Stroupe Christopher, Hickey Christopher M, Mima Joji, Burfeind Amy S, Wickner William
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17626-33. doi: 10.1073/pnas.0903801106. Epub 2009 Oct 13.
Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.
Rab GTP酶及其效应蛋白介导对接，即双层膜融合之前细胞内膜的初始接触。然而，Rab蛋白和效应蛋白是否足以介导膜间相互作用尚不清楚。我们最近报道了重组膜融合，该过程需要酵母液泡SNARE蛋白、脂质以及同型融合和液泡蛋白分选（HOPS）/C类Vps复合体，后者是酵母液泡Rab GTP酶Ypt7p的效应蛋白和鸟嘌呤核苷酸交换因子。我们现在报道了无裂解膜融合的重组，该过程需要纯化的结合GTP的Ypt7p、HOPS复合体、液泡SNARE蛋白、ATP水解以及SNARE拆卸催化剂Sec17p和Sec18p。我们利用这个重组系统表明，SNARE蛋白和Sec17p/Sec18p以及Ypt7p和HOPS复合体是稳定膜间相互作用所必需的，并且三个液泡Q-SNARE蛋白足以介导这些相互作用。
