CAREER: Structural-functional Analysis of the V-ATPase Subunit d by Site-directed Mutagenesis and Overexpression in Yeast

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Vacuolar H+-ATPases (V-ATPases) are proton pumps required for normal cell growth, organelle acidification, and ion homeostasis, and they consist of a peripheral complex, V1, and a membrane-bound complex, V0. This research focuses on establishing the roles and functions of the subunit d of the V0 complex in the yeast Saccharomyces cerevisiae. Yeast provides an excellent system to explore V-ATPase assembly, catalysis, and regulation by using genetics, cellular, and biochemical techniques. Specifically, amino acid substitutions in conserved regions of subunit d will be used to probe the structural and functional importance of these regions. Subunit d interactions with V1 and V0 will be further explored by overexpressing subunit d in cells that carry intact V-ATPases (wild-type), V0 alone (vma2), and V1 alone (vma3). These studies will contribute to the elucidation of both the structure and regulatory mechanisms of V-ATPases.Broader Impact: This project is designed to make significant contributions to both the understanding of V-ATPases and to strengthening of undergraduate education. The latter goal includes enhancing the undergraduate curriculum by including research-based experiments in the biochemistry lab courses. This activity will provide opportunities for critical thinking and scientific discovery in the classroom. Student participation will be encouraged with possibilities for academic credit, stipends, awards, and co-authorship of resulting publications. Intrinsic to the project is the goal to enhance diversity by fostering the participation and retention of students that are underrepresented in sciences. The project will also involve summer research by high school students. These activities would enhance student retention at Ball State University and encourage diversity.