Regulation of V-ATPases by Phosphoinositides

项目总结 V-ATPase是高度保守的多亚单位质子泵，可使细胞器酸化，包括 所有真核细胞中的溶酶体、内体、高尔基体和分泌颗粒。这其中的每一个 细胞器需要将其管腔pH值调节到一个狭窄的范围才能发挥作用。然而，它并不完全 了解这些pH范围是如何维持的，或者不同细胞器中的V-ATPase在 环境条件对当地的pH梯度构成了挑战。我们的中心假设是，PI血脂提供了 细胞器特定的输入来局部调节V-ATPase的活性，从而可以影响 许多功能依赖于V-ATPase活性和细胞器酸化。在目标1中，我们使用井- 表征酵母V-ATPase系统以表征膜上胞浆结构域的结合 结合a亚基异构体Vph1和Stv1到磷脂酰肌醇(PI)脂类。我们发现了驻留在液泡中的 Vph1能够结合空泡信号脂PI(3，5)P2，且驻留在高尔基体内的Stv1更喜欢高尔基体富集 我们将量化和表征这些结合偏好，并探索如何结合特定的 脂类激活含有单个a-亚单位异构体的V-ATPase。基于最近的结构和同调 建模后，我们将为每种异构体开发脂结合突变体。最后，我们将评估PI绑定 四种人类α亚基亚基异构体的特异性。我们测试了四种人类亚型中的三种，发现 体外存在PI特异性相互作用的证据。我们将表征PI结合和亲和力的特异性和亲和力 确定潜在的结合部位。在目标2中，我们解决了PI脂结合的空间和时间后果 到含有全长Vph1和Stv1的完整V-ATPase。我们假设PI脂通过以下方式发挥空间控制作用 激活它们居留细胞器中的V-ATPase。具体来说，我们提出：1)PI(3，5)P2结合到 含有Vph1的V-ATPase诱导一种构象，稳定外周V1之间的相互作用 2)PI(4)P与Stv1-P结合 含有V-ATPase促进高尔基体中的滞留，并且3)PI(3)P促进酸化 与含有Vph1的V-ATPase相互作用的内吞途径。相反，高渗应激促进 PI(3，5)P2的快速和短暂的升高，可逆地激活液泡中含有Vph1的V-ATPase。我们 我将分析这种暂时性V-ATPase激活的机制及其在提供短期 对盐胁迫的适应。这些实验将PI和V-ATPase亚型含量联系在一起，这是两个基本特征 细胞器的身份。他们承诺为许多与缺陷有关的疾病提供新的见解 细胞器酸化，包括神经变性、癌症和分枝杆菌感染，并可能提示 新的治疗路线。

PROJECT SUMMARY V-ATPases are highly conserved, multisubunit proton pumps that acidify organelles including lysosomes, endosomes, Golgi apparatus, and secretory granules in all eukaryotic cells. Each of these organelles requires tuning of its luminal pH to a narrow range for its function. However, it is not fully understood how these pH ranges are maintained or how V-ATPases in different organelles respond when environmental conditions challenge local pH gradients. Our central hypothesis is that PI lipids provide organelle-specific inputs to locally regulate V-ATPase activity, and thus can impact individual subsets of the many functions dependent on V-ATPase activity and organelle acidification. In Aim 1, we use the well- characterized yeast V-ATPase system to characterize binding of the cytosolic domains of the membrane- bound a-subunit isoforms, Vph1 and Stv1, to phosphoinositide (PI) lipids. We have found that vacuole-resident Vph1 is able to bind the vacuolar signaling lipid PI(3,5)P2 and Golgi-resident Stv1 prefers the Golgi-enriched lipid PI(4)P. We will quantify and characterize these binding preferences and probe how binding of specific lipids activates V-ATPases containing individual a-subunit isoforms. Based on recent structures and homology modeling, we will then develop lipid-binding mutants for each isoform. Finally, we will evaluate PI binding specificities of the four human a-subunit isoforms. We have tested three of the four human isoforms and found evidence of PI-specific interactions in vitro. We will characterize specificity and affinities of PI binding and identify potential binding sites. In Aim 2, we address the spatial and temporal consequences of PI lipid binding to intact V-ATPases containing full-length Vph1 and Stv1. We hypothesize that PI lipids exert spatial control by activating V-ATPases in their organelles of residence. Specifically, we propose that: 1) PI(3,5)P2 binding to Vph1-containing V-ATPases induces a conformation that stabilizes interactions between the peripheral V1 sector and the integral membrane Vo sector of the V-ATPase, increasing activity, 2) PI(4)P binding to Stv1- containing V-ATPases promotes retention in the Golgi apparatus, and 3) PI(3)P promotes acidification of the endocytic pathway by interacting with Vph1-containing V-ATPases. In contrast, hyperosmotic stress promotes a rapid and transient rise in PI(3,5)P2 that reversibly activates Vph1-containing V-ATPases in the vacuole. We will analyze the mechanisms of this temporal V-ATPase activation and its role in providing short-term adaptation to salt stress. These experiments link PI and V-ATPase isoform content, two fundamental features of organelle identity. They promise to provide novel insights into the many diseases linked to defective organelle acidification, including neurodegeneration, cancer, and mycobacterial infection, and could suggest new therapeutic routes.