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.
| Lu, Ming; Ammar, David; Ives, Harlan et al. (2007) Physical interaction between aldolase and vacuolar H+-ATPase is essential for the assembly and activity of the proton pump. J Biol Chem 282:24495-503 |
| Lu, Ming; Sautin, Yuri Y; Holliday, L Shannon et al. (2004) The glycolytic enzyme aldolase mediates assembly, expression, and activity of vacuolar H+-ATPase. J Biol Chem 279:8732-9 |
