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

液泡H-ATPase(V-ATPase)是细胞正常生长、细胞器酸化和离子稳态所需的质子泵，由外周复合体V1和膜结合复合体V0组成。本研究旨在确定V0复合体亚基d在酿酒酵母中的作用和功能。酵母为利用遗传学、细胞学和生物化学技术探索V-ATPase的组装、催化和调控提供了一个极好的系统。具体地说，d亚基保守区域中的氨基酸替换将被用来探索这些区域的结构和功能重要性。亚基d与V1和V0的相互作用将通过在携带完整V-ATPase的细胞(野生型)、单独的V0(Vma2)和单独的V1(Vma3)中过表达来进一步探索。这些研究将有助于阐明V-ATPas的结构和调控机制。广泛影响：本项目旨在为理解V-ATPase和加强本科教育做出重大贡献。后一个目标包括通过在生物化学实验室课程中加入研究性实验来加强本科课程。这项活动将在课堂上提供批判性思维和科学发现的机会。将鼓励学生参与，并有可能获得学术学分、津贴、奖励，并与人合著出版相关出版物。该项目的内在目标是通过促进科学方面代表性不足的学生的参与和留住来增强多样性。该项目还将包括高中生的暑期研究。这些活动将提高鲍尔州立大学的学生留存率，并鼓励多样性。

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.