Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844.
Mol Biol Cell. 2023 Mar 1;34(3):ar17. doi: 10.1091/mbc.E22-10-0486. Epub 2023 Feb 3.
The conserved catalysts of intracellular membrane fusion are Rab-family GTPases, effector complexes that bind Rabs for membrane tethering, SNARE proteins of the R, Qa, Qb, and Qc families, and SNARE chaperones of the SM, Sec17/SNAP, and Sec18/NSF families. Yeast vacuole fusion is regulated by phosphatidylinositol-3-phosphate (PI3P). PI3P binds directly to the vacuolar Qc-SNARE and to HOPS, the vacuolar tethering/SM complex. We now report several distinct functions of PI3P in fusion. PI3P binds the N-terminal PX domain of the Qc-SNARE to enhance its engagement for fusion. Even when Qc has been preassembled with the Qa- and Qb-SNAREs, PI3P still promotes -SNARE assembly and fusion between these 3Q proteoliposomes and those with R-SNARE, whether with the natural HOPS tether or with a synthetic tether. With HOPS, efficient -SNARE complex formation needs PI3P on the 3Q-SNARE proteoliposomes, in to the Qc. PI3P is also needed for HOPS to confer resistance to Sec17/Sec18. With a synthetic tether, fusion is supported by PI3P on either fusion partner membrane, but this fusion is blocked by Sec17/Sec18. PI3P thus supports multiple stages of fusion: the engagement of the Qc-SNARE, -SNARE complex formation with preassembled Q-SNAREs, HOPS protection of SNARE complexes from Sec17/Sec18, and fusion per se after tethering and Q-SNARE assembly.
细胞内膜融合的保守催化剂是 Rab 家族 GTPases，它们是与 Rab 结合进行膜连接的效应物复合物，R、Qa、Qb 和 Qc 家族的 SNARE 蛋白，以及 SM、Sec17/SNAP 和 Sec18/NSF 家族的 SNARE 伴侣蛋白。酵母液泡融合受磷脂酰肌醇-3-磷酸（PI3P）调节。PI3P 直接结合液泡 Qc-SNARE 和 HOPS，即液泡连接/SM 复合物。我们现在报告了 PI3P 在融合中的几个不同功能。PI3P 结合 Qc-SNARE 的 N 端 PX 结构域，以增强其融合的参与。即使 Qc 已经与 Qa 和 Qb-SNARE 预组装，PI3P 仍然促进 -SNARE 组装，并且在这些 3Q 蛋白脂质体和那些带有 R-SNARE 的蛋白脂质体之间发生融合，无论是否带有天然的 HOPS 连接子还是带有合成的连接子。有了 HOPS，有效的 -SNARE 复合物形成需要 3Q-SNARE 蛋白脂质体上的 PI3P，而不是 Qc。PI3P 对于 HOPS 赋予对 Sec17/Sec18 的抗性也是必需的。对于合成的连接子，融合由融合伙伴膜上的 PI3P 支持，但这种融合被 Sec17/Sec18 阻断。因此，PI3P 支持融合的多个阶段：Qc-SNARE 的参与，与预组装的 Q-SNARE 形成 -SNARE 复合物，HOPS 保护 SNARE 复合物免受 Sec17/Sec18 的影响，以及在连接和 Q-SNARE 组装后本身发生融合。
