Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844.
Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844
Proc Natl Acad Sci U S A. 2020 Apr 7;117(14):7739-7744. doi: 10.1073/pnas.2000923117. Epub 2020 Mar 25.
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.
膜融合由保守蛋白 R、Qa、Qb 和 Qc SNARE 催化，这些蛋白在膜之间形成四聚体 RQaQbQc 复合物；SM、Sec17/αSNAP 和 Sec18/NSF 家族的 SNARE 伴侣；Rab-GTPases（Rabs）；和 Rab 效应器。Rabs 通过 C 末端异戊二烯基团锚定在膜上，但也可以在由非极性多肽锚定时发挥作用。Rabs 通过 GTPase 激活蛋白（GAPs）调节，激活结合 GTP 的水解。我们已经用来自酵母液泡的纯组分重新构建了融合，包括 SNARE、HOPS（同源融合和液泡蛋白分选）连接和 SNARE 组装复合物，以及 Rab Ypt7，通过 C 末端异戊二烯基团（Ypt7-pr）或重组跨膜锚（Ypt7-tm）锚定在膜上。我们现在报告说，HOPS 依赖性融合发生在 Ypt7 以两种方式锚定时，但只有 Ypt7-pr 需要 GTP 才能激活，并且在结合 GDP 或没有结合鸟嘌呤核苷酸时无活性。相比之下，Ypt7-tm 对 HOPS 依赖性融合具有组成型活性，与结合的鸟嘌呤核苷酸无关。GAP Gyp1-46 的融合抑制不仅限于结合 GTP 的 Ypt7-tm，表明该 GAP 具有调节融合的另一种模式。液泡激酶 Yck3 对 HOPS 的磷酸化使融合严格依赖于 GTP 激活的 Ypt7，无论其通过异戊二烯或跨膜锚定在膜上。GTP 或 GDP 的结合构成了 Ypt7 的选择性开关，但对于 Ypt7-tm，只有在其生理激酶 Yck3 将其磷酸化为 P-HOPS 后，该开关才由 HOPS 读取。Ypt7 的异戊二烯锚允许 HOPS 和 P-HOPS 都由 Ypt7 结合的鸟嘌呤核苷酸调节。
