A Rab prenyl membrane-anchor allows effector recognition to be regulated by guanine nucleotide.
A Rab prenyl membrane-anchor allows effector recognition to be regulated by guanine nucleotide.
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Rab 异戊二烯基膜锚允许鸟嘌呤核苷酸调节效应子识别。
DOI：
10.1073/pnas.2000923117
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发表时间：
2020
影响因子：
11.1
通讯作者：
Song,Hongki
中科院分区：
文献类型：
--
作者：
Lee,Miriam;Wickner,William;Song,Hongki
膜融合由保守蛋白R、Qa、Qb和Qc SNARE催化，它们在膜之间形成四聚体RQaQbQc复合物; SM、Sec 17/αSNAP和Sec 18/NSF家族的SNARE分子伴侣; Rab-GTP酶（Rabs）;和Rab效应物。Rabs通过C-末端异戊二烯基锚定到膜上，但也可以在通过非极性多肽锚定时起作用。Rabs受GTP酶激活蛋白（GAP）调节，激活结合GTP的水解。我们已经重建融合与纯成分从酵母液泡，包括SNARE，HOPS（同型融合和液泡蛋白分选）拴系和SNARE组装复合物，和Rab Ypt 7，结合到膜的C-末端异戊二烯基（Ypt 7-pr）或重组跨膜锚（Ypt 7-tm）。我们现在报道，HOPS依赖性融合发生与Ypt 7锚定的任何一种手段，但只有Ypt 7-pr需要GTP激活，是无活性的结合GDP或没有结合鸟嘌呤核苷酸。相反，Ypt 7-tm对于HOPS依赖性融合是组成型活性的，不依赖于结合的鸟嘌呤核苷酸。差距Gyp 1 -46对融合的抑制作用并不局限于结合GTP的Ypt 7-tm，表明该GAP具有调节融合的额外模式。液泡激酶Yck 3对HOPS的磷酸化使得融合严格依赖于GTP激活的Ypt 7，无论是通过异戊烯基还是跨膜锚结合到膜上。GTP或GDP的结合构成了Ypt 7的选择性开关，但对于Ypt 7-tm，该开关仅在通过其生理激酶Yck 3磷酸化为P-HOPS后被HOPS读取。Ypt 7的异戊烯基锚允许HOPS和P-HOPS两者被Ypt 7结合的鸟嘌呤核苷酸调节。
Membrane fusion is catalyzed by conserved proteins R, Qa, Qb, and Qc SNAREs, which form tetrameric RQaQbQc complexes between membranes; SNARE chaperones of the SM, Sec17/αSNAP, and Sec18/NSF families; Rab-GTPases (Rabs); and Rab effectors. Rabs are anchored to membranes by C-terminal prenyl groups, but can also function when anchored by an apolar polypeptide. Rabs are regulated by GTPase-activating proteins (GAPs), activating the hydrolysis of bound GTP. We have reconstituted fusion with pure components from yeast vacuoles including SNAREs, the HOPS (homotypic fusion and vacuole protein sorting) tethering and SNARE-assembly complex, and the Rab Ypt7, bound to membranes by either C-terminal prenyl groups (Ypt7-pr) or a recombinant transmembrane anchor (Ypt7-tm). We now report that HOPS-dependent fusion occurs with Ypt7 anchored by either means, but only Ypt7-pr requires GTP for activation and is inactive either with bound GDP or without bound guanine nucleotide. In contrast, Ypt7-tm is constitutively active for HOPS-dependent fusion, independent of bound guanine nucleotide. Fusion inhibition by the GAP Gyp1-46 is not limited to Ypt7-tm with bound GTP, indicating that this GAP has an additional mode of regulating fusion. Phosphorylation of HOPS by the vacuolar kinase Yck3 renders fusion strictly dependent on GTP-activated Ypt7, whether bound to membranes by prenyl or transmembrane anchor. The binding of GTP or GDP constitutes a selective switch for Ypt7, but with Ypt7-tm, this switch is only read by HOPS after phosphorylation to P-HOPS by its physiological kinase Yck3. The prenyl anchor of Ypt7 allows both HOPS and P-HOPS to be regulated by Ypt7-bound guanine nucleotide.