Localization of Rab3A-binding site on C2A domain of synaptotagmin I to reveal its regulatory mechanism

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• 作者：Xia Tang ; Chunliang Xie ; Ying Wang ; Xianchun Wang ; ^{wang_xianchun@263.net}
• 关键词：SDS-PAGE ; sodium dodecyl sulfate polyacrylamide gel electrophoresis ; SNARE ; soluble N-ethylmaleimide-sensitive factor attachment proteins receptor ; SNAP-25 ; Synaptosomal-associated protein 25 ; Syt I ; synaptotagmin I ; C2A ; C2A domain of Syt I ; C2B ; C2&thinsp ; B domain of Syt I ; Wild-type C2A ; C2A domain without mutation ; YVK180AAA ; Residues Y180V181K182 were mutated into AAA ; C2A 143&ndash ; 206 ; truncated mutant of C2A ; consisting of amino acid residues 143&ndash ; 206 ; C2A 207&ndash ; 244 ; truncated m
• 刊名：International Journal of Biological Macromolecules
• 出版年：2017
• 出版时间：March 2017
• 年：2017
• 卷：96
• 期：Complete
• 页码：736-742
• 全文大小：1185 K
• 卷排序：96

文摘

Synaptotagmin I (Syt I) functions in the regulation of neurotransmitter release and multiple other cellular processes through its C2 domain binding to other molecules. Our previous study demonstrated that Rab3A, a small GTP-binding protein, is a new interacting partner of Syt I and could bind to both of the C2 domains; the polylysine motif in C2B is a key site for Rab3A binding, but the binding site on C2A is not clear. In order to localize Rab3-binding site on C2A and reveal the relevant regulatory mechanism, in the present study we investigated the interaction between recombinant Rab3A and various C2A mutants. The results showed that a key Rab3A-binding site on C2A is located at R199K200 in the flexible loop 2 of the domain, and the site does not overlap with most of the known functional sites or residues. It was speculated that the interaction between Rab3A and C2A is not simply based on electrostatic force, and Rab3A regulates C2A-mediated vesicle-presynaptic membrane fusion mainly through affecting the C2A binding to phospholipids in the presynaptic membrane. These results have contributed to the comprehension of action mechanism of Rab3 and synaptotagmin in the regulation of synaptic vesicle exocytosis.