Khan Md Murad, Ebrahimi Roshanak, Oot Rebecca A, Wilkens Stephan
Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210, USA.
bioRxiv. 2025 May 24:2025.05.24.655954. doi: 10.1101/2025.05.24.655954.
The eukaryotic vacuolar H-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V-ATPase and V proton channel subcomplexes, a mode of regulation conserved from yeast to humans. While signals that govern V-ATPase assembly have been studied in the cellular context, the molecular mechanisms of the process at the level of the enzyme remain poorly understood. We recently discovered that Oxr1p, one of the two TLDc domain proteins in yeast, is essential for rapid V-ATPase . How the second TLDc protein, Rtc5p, functions in reversible disassembly, however, is less clear. Here we find that Rtc5p promotes assembly of functional holo V-ATPase from purified V and V subcomplexes . CryoEM structures show that Rtc5p's TLDc domain binds the C-terminal domain of the V-B subunit, with Rtc5p's C-terminal α-helix inserting into the catalytic hexamer, thereby opening a second catalytic site distal to its binding site. Unlike Oxr1p, however, which when deleted produces a distinct phenotype, Rtc5p does not appear to be essential for glucose driven enzyme (re)assembly, hinting at the presence of multiple assembly pathways .
真核生物液泡H⁺-ATP酶（V-ATP酶）通过可逆性解离为自抑制的V-ATP酶和V质子通道亚复合体进行调控，这种调控模式在从酵母到人类中都是保守的。虽然在细胞环境中已经研究了控制V-ATP酶组装的信号，但在酶水平上该过程的分子机制仍知之甚少。我们最近发现，酵母中两个TLDc结构域蛋白之一的Oxr1p对于快速组装V-ATP酶至关重要。然而，第二个TLDc蛋白Rtc5p在可逆解离中如何发挥作用尚不清楚。在这里，我们发现Rtc5p促进从纯化的V和V亚复合体组装功能性全酶V-ATP酶。冷冻电镜结构显示，Rtc5p的TLDc结构域与V-B亚基的C末端结构域结合，Rtc5p的C末端α螺旋插入催化六聚体，从而在其结合位点远端打开第二个催化位点。然而，与缺失时产生不同表型的Oxr1p不同，Rtc5p对于葡萄糖驱动的酶（重新）组装似乎不是必需的，这暗示存在多种组装途径。
