POWRE: Molecular Genetic Approaches to Study Organellar Membrane Proteins

POWRE：研究细胞器膜蛋白的分子遗传学方法

• 批准号: 9752974
• 负责人: Sabeeha Merchant
• 金额: $ 1.89万
• 依托单位: University of California-Los Angeles
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9752974&HistoricalAwards=false
• 关键词: POWRE; Molecular; Genetic; Approaches; Study

9752974 Merchant This is a POWRE Visiting Professor award. The broad, long-term objective of the PI's research program is the understanding of the process of assembly of the chloroplast energy transducing membrane, with particular emphasis on how the cofactors and polypeptides are assembled to yield a specific functional arrangement in vivo. A recently-developed project deals with the c-type cytochromes, soluble c6 and membrane-anchored f of the b6/f complex, and cyt b6, an integral membrane subunit of the b6/f complex. This complex is not essential for heterotrophic growth in the experimental model organism Chlamydomonas reinhardtii, which allows exploitation of classical, molecular genetic, and biochemical approaches of the study of its assembly. Genetic analysis of C. reinhardtii cyt b6/f-deficient mutants has led to the definition of multiple loci which are required for chloroplast c-type cytochrome maturation (ccsA in the plastid genome, and CCS1 through CCS5 in the nuclear genome). Biochemical analysis of ccs strains indicates that the products of the CCS loci are required for conversion of apocytochromes to holocytochromes in the thylakoid lumen. The ccsA and Ccs1 genes have been cloned; their products encode low abundance thylakoid membrane proteins. A prime objective now is to undertake functional studies of these two newly-identified components of a putative thylakoid membrane-associated cytochrome c assembly complex. Of interest are the topology and orientation of these novel proteins, and the identification of other components of the complex. In the case of cyt b6, the products of at least four loci (CCB1-CCB4) are required specifically for post-translational heme insertion into one of the two heme binding sites. These are also envisioned to be thylakoid membrane proteins, since cyt b6 synthesis and assembly is entirely a membrane-associated process. The objective of this POWRE project is to access approaches and methodologies employed for the functional character ization of multi-component organellar membrane-protein complexes so that they may be applied to the study of products of the CCS and CCB loci. The experimental strategies employed for the definition of the mitochondrial protein translocases serve as precedents for the development of methods for the study of the thylakoid membrane c-type cytochrome synthesis complex. The PI will therefore engage in a 6-month collaboration with Dr. Carla Koehler in the laboratory of Dr. Gottfried Schatz at the Biozentrum, University of Basel, Switzerland. During this visit, the PI will participate in the functional analysis of genetically-identified components of a mitochondrial translocase required for the import of carrier proteins into the inner membrane. Direct involvement in this project will facilitate the acquisition of the expertise the PI needs to move her project on chloroplast cytochrome biogenesis beyond gene identification to the definition of the biochemistry of the gene products. ***

9752974商人这是POWRE客座教授奖。PI研究计划的广泛和长期目标是了解叶绿体能量转导膜的组装过程，特别强调辅因子和多肽如何在体内组装以产生特定的功能排列。最近开发的一个项目涉及b6/f复合体的c型细胞色素、可溶性c6和膜锚定的f，以及b6/f复合体的一个完整的膜亚单位Cyt b6。这种复合体对于实验模式生物莱茵衣藻的异养生长并不是必不可少的，它允许利用经典的、分子遗传学和生化方法来研究其组装。对莱茵梭菌细胞色素b6/f缺失突变体的遗传分析已经确定了叶绿体c型细胞色素成熟所需的多个基因座(叶绿体基因组中的CCSA，以及核基因组中的CCS1至CCS5)。对CCS菌株的生化分析表明，CCS基因座的产物是类囊体腔内脱细胞色素向全细胞色素转化所必需的。CcsA和Ccs1基因已被克隆，其产物编码低丰度的类囊体膜蛋白。现在的一个主要目标是对这两个新发现的与类囊体膜相关的细胞色素c组装复合体的成分进行功能研究。令人感兴趣的是这些新蛋白质的拓扑结构和方向，以及复合体的其他成分的鉴定。在Cyt b6的情况下，至少四个基因座(CCB1-CCB4)的产物是翻译后血红素插入两个血红素结合位点之一所必需的。这些也被认为是类囊体膜蛋白，因为细胞色素b6的合成和组装完全是一个与膜相关的过程。本POWRE项目的目的是获得用于研究多组分细胞膜-蛋白质复合体功能特性的途径和方法，以便将其应用于CCS和CCB基因座的产物研究。用来定义线粒体蛋白转位酶的实验策略为研究类囊体膜C型细胞色素合成复合体的方法提供了先例。因此，PI将与Carla Koehler博士在瑞士巴塞尔大学Biozentum的Gottfred Schatz博士的实验室进行为期6个月的合作。在这次访问期间，PI将参与对线粒体转位酶基因识别成分的功能分析，线粒体转位酶是将载体蛋白输入内膜所需的。直接参与这一项目将有助于PI获得所需的专业知识，将她的叶绿体细胞色素生物发生项目从基因鉴定转移到基因产品生物化学的定义上。***