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. ***
