Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13219 Life Sciences Institute and Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor MI 48109.
Mol Biol Cell. 2014 Apr;25(8):1251-62. doi: 10.1091/mbc.E13-10-0563. Epub 2014 Feb 12.
Vacuolar proton-translocating ATPases (V-ATPases) are highly conserved, ATP-driven proton pumps regulated by reversible dissociation of its cytosolic, peripheral V1 domain from the integral membrane V(o) domain. Multiple stresses induce changes in V1-V(o) assembly, but the signaling mechanisms behind these changes are not understood. Here we show that certain stress-responsive changes in V-ATPase activity and assembly require the signaling lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). V-ATPase activation through V1-V(o) assembly in response to salt stress is strongly dependent on PI(3,5)P2 synthesis. Purified V(o) complexes preferentially bind to PI(3,5)P2 on lipid arrays, suggesting direct binding between the lipid and the membrane sector of the V-ATPase. Increasing PI(3,5)P2 levels in vivo recruits the N-terminal domain of V(o)-sector subunit Vph1p from cytosol to membranes, independent of other subunits. This Vph1p domain is critical for V1-V(o) interaction, suggesting that interaction of Vph1p with PI(3,5)P2-containing membranes stabilizes V1-V(o) assembly and thus increases V-ATPase activity. These results help explain the previously described vacuolar acidification defect in yeast fab1 and vac14 mutants and suggest that human disease phenotypes associated with PI(3,5)P2 loss may arise from compromised V-ATPase stability and regulation.
液泡质子转运 ATP 酶（V-ATPases）高度保守，是受细胞溶质、外周 V1 结构域与完整膜 V(o)结构域可逆解离调控的 ATP 驱动质子泵。多种应激诱导 V1-V(o)组装的变化，但这些变化背后的信号机制尚不清楚。在这里，我们表明 V-ATPase 活性和组装的某些应激响应变化需要信号脂质磷脂酰肌醇 3,5-二磷酸（PI(3,5)P2）。V1-V(o)组装引起的 V-ATPase 激活对盐胁迫下的 PI(3,5)P2 合成有强烈的依赖性。在脂质阵列上，纯化的 V(o)复合物优先结合 PI(3,5)P2，这表明脂质与 V-ATPase 的膜区之间存在直接结合。体内增加 PI(3,5)P2 水平会使 V(o)-结构域亚基 Vph1p 的 N 端结构域从细胞质招募到膜上，而不依赖于其他亚基。该 Vph1p 结构域对于 V1-V(o)相互作用至关重要，这表明 Vph1p 与含有 PI(3,5)P2 的膜相互作用可稳定 V1-V(o)组装，从而增加 V-ATPase 活性。这些结果有助于解释先前描述的酵母 fab1 和 vac14 突变体中液泡酸化缺陷，并表明与 PI(3,5)P2 缺失相关的人类疾病表型可能源于 V-ATPase 稳定性和调节受损。
