Regulation of mitochondrial fission

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DESCRIPTION:(provided by applicant) Mitochondria perform essential cellular functions that are influenced by the morphology and dynamics of the organelle. The goal of the proposed work is to understand the mechanisms involved in the fundamental and topologically complex process of mitochondrial fission, using the model eukaryote, S. cerevisiae. In yeast, we have shown that mitochondrial fission is mediated by the dynamin-related GTPase, Dnmlp, which is concentrated in punctate structures at sites where mitochondrial membrane constriction and fission occur. Human and C. elegans Dnm1p homologs also have been shown to control mitochondrial fission. Thus, elucidating the mechanism of mitochondrial fission in yeast will be relevant to fission in human cells and will ultimately help us to understand how changes in mitochondrial morphology and copy number contribute to the development of diseases. To date, additional components of the mitochondrial fission machinery have not been reported. We have isolated three novel nuclear genes required for mitochondrial fission, MDV1-MDV3 for Mitochondrial Division. Mdvlp is a predicted soluble cytosolic protein, containing at least three distinct regions: a novel NH2-terminal region; a middle region predicted to form a coiled-coil structure; and a C-terminal region containing 7 WD repeats. Four sets of data suggest that Mdvlp interacts with Dnm1p to mediate mitochondrial fission: 1)mdvl and dnm1 alleles genetically interact, 2) two-hybrid analysis reveals a strong interaction between Mdvlp and Dmnlp, 3) Mdvlp localizes to punctate structures associated with mitochondria, a pattern similar to Dnmlp's, and 4) localization of Mdvlp to punctate structures, but not to the mitochondrial membrane, requires Dnm1p function. In dmdv1 cells, Dnm1p is associated with punctate structures on mitochondrial membranes, but these structures are not able to mediate fission. Our data, therefore, suggest that Mdvlp functions relatively late in the fission process to stimulate Dnmlp-dependent mitochondrial membrane constriction and/or fission. The experiments proposed in this grant will test this hypothesis and determine the role of additional fission components using a combination of cytological, genetic and biochemical approaches. Specifically, we will characterize the step at which Mdvlp acts in the fission process, determine the structural features of Mdv1p required for mitochondrial fission, Mdv1p's interaction with Dnm1p, and the mitochondrial localization of Mdv1p in delta dnm1 cells, identify and characterize Mdvlp binding partners and, clone and characterize novel fission components. In the long term, our goal is to determine the molecular mechanism of mitochondrial fission by reconstituting this process in vitro.