SUBUNIT STRUCTURE AND FUNCTION IN VACUOLAR H+/ATPASES

拟议研究的目的是定义亚基结构和 液泡质子转运ATPase(H~+-ATPase)的功能关系 ATPase)。空泡H+-ATPase存在于所有真核细胞中，其中 他们既可以扮演结构性角色，也可以扮演特殊角色。构成要素 细胞器酸化参与了受体介导的内吞作用， 靶向生物合成途径中的蛋白质，激活 酵素、蛋白质降解、离子动态平衡和碱性吸收 化合物变成受调节的分泌颗粒。专门化的细胞 肾脏和骨骼的质膜上含有空泡H+-ATPase， 在那里它们将质子泵出细胞，导致尿液 酸化和骨吸收。构成细胞器酸化 也被病毒利用，如流感、毒素，包括 白喉毒素和细胞病原体，如沙门氏菌，在 对宿主细胞的入侵，表明有能力调节 隔室酸化可能会对多种类型产生影响 人类疾病的状态。尽管细胞调控对细胞的影响 酸化相当复杂，很明显，中心玩家是 液泡H+-ATPase。液泡H+-ATPase在 真菌、植物和动物，包括总体结构和 亚基的初级氨基酸序列。这项提案的重点是 酿酒酵母液泡型H~+-ATPase 目标是将有关该酶的遗传信息连接到 酵母菌及其衍生的生物化学特性 单元类型。拟议中的实验还将延长 几个亚基的特征一直不是很好- 学习。将产生亚基特异性抗体的集合 并在旨在定义亚基的生化研究中用作探针 天然酶中的相互作用。为了解决结构问题-- 酶的外周部分的功能关系，这是 包含三磷酸腺苷水解酶的催化部位，结构基因 其中三个外周亚基将被随机诱变并 影响酶的组装和功能的突变将是 已确认身份。膜部门的职能关系， 含有质子孔的，将通过生物化学来阐明 确定膜亚单位和区域之间的相互作用 这些亚基对结合外周扇区和 三磷酸腺苷水解与质子转运的耦合。结构模型 由生化和遗传方法产生的 通过亚基区域的定向突变进行遗传测试 被认为具有重要功能的基因。

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.