POWRE: Molecular Genetic Approaches to Study Organellar Membrane Proteins

9752974商人这是power客座教授奖。PI的研究计划的广泛，长期目标是了解叶绿体能量转导膜的组装过程，特别强调如何在体内组装辅因子和多肽以产生特定的功能安排。最近开发的一个项目涉及c型细胞色素，b6/f复合物的可溶性c6和膜锚定f，以及b6/f复合物的完整膜亚基cyt b6。这种复合物对于实验模式生物莱茵衣藻的异养生长并不是必需的，这允许利用经典的、分子遗传学的和生物化学的方法来研究它的组装。通过对莱茵梭菌胞浆b6/f缺陷突变体的遗传分析，确定了叶绿体c型细胞色素成熟所需的多个基因座（质体基因组中的ccsA，核基因组中的CCS1至CCS5）。ccs菌株的生化分析表明，ccs基因座的产物是类囊体腔内单核细胞色素向全细胞色素转化所必需的。已克隆出ccsA和Ccs1基因；它们的产物编码低丰度的类囊体膜蛋白。现在的主要目标是对假定的类囊体膜相关细胞色素c组装复合物的这两种新鉴定的组分进行功能研究。令人感兴趣的是这些新蛋白质的拓扑结构和取向，以及该复合物的其他组分的鉴定。在cyt b6中，至少需要四个位点（CCB1-CCB4）的产物才能在翻译后将血红素插入到两个血红素结合位点之一。这些也被设想为类囊体膜蛋白，因为cyt b6的合成和组装完全是一个与膜相关的过程。这个power项目的目标是获取用于多组分细胞器膜-蛋白质复合物功能特征的方法和方法，以便它们可以应用于CCS和CCB位点的产物的研究。用于定义线粒体蛋白转位的实验策略为研究类囊体膜c型细胞色素合成复合物的方法的发展提供了先例。因此，PI将与瑞士巴塞尔大学生物中心Gottfried Schatz博士实验室的Carla Koehler博士进行为期6个月的合作。在这次访问中，PI将参与线粒体转位酶的遗传鉴定成分的功能分析，该转位酶是将载体蛋白导入内膜所必需的。直接参与该项目将有助于获得PI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物的生物化学定义。＊＊＊

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. ***