Orr Amy, Song Hongki, Rusin Scott F, Kettenbach Arminja N, Wickner William
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755.
Norris Cotton Cancer Center, Lebanon, NH 03766.
Mol Biol Cell. 2017 Apr 1;28(7):975-983. doi: 10.1091/mbc.E16-10-0743. Epub 2017 Feb 1.
Rab GTPases, their effectors, SNAREs of the R, Qa, Qb, and Qc families, and SM SNARE-binding proteins catalyze intracellular membrane fusion. At the vacuole/lysosome, they are integrated by the homotypic fusion and vacuole protein sorting (HOPS) complex. Two HOPS subunits bind vacuolar Rabs for tethering, another binds the Qc SNARE, and a fourth HOPS subunit, an SM protein, has conserved grooves that bind R- and Qa-SNARE domains. Spontaneous quaternary SNARE complex assembly is very slow. We report an assay of SNARE complex assembly that does not rely on fusion and for which tethering does not coenrich the four SNAREs. HOPS is required in this assay for rapid SNARE complex assembly. Optimal assembly needs HOPS, lipid membranes to which the R- or Qa-SNARE and Ypt7:GTP are integrally bound, and each of the other three SNAREs. Each SNARE assembles into this complex relying on the others, suggesting four-SNARE complex assembly rather than direct binding of each to HOPS. SNAREs can be disassociated by Sec 17/Sec 18/ATP, completing a catalyzed cycle of SNARE assembly and disassembly.
Rab GTP酶、其效应蛋白、R、Qa、Qb和Qc家族的SNARE蛋白以及SM SNARE结合蛋白催化细胞内膜融合。在液泡/溶酶体处，它们由同型融合和液泡蛋白分选（HOPS）复合体整合。两个HOPS亚基结合液泡Rab蛋白用于拴系，另一个结合Qc SNARE蛋白，第四个HOPS亚基是一种SM蛋白，具有保守的凹槽，可结合R-和Qa-SNARE结构域。自发的四聚体SNARE复合体组装非常缓慢。我们报告了一种不依赖融合的SNARE复合体组装检测方法，并且在该检测中拴系不会使四种SNARE蛋白共同富集。在该检测中，快速的SNARE复合体组装需要HOPS。最佳组装需要HOPS、R-或Qa-SNARE以及Ypt7:GTP与之整体结合的脂质膜，以及其他三种SNARE蛋白中的每一种。每种SNARE蛋白依赖其他蛋白组装成该复合体，这表明是四聚体SNARE复合体组装，而不是每种蛋白直接与HOPS结合。SNARE蛋白可被Sec 17/Sec 18/ATP解离，从而完成SNARE组装和解离的催化循环。
