The Major Role of the Rab Ypt7p in Vacuole Fusion Is Supporting HOPS Membrane Association
The Major Role of the Rab Ypt7p in Vacuole Fusion Is Supporting HOPS Membrane Association
复制标题
DOI：
10.1074/jbc.m109.000737
复制
发表时间：
2009-06-12
影响因子：
4.8
通讯作者：
Wickner, William
中科院分区：
文献类型：
--
作者：
Hickey, Christopher M.;Stroupe, Christopher;Wickner, William
酵母空泡融合需要可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）、Rab GT3 Ypt 7 p、空泡脂质Sec 17 p和Sec 18 p以及同型融合和空泡蛋白分选复合物（HOPS）。HOPS是一种多亚基蛋白，与SNARE、液泡脂质和Ypt 7 p的GP结合形式具有直接亲和力;这些亲和力中的每一种都有助于HOPS与细胞器的结合。使用全纯化的组分，我们重建了融合，但不需要Rab Ypt 7 p。我们现在报道，由液泡激酶Yck 3 p磷酸化的HOPS块HOPS结合到液泡脂质，使HOPS膜协会和随后的融合依赖于Ypt 7 p的存在。与重构融合反应中的这一发现雅阁的是，当Yck 3 p存在并具有活性时，GTP酶激活蛋白Gyp 1 - 46 p对Ypt 7 p的失活仅阻断纯化液泡的融合。因此，虽然Ypt 7 p可能有助于其他融合功能，但其核心作用是将HOPS结合到膜上。
Yeast vacuole fusion requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), the Rab GTPase Ypt7p, vacuolar lipids, Sec17p and Sec18p, and the homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a multisubunit protein with direct affinities for SNAREs, vacuolar lipids, and the GTP-bound form of Ypt7p; each of these affinities contributes to HOPS association with the organelle. Using all-purified components, we have reconstituted fusion, but the Rab Ypt7p was not required. We now report that phosphorylation of HOPS by the vacuolar kinase Yck3p blocks HOPS binding to vacuolar lipids, making HOPS membrane association and the ensuing fusion depend on the presence of Ypt7p. In accord with this finding in the reconstituted fusion reaction, the inactivation of Ypt7p by the GTPase-activating protein Gyp1-46p only blocks the fusion of purified vacuoles when Yck3p is present and active. Thus, although Ypt7p may contribute to other fusion functions, its central role is to bind HOPS to the membrane.