Molecular Mechanisms of Mitochondrial DNA Inheritance

线粒体DNA遗传的分子机制

• 批准号: 9724143
• 负责人: Jodi Nunnari
• 金额: $ 10.95万
• 依托单位: University of California-Davis
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-10-01 至 2001-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9724143&HistoricalAwards=false
• 关键词: Molecular; Mechanisms; Mitochondrial; DNA; Inheritance

9724143 Nunnari Accurate transmission of subcellular organelles during eukaryotic cell division is an essential and regulated process. In the case of the semi-autonomous respiratory organelle, the mitochondrion, this process is more complicated because, in order for progeny cells to be respiratory competent, both the organelle and its genome must be accurately transmitted. Although it is widely accepted that during cell division the partitioning of mitochondria is an active process dependent on the cytoskeleton, one fundamental question that remains unanswered is what additional mechanisms are required for the partitioning and distribution of mitochondrial DNA (mtDNA) both within the organelle and to daughter cells. The long term goal of the proposed work is to determine the cellular and molecular mechanisms responsible for mtDNA inheritance. These questions will be addressed using the simple eukaryote, Saccharomyces cerevisiae (budding yeast). From a wealth of genetic data obtained using this organism, it is clear that mtDNA inheritance is a non-random process. The ability to visualize both the organelle and mtDNA in vivo, and the opportunity to combine both genetic and biochemical approaches to understanding function, make S. cerevisiae an excellent model system for examining the cellular and molecular mechanisms involved in mitochondrial inheritance. It has recently been shown that when S. cerevisiae haploid cells fuse, mitochondrial fusion also occurs and a single, continuous dynamic mitochondrial reticulum is created in the zygote. While mitochondrial matrix proteins are able to freely diffuse within this reticulum, the diffusion of inner membrane-associated mtDNA is severely restricted. Although the movement of mtDNA within the organelle is restricted, mtDNA does enter emerging buds with the mitochondria. Elucidating the molecular mechanisms underlying mtDNA limited diffusion is critical to understanding the exact mechanisms for mtDNA inheri tance. The specific aims of this proposal are directed at further characterizing this mechanism and at identifying and determining the mechanisms of action of the proteins in S. cerevisiae that mediate mtDNA inheritance. Specifically, mtDNA movement within cells will be characterized by time lapsed imaging of a fluorescently tagged mtDNA-binding protein. The molecular components responsible for the limited diffusion and segregation of mtDNA will be identified using both genetic and biochemical approaches. Conditional mutants that are unable to stably maintain the mitochondrial genome during mitosis will be isolated. The mtDNA-protein complexes will be purified and characterized biochemically. Associated proteins will be identified using tandem mass spectrometry. The role of these proteins in mtDNA segregation will be determined using genetic, biochemical, and microscopic approaches. When cells divide, the various essential components (genes, cytoplasm, organelles) of the cell must be apportioned among the two daughter cells. In the case of the mitochondrion, the organelle possesses its own genome (in the form of multiple copies of mitochondrial DNA), and this must also be apportioned. Prior work has shown that the mtDNA distributes non-randomly, suggesting that mechanisms exist to somehow tether the DNA so that the copies do not intermix spatially within the organelle. This project will shed light on just how this occurs. ***

9724143 Nunnari在真核细胞分裂过程中，亚细胞器的准确传递是一个必要的和受调控的过程。对于半自主的呼吸细胞器，即线粒体，这个过程更加复杂，因为为了让后代细胞具有呼吸能力，细胞器及其基因组都必须被准确地传递。尽管人们普遍认为，在细胞分裂过程中，线粒体的分裂是一个依赖于细胞骨架的活跃过程，但一个基本的问题是，线粒体DNA(MtDNA)在细胞器内和子细胞中的分裂和分布需要什么额外的机制才能解决。这项拟议工作的长期目标是确定线粒体DNA遗传的细胞和分子机制。这些问题将用简单的真核生物--酿酒酵母(芽殖酵母)来解决。从利用这种有机体获得的大量遗传数据中，很明显线粒体DNA的遗传是一个非随机的过程。在体内显示细胞器和线粒体DNA的能力，以及结合遗传和生化方法了解功能的机会，使酿酒酵母成为研究线粒体遗传所涉及的细胞和分子机制的极好的模型系统。最近的研究表明，当酿酒酵母单倍体细胞融合时，线粒体也会发生融合，并在受精卵中产生一个单一的、连续的动态线粒体网。虽然线粒体基质蛋白能够在这个网状结构内自由扩散，但与内膜相关的mtDNA的扩散受到严格限制。尽管线粒体DNA在细胞器内的运动受到限制，但线粒体确实进入了萌芽。阐明线粒体DNA限制性扩散的分子机制对于理解线粒体DNA遗传的确切机制至关重要。这项建议的具体目的是为了进一步表征这一机制，并识别和确定酿酒酵母中介导线粒体DNA遗传的蛋白质的作用机制。具体地说，线粒体DNA在细胞内的移动将通过对带有荧光标记的线粒体DNA结合蛋白的延时成像来表征。将使用遗传和生化方法确定造成线粒体DNA有限扩散和分离的分子成分。在有丝分裂过程中不能稳定保持线粒体基因组的条件突变体将被分离。线粒体DNA-蛋白质复合体将被提纯并进行生化表征。相关蛋白质将使用串联质谱仪进行鉴定。这些蛋白质在线粒体DNA分离中的作用将通过遗传、生化和显微方法来确定。当细胞分裂时，细胞的各种基本成分(基因、细胞质、细胞器)必须在两个子细胞之间分配。就线粒体而言，细胞器拥有自己的基因组(以线粒体DNA的多个副本的形式)，这也必须被分配。先前的工作已经表明，线粒体DNA是非随机分布的，这表明存在以某种方式拴住DNA的机制，以便拷贝不会在细胞器内在空间上混合。这个项目将阐明这种情况是如何发生的。***