Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755-3844.
Mol Biol Cell. 2024 May 1;35(5):ar71. doi: 10.1091/mbc.E24-02-0060. Epub 2024 Mar 27.
Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in , impaired Qc recognition by limiting HOPS without Sec18 still allows substantial -SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.
膜融合受 Rab GTPases 调控，其连接效应物如 HOPS、每个融合伴侣上的 SNARE 蛋白、催化 SNARE 组装的 SM 蛋白、Sec17（SNAP）和 Sec18（ NSF）。尽管浓缩的 HOPS 可以在没有 Sec18 的情况下支持融合，但我们现在报告说，在较低的 HOPS 水平下，融合急剧下降，而 Sec18 直接与 HOPS 结合可恢复融合。这种 Sec18 依赖的融合需要腺嘌呤核苷酸，但不需要 ATP 水解或 Sec17。Sec18 增强了 HOPS 对 Qc-SNARE 的识别。在高浓度的 HOPS 下，Qc 融合的 Km 值为几个 nM。要么降低 HOPS 水平，要么用合成的系绳替代 HOPS，会显著增加 Qc 的 Km 值到几百 nM。在稀 HOPS 下，Sec18 将 Qc 的 Km 值恢复到低 nM。相比之下，HOPS 浓度和 Sec18 对 Qb-SNARE 的识别没有影响。正如 Qc 是融合所必需的，而不是在 中 SNARE 最初组装所必需的，在没有 Sec18 的情况下限制 HOPS 对 Qc 的识别仍然允许大量的 -SNARE 组装。因此，除了已知的 Sec18 功能，如拆开 SNARE 复合物、寡聚 Sec17 以利于膜结合，以及允许 Sec17 在不完全 SNARE 拉链的情况下驱动融合外，我们还报告了 Sec18 的第四个功能，即 Sec18 与 HOPS 的独立结合，以增强功能性 Qc-SNARE 结合。
