Structure, Mechanism and Regulation of the V-ATPases

本提案的长期目标是确定液泡(H+)- atp酶（或V- atp酶）的结构、机制和调控。v - atp酶负责真核细胞胞内区室的酸化，并在多种细胞过程中发挥重要作用，包括受体介导的内吞作用、细胞膜内运输、蛋白质加工和降解以及小分子的偶联运输。特化细胞质膜上的v - atp酶也在肾脏酸化、pH稳态、骨吸收和肿瘤转移中起作用。因此，了解v - atp酶是如何调控的对于理解许多疾病过程至关重要，包括病毒侵入、骨质疏松和转移。v -ATP酶被组织成两个功能域：负责ATP水解的外周V1结构域和负责质子易位的整体V0结构域。v - atp酶复合物的电子显微镜图像显示V1和V0结构域之间存在多种连接。为了确定v - atp酶复合物内亚基的排列，将独特的半胱氨酸残基引入B亚基并用作光激活交联剂的附着位点。此外，用亚单位特异性抗体修饰的复合物的电子显微镜将被执行。催化a亚基的一个独特结构域的功能将通过删除和随机诱变来解决。100 kDa a亚基的结构及其与V0结构域蛋白脂亚基的相互作用将通过半胱氨酸诱变、化学标记和二硫键形成来确定。最后，将研究v - atp酶复合物的体内解离，这被认为是一个重要的调节机制。将比较位于不同细胞内区室的v - atp酶对葡萄糖消耗的解离反应，并选择和分析解离缺陷突变体。这些研究将进一步深入了解这一重要的(H+)- atp酶家族的结构和调控。

The long term objectives of this proposal are to determine the structure, mechanism and regulation of the vacuolar (H+)-ATPases (or V- ATPases). The V-ATPases are responsible for acidification of intracellular compartments in eukaryotic cells and serve an important function in a variety of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, protein processing and degradation and coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in renal acidification, pH homeostasis, bone resorption and tumor metastasis. Understanding how V-ATPases are regulated is thus crucial to understanding many disease processes, including viral entry, osteoporosis and metastasis. The V-ATPases are organized into two functional domains: a peripheral V1 domain responsible for ATP hydrolysis and a integral V0 domain responsible for proton translocation. Electron microscopic images of the V-ATPase complex reveal multiple connections between the V1 and V0 domains. To determine the arrangement of subunits within the V-ATPase complex, unique cysteine residues will be introduced into the B subunit and used as sites of attachment of a photoactivated crosslinker. In addition, electron microscopy of complexes decorated with subunit- specific antibodies will be performed. The function of a unique domain of the catalytic A subunit will be addressed by deletion and random mutagenesis. The structure of the 100 kDa a subunit and its interactions with the proteolipid subunits of the V0 domain will be determined using cysteine mutagenesis, chemical labeling and disulfide bond formation. Finally, the in vivo dissociation of the V-ATPase complex, which has been proposed to be an important regulatory mechanism, will be investigated. Dissociation in response to glucose depletion will be compared in V-ATPases located in different intracellular compartments and mutants defective in dissociation will be selected and analyzed. These studies should provide further insight into the structure and regulation of this important family of (H+)-ATPases.