Mitochondrial rhomboid function in yeast

酵母中线粒体菱形功能

• 批准号: 7981030
• 负责人: DONNA M GORDON
• 金额: $ 41.47万
• 依托单位: MISSISSIPPI STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7981030
• 关键词: Academic Research Enhancement Awards; Alleles; Apoptosis; Biochemical; Biochemical Genetics; Biogenesis; Biological Assay; Biological Models; Cell Survival; Cells; Code; Complement; Complex; Data; Dependence; Dynamin; Ensure; Event; Future; Generations; Genes; Genetic; Goals; Human; Inner mitochondrial membrane; Link; Lipids; Maintenance; Measures; Membrane; Methodology; Methods; Mission; Mitochondria; Modeling; Molecular; Morphology; Nature; Neuropathy; Organelles; Pathway interactions; Peptide Hydrolases; Physiology; Population; Positioning Attribute; Precipitation; Process; Property; Protein Import; Protein Subunits; Proteins; Role; Saccharomyces cerevisiae; Scaffolding Protein; Site; Steroids; Structure; Students; Sucrose; Techniques; Temperature; Testing; Transport Process; Ultracentrifugation; Western Blotting; Yeasts; aminoacid biosynthesis; base; design; fatty acid metabolism; migration; mutant; numb protein; overexpression; protein complex; public health relevance; rhomboid; tool

DESCRIPTION (provided by applicant): Mitochondria are essential organelles within cells. They are positioned at the center of numerous cellular events including ATP generation, steroid and amino acid biosynthesis, fatty acid metabolism, Fe-S cluster assembly and apoptosis. Maintenance of 'normal' mitochondrial biogenesis is therefore critical to cell viability. One feature that has emerged as a key regulated step in mitochondrial physiology is the maintenance of organelle morphology. A number of proteins identified for their role in controlling organelle dynamics have been shown to also influence the transport, processing, and assembly of mitochondrial preproteins. One such protein is the mitochondrial inner membrane peptidase Pcp1p. In yeast, Pcp1p has been linked to the processing of the dynamin-related protein Mgm1p. Mgm1p processing is dependent not only on Pcp1p activity but also on a functional TIM23- PAM translocation complex suggesting a functional link between the two. These results form the basis of the hypothesis tested in our proposal. Our preliminary data shows that Pcp1p is a subunit of a large protein complex. The broad objective of this proposal is to identify subunits of this complex, while directly testing for a direct physical linkage between Pcp1p and subunits of the TIM23 translocon. Using Saccharomyces cerevisiae as our model system, we will utilize two independent methodologies to identify and characterize protein components of the Pcp1p complex. In a biochemical approach, sucrose gradient ultracentrifugation and Blue-Native PAGE analysis will be carried out to characterize the biophysical properties of this complex. Co-migration of Pcp1p with components of the TIM23 translocon will be measured by Western blot analysis. Proteins shown to co-purify with Pcp1p will be identified by MALDI-TOF. These biochemical methods will be complemented by classic genetics approaches designed to identify genes that code for proteins involved in the formation of the Pcp1p complex. We propose to generate temperature sensitive alleles of PCP1 to be used for the isolation of overexpression suppressors. Putative candidates identified using both of these methods will likely include protein subunits of the Pcp1p complex, accessory proteins involved in substrate recognition, scaffolding proteins involved in Pcp1p complex assembly, and/or protein subunits of the TIM23 translocon. Our results will help build a model for the functional organization of the yeast Pcp1p peptidase within the mitochondrial inner membrane and its interactions with the preprotein translocation machinery. In accordance with the mission of the R15 program, the simplicity of both the model system and the techniques used throughout the proposal will ensure that undergraduate students will be able to make significant contributions to this proposal. PUBLIC HEALTH RELEVANCE: It is becoming increasingly evident that mitochondrial function is controlled by the intersection of multiple pathways including those involved in directing mitochondrial dynamics and preprotein transport. This study's aim is to provide evidence for a physical link between components of the preprotein transport machinery and proteins involved in regulating organelle structure. Understanding the molecular nature of these interactions will provide us with a basis for the design of future treatments targeting various mitochondrial neuropathies in humans.

描述（由申请人提供）：线粒体是细胞内的重要细胞器。它们处于许多细胞事件的中心，包括 ATP 生成、类固醇和氨基酸生物合成、脂肪酸代谢、Fe-S 簇组装和细胞凋亡。因此，维持“正常”线粒体生物发生对于细胞活力至关重要。作为线粒体生理学中关键调节步骤的一个特征是细胞器形态的维持。许多在控制细胞器动力学中发挥作用的蛋白质已被证明也影响线粒体前蛋白的运输、加工和组装。其中一种蛋白质是线粒体内膜肽酶 Pcp1p。在酵母中，Pcp1p 与动力相关蛋白 Mgm1p 的加工有关。 Mgm1p 加工不仅取决于 Pcp1p 活性，还取决于功能性 TIM23-PAM 易位复合物，表明两者之间存在功能联系。这些结果构成了我们提案中测试的假设的基础。我们的初步数据表明，Pcp1p 是大型蛋白质复合物的一个亚基。该提案的主要目标是识别该复合物的亚基，同时直接测试 Pcp1p 和 TIM23 易位子亚基之间的直接物理联系。使用酿酒酵母作为我们的模型系统，我们将利用两种独立的方法来识别和表征 Pcp1p 复合物的蛋白质成分。在生化方法中，将进行蔗糖梯度超速离心和 Blue-Native PAGE 分析来表征该复合物的生物物理特性。 Pcp1p 与 TIM23 易位子成分的共迁移将通过蛋白质印迹分析来测量。显示与 Pcp1p 共纯化的蛋白质将通过 MALDI-TOF 进行鉴定。这些生化方法将得到经典遗传学方法的补充，这些方法旨在识别编码参与 Pcp1p 复合物形成的蛋白质的基因。我们建议生成 PCP1 的温度敏感等位基因，用于分离过表达抑制因子。使用这两种方法鉴定的假定候选者可能包括 Pcp1p 复合物的蛋白质亚基、参与底物识别的辅助蛋白、参与 Pcp1p 复合物组装的支架蛋白和/或 TIM23 易位子的蛋白质亚基。我们的结果将有助于建立线粒体内膜内酵母 Pcp1p 肽酶的功能组织及其与前蛋白易位机制相互作用的模型。根据 R15 计划的使命，模型系统和整个提案中使用的技术的简单性将确保本科生能够对此提案做出重大贡献。 公共健康相关性：越来越明显的是，线粒体功能是由多种途径的交叉控制的，包括那些参与指导线粒体动力学和前蛋白转运的途径。这项研究的目的是为前蛋白转运机制的组成部分和参与调节细胞器结构的蛋白质之间的物理联系提供证据。了解这些相互作用的分子性质将为我们设计未来针对人类各种线粒体神经病的治疗方法奠定基础。