SUBUNIT STRUCTURE AND FUNCTION IN VACUOLAR H+/ATPASES

该研究的目的是确定亚基结构， 液泡质子转运ATP酶（H ~+-ATP酶）的功能关系 ATP酶）。在所有的真核细胞中发现了极性H+-ATP酶，其中 它们既可发挥组织作用，也可发挥专门作用。本构 细胞器酸化参与受体介导的内吞作用， 生物合成途径中蛋白质的靶向， 酶原、蛋白质降解、离子稳态和碱性磷酸酶的摄取 调节分泌颗粒。特化细胞 肾和骨在质膜上含有空泡H+-ATP酶， 它们将质子泵出细胞， 酸化和骨吸收。组成性细胞器酸化 也被病毒利用，如流感，毒素， 白喉毒素和细胞病原体，如沙门氏菌， 入侵宿主细胞，这表明调节的能力， 隔室酸化可能对各种各样的 人类疾病状态。虽然细胞调节 酸化是相当复杂的，很明显，核心球员是 液泡H+ ATP酶。两种细胞中的H+-ATP酶非常相似， 真菌，植物和动物，无论是在整体结构和在 亚基的一级氨基酸序列。该提案的重点是 酿酒酵母的液泡H+-ATP酶， 目标是将有关酶的遗传信息连接起来， 酵母和生化特性来自其他工作 细胞类型。这些实验也将扩大 几个亚单位的特征还没有很好地- 研究了将产生亚基特异性抗体的集合 并在生物化学研究中用作探针， 天然酶的相互作用。为了解决结构问题- 在酶的外围部分的功能关系， 含有ATP水解的催化位点，结构基因 对于三个外周亚基将被随机诱变， 影响酶组装和功能的突变将被 鉴定膜部门的功能关系， 含有质子孔，将通过生物化学方法阐明 定义膜亚单位和细胞膜区域之间的相互作用， 这些亚单位对外周部分的结合很重要， ATP水解和质子转运的耦合。结构模型 由生物化学和遗传学方法产生的基因将是 通过亚基区域的定向诱变进行遗传学测试， 被认为是功能重要的基因。

The aim of the proposed research is to define the subunit structure and function relationships in vacuolar proton-translocating ATPases (H+- ATPases). Vacuolar H+-ATPases are found in all eukaryotic cells, where they may play both constitutive and specialized roles. Constitutive organelle acidification is involved in receptor-mediated endocytosis, targeting of proteins in the biosynthetic pathway, activation of zymogens, protein degradation, ion homeostasis, and uptake of basic compounds into regulated secretory granules. Specialized cells of the kidney and bone contain vacuolar H+-ATPases at the plasma membrane, where they pump protons out of the cell, resulting in urinary acidification and bone resorption. Constitutive organelle acidification is also exploited by viruses, such as influenza, toxins, including diphtheria toxin, and cellular pathogens, such as Salmonella,in invasion of the host cell, suggesting that the ability to regulate compartment acidification could have implications for a wide variety of human disease states. Although the cellular regulation of acidification is quite complex, it is clear that the central player is the vacuolar H+ATPase. Vacuolar H+-ATPases are remarkably similar in fungi, plants, and animals, both in overall structure and in the primary amino acid sequences of the subunits. This proposal focuses on the vacuolar H+-ATPase of the yeast Saccharomyces cerevisiae, with the goal of bridging the genetic information available about the enzyme in yeast and the biochemical characterization derived from work on other cell types. The experiments proposed will also extend the characterization of several of the subunits that have not been well- studied. A collection of subunit-specific antibodies will be generated and used as probes in biochemical studies aimed at defining subunit interactions in the native enzyme. In order to address structure- function relationships in the peripheral sector of the enzyme, which contains the catalytic sites for ATP hydrolysis, the structural genes for three of the peripheral subunits will be randomly mutagenized and mutations affecting assembly and function of the enzyme will be identified. The functional relationships in the membrane sector, containing the proton pore, will be elucidated by biochemicaIly defining interactions between the membrane subunits and the regions of these subunits important for binding of the peripheral sector and coupling of ATP hydrolysis and proton transport. Structural models generated by both the biochemical and genetic approaches will be genetically tested by directed mutagenesis of regions of the subunit genes believed to be functionally important.