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

职业：通过酵母中的定点诱变和过表达对 V-ATP 酶亚基 d 进行结构功能分析

• 批准号: 0728833
• 负责人: Karlett Parra
• 金额: --
• 依托单位: University of New Mexico Health Sciences Center
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-10-31 至 2009-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0728833&HistoricalAwards=false
• 关键词: CAREER; Structural; functional; Analysis; ATPase

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.

液泡H+- atp酶（v - atp酶）是正常细胞生长、细胞器酸化和离子稳态所必需的质子泵，它们由外周复合物V1和膜结合复合物V0组成。本研究的重点是确定V0复合体的亚基d在酿酒酵母中的作用和功能。酵母提供了一个很好的系统来探索v - atp酶的组装、催化和调控，通过遗传学、细胞和生化技术。具体来说，将使用亚基d保守区域的氨基酸替换来探测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-ATPases（野生型）、V0单独（vma2）和V1单独（vma3）的细胞中过表达亚基d进一步探索。这些研究将有助于阐明v - atp酶的结构和调控机制。更广泛的影响：该项目旨在对v - atp酶的理解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。鼓励学生参与，并有可能获得学分、津贴、奖励和合作出版的成果。该项目的内在目标是通过促进参与和保留在科学领域代表性不足的学生来增强多样性。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保留率，并鼓励多样性。