Aldolase-mediated Regulation of V-ATPase

醛缩酶介导的 V-ATP 酶调节

• 批准号: 6804964
• 负责人: MING LU
• 金额: $ 15.15万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6804964
• 关键词: SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; aldehyde lyase; autoradiography; chemical stability; densitometry; enzyme activity; fungal genetics; gene deletion mutation; immunoprecipitation; membrane transport proteins; molecular assembly /self assembly; nucleic acid sequence; protein protein interaction; protein structure function; protein transport; site directed mutagenesis; vesicle /vacuole; yeast two hybrid system

DESCRIPTION (provided by applicant): Vacuolar H+ATPases (V-ATPases or H+ATPases) are highly conserved proton pumps that couple hydrolysis of ATP to proton transport out of the cytosol. They are essential for renal acid-base homeostasis, for sorting of newly synthesized proteins in the Golgi, and for acidification and normal function of the yeast vacuole. Although a central question in the field is how V-ATPase is regulated under physiological conditions, until recently little was known about the underlying mechanisms. The glycolytic enzyme aldolase has been identified to interact with three subunits of V-ATPase by our lab. This represents the first example of physical association between the ATP-generating glycolytic pathway and an ATP-hydrolyzing ion pump. Deletion of the aldolase gene in yeast cells results in complete disassembly of and a dramatic reduction in V-ATPase. These abnormalities can be fully restored by aldolase complementation. Our data suggest that disruption of the interaction between aldolase and V-ATPase results in malfunction of V-ATPase, which leads to renal tubular acidosis found in patients with hereditary fructose intolerance, an autosomal recessive disorder caused by mutations in an isoform of aldolase. In this proposal, we will carry out molecular genetic analysis in yeast cells to examine the structural basis and regulation of the interaction between aldolase and V-ATPase, and test the hypothesis that aldolase mediates V-ATPase assembly, function and stability. The specific aims of this proposal are: 1) to generate aldolase and V-ATPase subunit mutants that lack binding for a specific interaction but retain aldolase enzymatic activity and/or binding to other V-ATPase subunits; 2) to express the aldolase and V-ATPase subunit mutants in yeast deletion mutant strains lacking either aldolase or a single subunit of V-ATPase, and examine the effects on V-ATPase assembly, function and stability; 3) to examine the parameters required for aldolase to bind intact V-ATPase and disassembled V-ATPase sectors. These studies will provide important insight into the molecular basis for metabolic control of proton transport.

描述（由申请人提供）：液泡H+ATPases（V-ATPase或H+ATPases）是高度保守的质子泵，将ATP水解二回到质子中，从细胞溶胶中输出。它们对于肾脏酸碱稳态，对于高尔基体中新合成的蛋白质以及酵母液泡的酸化和正常功能至关重要。尽管该领域的一个核心问题是如何在生理条件下调节V-ATPase，直到最近对基本机制知之甚少。我们的实验室已鉴定出糖酵解酶藻酶与V-ATPase的三个亚基相互作用。这代表了ATP生成的糖酵解途径和ATP - 氢化离子泵之间物理关联的第一个例子。酵母细胞中醛糖酶基因的缺失导致V-ATPase的完全拆卸和显着降低。这些异常可以通过醛酶互补完全恢复。我们的数据表明，醛糖酶和V-ATPase之间相互作用的破坏会导致V-ATPase的故障，这导致肾上腺果糖不耐症患者发现肾小管酸中毒，这是一种由藻酶同一个酶的同一个糖酶突变引起的常染色体隐性隐性疾病。在该建议中，我们将在酵母细胞中进行分子遗传分析，以检查醛糖酶与V-ATPase之间相互作用的结构基础和调节，并检验醛糖酶介导V-ATPase组装，功能和稳定性的假设。该提案的具体目的是：1）生成缺乏特定相互作用的结合但保留醛糖酶酶活性和/或与其他V-ATPase亚基结合的醛酶和V-ATPase亚基突变体； 2）在酵母缺失突变体中表达缺乏醛糖酶或单个V-ATPase亚基的酵母缺失突变体中的醛酶和V-ATPase亚基突变体，并检查对V-ATPase组装，功能和稳定性的影响； 3）检查藻酶结合完整的V-ATPase和拆卸V-ATPase扇区所需的参数。这些研究将为质子转运代谢控制的分子基础提供重要的见解。