Regulation and Cellular Functions of V-ATPases

项目总结 V-ATPase是一种多功能、高度保守的多亚单位质子泵，负责细胞器的酸化 在几乎所有的真核细胞中。V-ATPase活性的完全丧失在除真菌以外的所有真核生物中都是致命的，但 亚基异构体的突变与远端肾小管酸中毒、不孕症、耳聋和骨化症有关。 V-ATPase活性在癌症中被颠覆，以支持对细胞pH动态平衡的额外需求 通过创造酸性的细胞外环境来促进转移。V-ATPase引起的内体酸化 也促进了许多病毒的进入。我们迫切需要了解V-的作用和调节。 靶向V-ATPase的ATPase亚基亚型和酶亚群 从治疗上讲。V-ATPase由外周V1亚复合体的可逆解离调节 整体膜Vo亚复合体和RAVE/RabConnectin-3复合体在这一过程中起着关键作用。 我们将研究酵母狂欢和哺乳动物的结构、机制和亚基组成。 RabConnectin-3复合体，它似乎以特定的V-ATPase亚单位亚基为靶标作为其活性的一部分。 重要的是，RabConnectin-3复合体的突变与癫痫和 神经退化，但潜在的疾病机制尚不清楚。我们之前在酵母菌中展示了 富含细胞器的磷脂酰肌醇与两种α-亚基的不同结合。 VO亚复合体，为V-ATPase的定位和活性提供细胞器特异性的输入。类脂 在体外观察到与哺乳动物a亚基异构体的相互作用，我们将把这些研究扩展到 描述在培养的哺乳动物细胞中相互作用的影响。最后，尽管V-ATPase必须 与pH动态平衡的其他细胞机制协同作用，其潜在机制 协调是不被理解的。我们将在酵母中解决这个问题，我们在那里发现了急性 或V-ATPase活性的慢性丧失触发主要H+输出泵Pma1的一部分内吞作用 质膜。我们将确定这个液泡对质膜pH串扰的机制。 减少的空泡/溶酶体酸化是酵母和哺乳动物细胞衰老的早期阶段。我们会 评估老化的酵母细胞是否表现出协调pH动态平衡的丧失或V- ATPase本身，并确定这些进程是否可以被操纵。

PROJECT SUMMARY V-ATPases are versatile, highly conserved, multi-subunit proton pumps responsible for organelle acidification in virtually all eukaryotic cells. Complete loss of V-ATPase activity is lethal in all eukaryotes except fungi, but mutations in subunit isoforms are linked to distal renal tubule acidosis, infertility, deafness, and osteopetrosis. V-ATPase activity is subverted in cancer to support additional demands on cellular pH homeostasis and promote metastasis by creating an acidic extracellular environment. Endosomal acidification by V-ATPases also promotes entry of many viruses. There is a crucial need to understand the roles and regulation of V- ATPase subunit isoforms and enzyme subpopulations in order to target of V-ATPases in specific locations therapeutically. V-ATPases are regulated by reversible disassembly of the peripheral V1 subcomplex from the integral membrane Vo subcomplex, and RAVE/Rabconnectin-3 complexes play a critical role in this process. We will investigate the structure, mechanism, and subunit composition of the yeast RAVE and mammalian Rabconnectin-3 complexes, which appear to target specific V-ATPase subunit isoforms as part of their activity. Importantly, mutations in Rabconnectin-3 complexes have been associated with epilepsy and neurodegeneration but the underlying disease mechanism is unclear. We previously demonstrated in yeast that organelle-enriched phosphoinositide phospholipids bind differentially to the two a-subunit isoforms of the Vo subcomplex, providing organelle-specific inputs into V-ATPase localization and activity. Similar lipid interactions are observed with mammalian a-subunit isoforms in vitro, and we will extend these studies to characterizing the effects of the interactions in cultured mammalian cells. Finally, although V-ATPases must function in concert with other cellular mechanisms of pH homeostasis, the underlying mechanisms of this coordination are not understood. We will address this question in yeast, where we have discovered that acute or chronic loss of V-ATPase activity triggers endocytosis of a portion of the major H+ export pump, Pma1, from the plasma membrane. We will determine the mechanism of this vacuole to plasma membrane pH crosstalk. Reduced vacuole/lysosome acidification is an early step in aging in both yeast and mammalian cells. We will assess whether aging yeast cells display a loss in coordinated pH homeostasis or emerging defects in the V- ATPase itself and determine whether these processes can be manipulated.