Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion
Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion
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液泡激酶 Yck3p 对效应复合物 HOPS 的磷酸化赋予 Rab 核苷酸对液泡对接和融合的特异性
DOI：
10.1091/mbc.e12-04-0279
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发表时间：
2012
影响因子：
3.3
通讯作者：
Wickner W
中科院分区：
文献类型：
--
作者：
Zick M;Wickner W
酵母空泡的同型融合需要Rab-family GT3 Ypt 7 p及其效应子复合物、同型融合和空泡蛋白分选复合物（HOPS）。虽然液泡激酶Yck 3 p是液泡融合对调节Rab GTP环-Gdi 1 p（GDP解离抑制剂[GDI]）或Gyp 1 p/Gyp 7 p（GTP酶激活蛋白）的蛋白质的敏感性所必需的，但这种激酶磷酸化HOPS而不是Ypt 7 p。我们解决了这个难题，在重组蛋白脂质体融合反应与所有纯化的成分。在HOPS和Sec 17 p/Sec 18 p的存在下，当它们具有携带GDP或GTP的Ypt 7 p时，存在4-SNARE（可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体）蛋白脂质体的可比融合，这是Ras超家族GTP酶的仅GTP结合形式具有活性构象的规则的显著例外。然而，HOPS的磷酸化重组Yck 3 p赋予了一个严格的要求GTP结合Ypt 7 p结合磷酸化的HOPS，最佳的膜拴系，和蛋白脂质体融合。添加的GTP酶激活蛋白促进Ypt 7 p的GTP水解，并且添加的GDI在核苷酸循环期间捕获处于GDP结合状态的Ypt 7 p。在任一情况下，Ypt 7：GTP至Ypt 7：GDP的净转化对HOPS结合或活性没有影响，但阻断由磷酸化HOPS介导的融合。因此，鸟嘌呤核苷酸特异性的空泡融合Rab Ypt 7 p是通过其效应复合物的下游翻译后修饰。
The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle—Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)—this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (solubleN-ethylmaleimide–sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.
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DOI：
10.1042/bj20110687
发表时间：
2012
期刊：
The Biochemical journal
影响因子：
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