STRUCTURE AND FUNCTION OF THE VACUOLAR ATPASE

液泡ATP酶是一种存在于内膜中的大型复合酶 真核细胞系统。 它的作用是酸化 几个细胞器，例如，溶酶体和分泌囊泡，并产生 电化学梯度驱动跨细胞器的运输 膜的 我们已经描述了液泡膜中ATP酶的特征， 他们已经分离出编码三种主要的 亚单位。 该酶似乎至少有六个额外的亚基。 本研究的目的是：（1）分离其他多肽 与ATP酶活性共纯化，获得部分氨基酸序列， 构建寡核苷酸探针，分离相应基因。 (2)利用遗传学来确定基因的数量， 合成液泡ATP酶。 (3)为了检查低聚物 电子显微镜下的ATP酶结构，使用抗体进行映射 几种多肽的位置。 (4)检查染色体 位置，外显子/内含子结构，以及 液泡ATP酶基因的转录本。 对这种酶的研究是重要的，因为它的核心作用是 在许多细胞器中发挥作用。 配体的解偶联和 内体中的受体，分泌中神经递质的包装， 囊泡和溶酶体中酶的水解作用都依赖于 对液泡ATP酶活性的影响。 此外，空泡型 据报道，ATP酶位于破骨细胞的质膜上， 它在骨重建中起着重要作用， 在某些肾细胞中，它在尿液酸化中起作用。

The vacuolar ATPase is a large complex enzyme found in the endomembrane system of eucaryotic cells. It functions to acidify the interior of several organelles, e.g., lysosomes and secretory vesicles, and generates an electrochemical gradient to drive transport across the organellar membrane. We have characterized the ATPase in vacuolar membranes from Neurospora and have isolated the genes that encode three of the major subunits. The enzyme appears to have at least six additional subunits. The goals of this proposal are: (1) To isolate the other polypeptides which copurify with ATPase activity, obtain partial amino acid sequence, construct oligonucleotide probes, and isolate the corresponding genes. (2) To use genetics to determine the number of genes essential for synthesizing the vacuolar ATPase. (3) To examine the oligomeric structure of the ATPase by electron microscopy, using antibodies to map the location of several polypeptides. (4) To examine the chromosomal location, exon/intron structure, and the abundance and size of transcripts of vacuolar ATPase genes. Investigation of this enzyme is important because of the central role it plays in the function of many organelles. The uncoupling of ligands and receptors in endosomes, the packaging of neurotransmitters in secretory vesicles, and the hydrolytic action of enzymes in lysosomes all depend on the activity of the vacuolar ATPase. Furthermore, a vacuolar-type ATPase has been reported on the plasma membranes of osteoclasts, where it plays an important role in bone remodeling, and on the plasma membrane of some renal cells, where it plays a role in urinary acidification.