COATED VESICLE PROTON PUMP

这项建议的长期目标是了解 (H)-空泡家族的结构、机制及调控 ATPase(或V-ATPase)。V-ATPase负责酸化 在真核细胞中的细胞内隔间，并为重要的 在许多细胞过程中发挥作用，包括受体介导的 内吞作用、细胞内膜运输、大分子降解 以及小分子的加工和耦合传输。我们的 以往的研究主要集中在V-ATPase的结构分析上 并提供了对它的洞察力 胞内分布。我们也已经开始解决这一功能 V-ATPase复合体的亚基组成和特定残基在细胞内的作用 催化和调节。 我们先前已经确定73 kDa A亚基中的Cys254是 V-ATPase对巯基敏感的残基 试剂，并通过以下途径提供其参与的证据 V-ATPase活性调节中二硫键的形成。为了进一步 确定位于A上的核苷酸结合位点的结构 亚基，Cys254的二硫键结合伙伴将被鉴定并 将确定与[~(32)P]-2-叠氮基-三磷酸腺苷的反应部位。B类 用[32P]-BZ-ATP标记的亚基多肽也将被鉴定。这个 外周V1结构域中特定亚基的功能将为 通过使用蛋白质分离解离的V1多肽来解决 化学和免疫化学技术与分离的重组 利用V0整合域的不同组合中的多肽 这个方案以前是在这个实验室制定的。重新组装 还将执行将V0域从其分离的组件中积分出来 为了解决特定的V0亚基在质子传导中的作用 以及被动质子通量的调节机制。实验 还将进行巴菲罗星结合部位的定位。 最后，V-ATPase在MDBK细胞中的组装途径为 用[35S]蛋氨酸对V-ATPase进行代谢标记 和标记泵的免疫沉淀，使用可用的 抗人工合成单抗和多克隆抗血清的制备 多肽。这些研究应该会为以下方面提供重要的新见解 这类重要的(H)-ATPase。

The long-term objectives of this proposal are to understand the structure, mechanism and regulation of the vacuolar family of (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 number of cellular processes, including receptor-mediated endocytosis, intracellular membrane traffic, macro-molecular degradation and processing and the coupled transport of small molecules. Our previous studies have focused on a structural analysis of the V-ATPase of clathrin-coated vesicles and have provided insight into its intracellular distribution. We have also begun to address the function of subunits in the V-ATPase complex and the role of specific residues in catalysis and regulation. We have previously identified Cys254 of the 73 kDa A subunit as the residue responsible for the sensitivity of the V-ATPases to sulfhydryl reagents and have presented evidence for its involvement through disulfide bond formation in regulation of V-ATPase activity. To further define the structure of the nucleotide binding site located on the A subunit, the disulfide bonded partner of Cys254 will be identified and the sites of reaction with [32P]-2-azido-ATP will be determined. B subunit peptides labeled by [32P]-Bz-ATP will also be identified. The function of specific subunits in the peripheral V1 domain will be addressed by separation of the dissociated V1 polypeptides using protein chemical and immunochemical techniques and reassembly of the separated polypeptides in different combinations with the integral V0 domain using the protocol previously worked out in this laboratory. Reassembly of the integral V0 domain from its separated components will also be carried out in order to address the role of specific V0 subunits in proton conduction and the mechanism by which passive proton flux is regulated. Experiments to localize the site of bafilomycin binding will also be performed. Finally, the assembly pathway of the V-ATPase in MDBK cells will be determined by metabolic labeling of the V-ATPase with [35S] methionine and immunoprecipitation of the labeled pumps using both available monoclonal antibodies and polyclonal antisera prepared against synthetic peptides. These studies should provide significant new insights into this important class of (H+)-ATPases.