High‐Throughput Yeast Screen to Identify Vacuolar H+‐ATPase subunit a specific inhibitors

Vacuolar H+‐ATPases (V‐ATPases) are multi‐subunit proteins critical for acidification of intracellular compartments. V‐ATPases are important for biological processes such as bone resorption and disease related processes including metastasis. V‐ATPase localization is determined by the subunit a isoform. There are four a isoforms in humans, a1‐a4. V‐ATPases at the plasma membrane of osteoclasts have a3 and those at the plasma membrane of certain renal cells have a4. Genetic defects in a3 and a4 lead to osteopetrosis and renal tubular acidosis, respectively. We would like to exploit the specificity of a isoform function to develop isoform specific inhibitors that can be used as therapeutics for diseases. A high‐throughput screen will be developed in which each of the human a isoforms will be expressed in a yeast strain lacking the endogenous a subunit genes. These yeast strains will be grown in the presence of compounds from small molecule libraries. Isoform‐specific inhibitors will be identified by their ability to selectively block growth at neutral pH in yeast strains expressing the appropriate a isoforms. Thus far our experiments indicate that expression of the human a subunit alone does not complement the loss of the yeast a subunits. Thus, to develop the inhibitor screen we must determine the minimal number of mammalian V0 subunits necessary to have a functional V‐ATPase containing the human a subunit.