Structure, Mechanism and Regulation of the V-ATPases

V-ATP酶的结构、机制和调控

• 批准号: 6615777
• 负责人: MICHAEL D FORGAC
• 金额: $ 46.85万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-08-30 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6615777
• 关键词: Saccharomyces cerevisiae; cell growth regulation; crosslink; electron microscopy; enzyme complex; fungal genetics; gene expression; high performance liquid chromatography; hydrogen transporting ATP synthase; intermolecular interaction; isozymes; laboratory rabbit; lipid bilayer membrane; mass spectrometry; model design /development; molecular assembly /self assembly; molecular genetics; molecular site; physical model; protein localization; protein structure function; proteolipids; site directed mutagenesis; structural biology; vesicle /vacuole; western blottings

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.

本计划的长期目标是确定液泡（H+）-ATP酶（或V-ATP酶）的结构、机制和调节。V-ATP酶负责真核细胞中细胞内区室的酸化，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、细胞内膜运输、蛋白质加工和降解以及小分子的偶联转运。特化细胞质膜中的V-ATP酶也在肾酸化、pH稳态、骨吸收和肿瘤转移中起作用。因此，了解V-ATP酶是如何调节的对于了解许多疾病过程至关重要，包括病毒进入，骨质疏松症和转移。V-ATP酶被组织成两个功能结构域：负责ATP水解的外周V1结构域和负责质子转运的完整V0结构域。电子显微镜图像的V-ATP酶复合物揭示了V1和V0域之间的多重连接。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亚基中并用作光活化交联剂的连接位点。此外，将对亚单位特异性抗体修饰的复合物进行电子显微镜检查。催化A亚基的独特结构域的功能将通过缺失和随机诱变来解决。将使用半胱氨酸诱变、化学标记和二硫键形成来确定100 kDa α亚基的结构及其与V0结构域的蛋白脂质亚基的相互作用。最后，在体内解离的V-ATP酶复合物，这已被提出是一个重要的监管机制，将进行调查。将在位于不同细胞内区室的V-ATP酶中比较响应于葡萄糖消耗的解离，并将选择和分析解离缺陷的突变体。这些研究将为进一步了解这个重要的（H+）-ATP酶家族的结构和调控提供帮助。