Functional Analysis of the Oxa1p Export Machinery of Yeast Mitochondria

线粒体Oxa 1蛋白是保守的Alb 3/Oxa 1/YidC蛋白家族的成员，其参与将蛋白质插入整个原核生物和真核生物的生物膜中。Oxa 1的存在是插入来自线粒体基质并整合到内膜中的一组细胞核和细胞膜编码蛋白所必需的。细胞色素氧化酶（考克斯）亚基，特别是Cox 1和Cox 2，强烈依赖于Oxa 1的插入。Oxa 1在其膜插入中的直接作用通过以下证明而提出：Oxa 1在其作为新生多肽链的合成期间与考克斯亚基物理相互作用，即以共翻译的方式。Stuart博士的实验室最近提供了Oxa 1和线粒体之间直接物理相互作用的证据，并提出这种密切的联系确保了翻译和膜插入事件之间的紧密耦合。Oxa 1与核糖体相互作用的能力已被发现是至关重要的共同翻译膜插入的翻译编码的考克斯亚基。此外，Stuart博士已经能够证明线粒体大核糖体亚基的两个亚基Mrp 20和MrpL 40与Oxa 1非常接近。Mrp 20与细菌核糖体蛋白L23同源，位于核糖体的新生链出口位点，并参与陪伴和将新生链靶向Sec易位子。Stuart博士的分析表明，MrpL 40可能与细菌L24蛋白同源，L24蛋白是位于新生链出口位点的另一个核糖体亚基。这里提出的初步证据表明，MrpL 40不仅在物理上，而且在功能上，与Oxa 1蛋白相互作用。阐明Oxa 1在线粒体DNA编码的考克斯亚基的膜插入及其与线粒体的相互作用中的作用是该研究的主要焦点。 在这个项目中，Stuart博士将进一步分析Oxa 1-核糖体相互作用，并旨在识别和表征与Oxa 1物理相互作用的线粒体蛋白。这些核糖体蛋白在靶向翻译核糖体的Oxa 1位点和翻译和膜插入事件的耦合中的作用将被探索。该项目的目标是：1。探讨Oxa 1与核糖体大亚基蛋白Mrp 20和MrpL 40之间的物理和功能相互作用。2.对Oxa 1与核糖体相互作用的重要区域进行遗传表征。3.分析其他预测位于核糖体新生链出口位点的蛋白质是否可能与Oxa 1发生功能性相互作用。拟定活性的知识价值：Oxa 1蛋白是进化上保守的Oxa 1/YidC/Alb 3蛋白家族的成员，参与蛋白质插入不同细胞膜。阐明线粒体Oxa 1复合物的作用机制对于进一步理解线粒体中的膜蛋白插入事件是重要的。此外，由于Oxa 1/YidC/Alb 3家族成员的保守性，从Oxa 1的拟议研究中获得的信息将为细菌和类囊体膜中的蛋白质插入事件提供重要的信息。拟议活动产生的更广泛影响：蛋白质靶向和膜插入事件机制的阐明对生物膜如何建立和保持其特异性和特性的基本细胞问题具有重要意义。拟议工作的结果将在国际科学会议上传播，并在同行评审的科学期刊上发表。私家侦探坚定地致力于教育和培养未来的科学家，无论是在本科和研究生水平。因此，她有意识地选择领导一个面向博士生和本科生的研究小组。到目前为止，许多本科生和研究生都在P.I.的实验室接受了培训，这个项目将作为进一步培训学生的工具。 私家侦探也致力于多样性，并已招募，目前正在指导少数民族（非洲裔美国人）博士生。

The mitochondrial Oxa1 protein is a member of the conserved Alb3/Oxa1/YidC protein family involved in the insertion of proteins into biological membranes throughout prokaryotes and eukaryotes. The presence of Oxa1 is required for the insertion of a subset of nuclearly- and mitochondrially-encoded proteins, coming from the mitochondrial matrix and integrating into the inner membrane. The mitochondrially encoded cytochrome oxidase (COX) subunits, Cox1 and Cox2 in particular, are strongly dependent on Oxa1 for their insertion. A direct role for Oxa1 in their membrane insertion was suggested by the demonstration that Oxa1 physically interacts with the COX subunits during their synthesis as nascent polypeptide chains, i.e. in a co-translational fashion. Dr. Stuart's laboratory has recently provided evidence for a direct physical interaction between Oxa1 and the mitoribosome and propose that this close association ensures a tight coupling between the translation and membrane insertion events. The ability of Oxa1 to interact with the ribosome has found to be critical for the co-translational membrane insertion of the mitochondrially encoded COX subunits. Moreover, Dr. Stuart has been able to show that two subunits of the mitochondrial large ribosomal subunit, Mrp20 and MrpL40, exist in close proximity to Oxa1. Mrp20 is homologous to bacterial ribosome protein L23, located at the nascent chain exit site of the ribosome and is involved in chaperoning and targeting the emerging nascent chain to the Sec translocon. Dr. Stuart's analysis indicates that MrpL40 may be homologous to the bacterial L24 protein, another ribosomal subunit located at the nascent chain exit site. Preliminary evidence presented here demonstrates that MrpL40 not only physically, but also functionally, interacts with the Oxa1 protein. The elucidation of the role of Oxa1 in the membrane insertion of the mtDNA encoded COX subunits and its interaction with the mitoribosome is a major focal point of the proposed research. In this project, Dr. Stuart will further analyze the Oxa1-ribosome interaction and aim to identify and characterize mitoribosome proteins which physically interact with Oxa1. The role of these ribosomal proteins in targeting the translating ribosome to the Oxa1 site and in the coupling of translation and membrane insertion events shall be explored. The goals of the project are: 1. To explore the physical and functional interaction between Oxa1 and the ribosomal large subunit proteins Mrp20 and MrpL40. 2. To genetically characterize regions of Oxa1 important for interacting with the ribosome. 3. To analyze if other proteins predicted to be located at the nascent chain exit site of the ribosome may functionally interact with Oxa1. Intellectual merit of proposed activity: The Oxa1 protein is a member of the evolutionarily conserved Oxa1/YidC/Alb3 protein family involved in the insertion of proteins into diverse cellular membranes. Elucidation of the mechanism of action of the mitochondrial Oxa1 complex is important to further the understanding of membrane protein insertion events in mitochondria. Moreover, information gained from the proposed study of Oxa1 will shed important light on protein insertion events in the bacterial and thylakoid membranes, due to the conservation of Oxa1/YidC/Alb3 family members. Broader impacts resulting from the proposed activity: The elucidation of mechanisms of protein targeting and membrane insertion events have important implications for the fundamental cellular question of how biological membranes establish and maintain their specificity and identity. The results from the proposed work will be disseminated both at international scientific meetings and published in peer-reviewed scientific journals. The P.I. is strongly committed to the education and training of future scientists, both at undergraduate and post-graduate levels. She has therefore consciously chosen to lead a PhD student- and undergraduate-oriented research group. To date, many undergraduate and graduate students have received training in the lab of the P.I., and this project will serve as a vehicle for further student training. The P.I. is also committed to diversity and has recruited and is currently mentoring a minority (African American) PhD student.