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

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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.