenue, SMI 129, Madison, Wisconsin, USA.
Nat Struct Mol Biol. 2006 Apr;13(4):323-30. doi: 10.1038/nsmb1076. Epub 2006 Mar 26.
In nerve terminals, exocytosis is mediated by SNARE proteins and regulated by Ca(2+) and synaptotagmin-1 (syt). Ca(2+) promotes the interaction of syt with anionic phospholipids and the target membrane SNAREs (t-SNAREs) SNAP-25 and syntaxin. Here, we have used a defined reconstituted fusion assay to determine directly whether syt-t-SNARE interactions couple Ca(2+) to membrane fusion by comparing the effects of Ca(2+)-syt on neuronal (SNAP-25, syntaxin and synaptobrevin) and yeast (Sso1p, Sec9c and Snc2p) SNAREs. Ca(2+)-syt aggregated neuronal and yeast SNARE liposomes to similar extents via interactions with anionic phospholipids. However, Ca(2+)-syt was able to bind and stimulate fusion mediated by only neuronal SNAREs and had no effect on yeast SNAREs. Thus, Ca(2+)-syt regulates fusion through direct interactions with t-SNAREs and not solely through aggregation of vesicles. Ca(2+)-syt drove assembly of SNAP-25 onto membrane-embedded syntaxin, providing direct evidence that Ca(2+)-syt alters t-SNARE structure.
在神经末梢，胞吐作用由可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）蛋白介导，并受钙离子（Ca(2+)）和突触结合蛋白-1（syt）调控。Ca(2+)促进syt与阴离子磷脂以及靶膜SNAREs（t-SNAREs）SNAP-25和 syntaxin的相互作用。在此，我们使用了一种明确的重组融合测定法，通过比较Ca(2+)-syt对神经元（SNAP-25、syntaxin和突触小泡蛋白）和酵母（Sso1p、Sec9c和Snc2p）SNAREs的影响，直接确定syt-t-SNARE相互作用是否将Ca(2+)与膜融合偶联起来。Ca(2+)-syt通过与阴离子磷脂的相互作用，使神经元和酵母SNARE脂质体聚集到相似程度。然而，Ca(2+)-syt仅能结合并刺激由神经元SNAREs介导的融合，对酵母SNAREs没有影响。因此，Ca(2+)-syt通过与t-SNAREs的直接相互作用而非仅仅通过囊泡聚集来调节融合。Ca(2+)-syt促使SNAP-25组装到膜嵌入的syntaxin上，提供了Ca(2+)-syt改变t-SNARE结构的直接证据。
