STRUCTURE OF THE VACUOLAR ATPase

描述（由申请人提供）：液泡atp酶（v - atp酶；v1vo - atp酶）是在真核生物的膜系统中发现的大型多亚基蛋白复合物，其功能是酸化亚细胞区室的内部。在高等真核生物的极化细胞中，液泡atp酶也可以在质膜中发挥作用，以便将质子泵送到细胞外。液泡atp酶的质子泵送作用涉及大量的细胞内和细胞间过程，如受体介导的内吞作用、蛋白质运输、pH稳态、代谢物的储存和神经递质释放。鉴于其广泛存在的性质，越来越多的基本疾病如糖尿病、癌症、骨质疏松症和艾滋病被发现与人类空泡atp酶缺陷有关就不足为奇了。我们正在通过电子显微镜、x射线晶体学和溶液核磁共振波谱学以及其他生物物理技术研究这种重要酶的结构。在这个项目的第一个资助期，我们已经生成了哺乳动物和酵母液泡atp酶的三维结构模型。此外，通过差分作图、免疫标记和x射线晶体结构的拟合，我们已经能够确定v - atp酶复合物的亚基结构。我们现在建议扩展我们的结构研究，并使用各种独立的技术来获得哺乳动物和酵母空泡atp酶以及来自古细菌的空泡样atp酶的高分辨率结构数据。此外，我们建议测试一些关于atp酶驱动的液泡atp酶的质子泵送活性在体内调节机制的假设。本研究的具体目标是：1)研究外周茎的结构和功能；2)阐明v1 - atp酶结构域活性沉默的机制；3)通过电子晶体学确定酵母VO结构域的亚基结构。从液泡型ATP酶的高分辨率结构数据将有望更好地理解ATP水解驱动质子泵送的催化机制和体内调节酶活性的可逆解离/再结合机制。

DESCRIPTION (provided by applicant): Vacuolar ATPases (V-ATPases; V1VO-ATPases) are large, multi subunit protein complexes found in the endomembrane system of eukaryotic organisms where they function to acidify the interior of subcellular compartments. In polarized cells of higher eukaryotes, the vacuolar ATPase can also function in the plasma membrane in order to pump protons to the outside of the cell. The proton pumping action of the vacuolar ATPase is involved in a large number of intra- and inter cellular processes such as receptor mediated endocytosis, protein trafficking, pH homeostasis, storage of metabolites and neurotransmitter release. Given its widespread nature, it is not surprising that more and more diseases as fundamental as diabetes, cancer, osteoporosis and AIDS are found to be associated with a defective human vacuolar ATPase. We are studying the structure of this important enzyme by electron microscopy, X-ray crystallography and solution nuclear magnetic resonance spectroscopy as well as other biophysical techniques. In the first funding period for this project, we have generated three-dimensional structural models of the mammalian- and yeast vacuolar ATPase. Furthermore, by using difference mapping, immuno labeling and fitting of X-ray crystal structures, we have been able to determine the subunit architecture of the V-ATPase complex. We are now proposing to extend our structural studies and to use a variety of independent techniques to obtain high resolution structural data for the mammalian- and yeast vacuolar ATPase and for the vacuolar like ATPase from Archaea. Furthermore, we propose to test a number of hypotheses regarding the mechanism by which the ATPase driven proton pumping activity of the vacuolar ATPase is regulated in vivo. The specific aims for this competing continuation proposal are: 1) to investigate the structure and function of the peripheral stalk(s), 2) to elucidate the mechanism of activity silencing in the V1-ATPase domain 3) to determine the subunit architecture of the yeast VO domain by electron crystallography. From the high resolution structural data for the vacuolar ATPase will hope to better understand the catalytic mechanism of ATP hydrolysis powered proton pumping and the mechanism of reversible dissociation/reassociation by which the enzyme's activity is regulated in vivo.