Genetic and cellular basis of mitochondrial genome inheritance and maintenance in yeast

线粒体基因组的遗传和维持对真核细胞的能量代谢起着重要作用。功能障碍导致神经退行性疾病和衰老症状。线粒体基因组遗传的遗传和细胞基础及其修复和质量控制机制知之甚少。酿酒酵母是研究这些过程的一个很好的模式生物，因为线粒体呼吸是兼性呼吸，酵母非常适合遗传和系统生物学方法。目前的建议有两个主要目标：1。对核基因与野生型或突变型线粒体DNA（mtDNA）的相互作用进行系统的全基因组分析; 2.在单细胞水平上研究mtDNA遗传的方法的发展和建立。对于第一个目标，我们将使用覆盖整个酵母基因组的突变体库进行合成遗传阵列（SGA），这些突变体库面临各种mtDNA变体。这些实验将系统地揭示对mtDNA遗传重要的基因，长期保持功能性mtDNA并保护其免受损伤。这些包括介导mtDNA复制、重组和分配的组分，以及参与保护、修复和消除缺陷mtDNA的因子。对于第二个目标，我们将荧光标记含有不同线粒体基因组的异质细胞中的mtDNA类核。这将通过分别用荧光蛋白或荧光原位杂交（FISH）标记类核蛋白来完成。此外，我们还将研究线粒体或胞质蛋白在线粒体DNA转运或分配中的作用。这可以通过光学和电子显微镜或荧光互相关光谱（FCCS）中的共定位来分析。在该项目的后期阶段，这两个主要目标将通过使用新建立的方法对全基因组筛选中新发现的成分进行功能分析来结合起来。这些实验承诺在一个简单的真核细胞中的线粒体基因组的遗传和细胞通路的详细了解。

Inheritance and maintenance of the mitochondrial genome play an important role for the energy metabolism of eukaryotic cells. Dysfunctions cause neurodegenerative diseases and symptoms of aging. The genetic and cellular basis of mitochondrial genome inheritance and its repair and quality control mechanisms are only poorly understood. Yeast Saccharomyces cerevisiae is an excellent model organism to study these processes, as mitochondrial respiration is facultative dispensable, and yeast is ideally suited for genetic and systems biology approaches. The current proposal has two major aims: 1. A systematic genome-wide analysis of the interaction of nuclear genes with wild type or mutant mitochondrial DNA (mtDNA); 2. the development and establishment of methods to study mtDNA inheritance at the single-cell level. For the first aim, we will perform synthetic genetic arrays (SGA) using mutant libraries covering the entire yeast genome that are confronted with various mtDNA variants. These experiments will systematically reveal the genes that are important for mtDNA inheritance, maintain functional mtDNA in the long-term and protect it against damage. These include components mediating replication, recombination, and partitioning of mtDNA and factors involved in protection, repair, and elimination of defective mtDNA. For the second aim, we will fluorescently label mtDNA nucleoids in heteroplasmic cells containing different mitochondrial genomes. This will be done by tagging of nucleoid proteins with fluorescent proteins or fluorescence in situ hybridization (FISH), respectively. Furthermore, we will examine the involvement of mitochondrial or cytosolic proteins in transport or partitioning of mtDNA. This can be analyzed by colocalization in light and electron microscopy or fluorescence cross-correlation spectroscopy (FCCS). In a later phase of the project both major aims will be combined by functional analysis of the components newly discovered in the genome-wide screens employing the newly established methods. These experiments promise a detailed understanding of the genetic and cellular pathways contributing to inheritance and maintenance of the mitochondrial genome in a simple eukaryotic cell.