Center for Integrative Physiology and Molecular Medicine, School of Medicine, University of Saarland, Homburg, Germany.
Elife. 2024 Nov 25;12:RP92438. doi: 10.7554/eLife.92438.
Complexin determines magnitude and kinetics of synchronized secretion, but the underlying molecular mechanisms remained unclear. Here, we show that the hydrophobic face of the amphipathic helix at the C-terminus of Complexin II (CpxII, amino acids 115-134) binds to fusion-promoting SNARE proteins, prevents premature secretion, and allows vesicles to accumulate in a release-ready state in mouse chromaffin cells. Specifically, we demonstrate that an unrelated amphipathic helix functionally substitutes for the C-terminal domain (CTD) of CpxII and that amino acid substitutions on the hydrophobic side compromise the arrest of the pre-fusion intermediate. To facilitate synchronous vesicle fusion, the N-terminal domain (NTD) of CpxII (amino acids 1-27) specifically cooperates with synaptotagmin I (SytI), but not with synaptotagmin VII. Expression of CpxII rescues the slow release kinetics of the Ca-binding mutant Syt I R233Q, whereas the N-terminally truncated variant of CpxII further delays it. These results indicate that the CpxII NTD regulates mechanisms which are governed by the forward rate of Ca binding to Syt I. Overall, our results shed new light on key molecular properties of CpxII that hinder premature exocytosis and accelerate synchronous exocytosis.
复合蛋白决定了分泌的幅度和动力学，但背后的分子机制仍不清楚。在这里，我们表明，复合蛋白 II（CpxII，氨基酸 115-134）的 C 端两性螺旋的疏水面与促进融合的 SNARE 蛋白结合，防止过早分泌，并使囊泡在释放准备状态下在小鼠嗜铬细胞中积累。具体来说，我们证明了一个不相关的两性螺旋可以替代 CpxII 的 C 端结构域（CTD），并且疏水面上的氨基酸取代会损害预融合中间产物的捕获。为了促进囊泡融合的同步性，CpxII 的 N 端结构域（NTD，氨基酸 1-27）与突触融合蛋白 I（SytI）特异性合作，但与突触融合蛋白 VII 不合作。CpxII 的表达挽救了 Ca 结合突变体 Syt I R233Q 的缓慢释放动力学，而 CpxII 的 N 端截断变体则进一步延迟了它。这些结果表明，CpxII 的 NTD 调节了由 Ca 与 Syt I 的结合正向速率控制的机制。总的来说，我们的研究结果揭示了复合蛋白 II 阻碍过早胞吐和加速同步胞吐的关键分子特性。
