Togneri John, Cheng Yi-Shan, Munson Mary, Hughson Frederick M, Carr Chavela M
Department of Pathology and Laboratory Medicine, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA.
Proc Natl Acad Sci U S A. 2006 Nov 21;103(47):17730-5. doi: 10.1073/pnas.0605448103. Epub 2006 Nov 7.
The Sec1/Munc-18 (SM) family of proteins is required for vesicle fusion in eukaryotic cells and has been linked to the membrane-fusion proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SM proteins may activate the target-membrane SNARE, syntaxin, for assembly into the fusogenic SNARE complex. In support of an activation role, SM proteins bind directly to their cognate syntaxins. An exception is the yeast Sec1p, which does not bind the yeast plasma-membrane syntaxin, Sso1p. This exception could be explained if the SM interaction motif were blocked by the highly stable closed conformation of Sso1p. We tested the possibility of a latent binding motif using sso1 mutants in yeast and reconstituted the Sec1p binding specificity observed in vivo with purified proteins in vitro. Our results indicate there is no latent binding motif in Sso1p. Instead, Sec1p binds specifically to the ternary SNARE complex, with no detectable binding to the binary t-SNARE complex or any of the three individual SNAREs in their uncomplexed forms. We propose that vesicle fusion requires a specific interaction between the SM protein and the ternary SNARE complex.
Sec1/Munc-18（SM）蛋白家族是真核细胞中囊泡融合所必需的，并且与被称为可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）的膜融合蛋白有关。SM蛋白可能会激活靶膜SNARE蛋白Syntaxin，使其组装成促融合SNARE复合体。为支持这一激活作用，SM蛋白直接与其同源的Syntaxin结合。一个例外是酵母Sec1p，它不与酵母质膜Syntaxin Sso1p结合。如果SM相互作用基序被Sso1p高度稳定的封闭构象所阻断，那么这个例外就可以得到解释。我们利用酵母中的sso1突变体测试了潜在结合基序的可能性，并在体外使用纯化蛋白重建了体内观察到的Sec1p结合特异性。我们的结果表明，Sso1p中不存在潜在的结合基序。相反，Sec1p特异性地结合三元SNARE复合体，而与二元t-SNARE复合体或任何一种未复合形式的三个单独SNARE均无可检测到的结合。我们提出，囊泡融合需要SM蛋白与三元SNARE复合体之间的特异性相互作用。
