From the Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755-3844.
From the Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire 03755-3844
J Biol Chem. 2018 Jan 26;293(4):1346-1352. doi: 10.1074/jbc.RA117.000791. Epub 2017 Dec 5.
Membrane fusion is essential for intracellular protein sorting, cell growth, hormone secretion, and neurotransmission. Rapid membrane fusion requires tethering and Sec1-Munc18 (SM) function to catalyze R-, Qa-, Qb-, and Qc-SNARE complex assembly in , as well as SNARE engagement by the SNARE-binding chaperone Sec17/αSNAP. The hexameric vacuolar HOPS (motypic fusion and vacuole rotein orting) complex in the yeast tethers membranes through its affinities for the membrane Rab GTPase Ypt7. HOPS also has specific affinities for the vacuolar SNAREs and catalyzes SNARE complex assembly, but the order of their assembly into a 4-SNARE complex is unclear. We now report defined assembly intermediates on the path to membrane fusion. We found that a prefusion intermediate will assemble with HOPS and the R, Qa, and Qc SNAREs, and that this assembly undergoes rapid fusion upon addition of Qb and Sec17. HOPS-tethered membranes and all four vacuolar SNAREs formed a complex that underwent an even more dramatic burst of fusion upon Sec17p addition. These findings provide initial insights into an ordered fusion pathway consisting of the following intermediates and events: 1) Rab- and HOPS-tethered membranes, 2) a HOPS:R:Qa:Qc -complex, 3) a HOPS:4-SNARE -complex, 4) an engagement with Sec17, and 5) the rapid lipid rearrangements during fusion. In conclusion, our results indicate that the R:Qa:Qc complex forms in the context of membrane, Ypt7, HOPS, and -SNARE assembly and serves as a functional intermediate for rapid fusion after addition of the Qb-SNARE and Sec17 proteins.
膜融合对于细胞内蛋白质分拣、细胞生长、激素分泌和神经递质传递至关重要。快速的膜融合需要连接和 Sec1-Munc18（SM）功能来催化 R-、Qa-、Qb-和 Qc-SNARE 复合物在 中的组装，以及 SNARE 结合伴侣 Sec17/αSNAP 与 SNARE 的结合。酵母中的六聚体液泡 HOPS（典型融合和液泡蛋白分选）复合物通过其与膜 Rab GTPase Ypt7 的亲和力来连接膜。HOPS 还对液泡 SNARE 具有特定的亲和力，并催化 SNARE 复合物的组装，但它们组装成 4-SNARE 复合物的顺序尚不清楚。我们现在报告了在膜融合过程中的明确组装中间体。我们发现，融合前中间体将与 HOPS 和 R、Qa 和 Qc SNARE 组装，并且当添加 Qb 和 Sec17 时，这种组装会迅速融合。HOPS 连接的膜和所有四个液泡 SNARE 形成了一个复合物，当添加 Sec17p 时，该复合物会发生更剧烈的融合爆发。这些发现为一个有序的融合途径提供了初步的见解，该途径包括以下中间体和事件：1）Rab 和 HOPS 连接的膜，2）HOPS：R：Qa：Qc-复合物，3）HOPS：4-SNARE-复合物，4）与 Sec17 的结合，以及 5）融合过程中的快速脂质重排。总之，我们的结果表明，R：Qa：Qc 复合物在膜、Ypt7、HOPS 和 -SNARE 组装的背景下形成，并在添加 Qb-SNARE 和 Sec17 蛋白后作为快速融合的功能中间体。
