Mitochondrial dynamics and morphology are controlled by balanced fission and fusion events, which are mediated by highly conserved dynamin-related proteins (DRPs). The importance of these events in cellular physiology is reflected by the altered mitochondrial dynamics and morphology observed in many age-related diseases, and that mutations in human fusion DRPs have been directly linked to neurodegenerative diseases and stroke. Furthermore, membrane fusion is a novel membrane remodeling function of DRPs and warrants detailed investigation. Previous work utilizing the powerful genetic tools available in S. cerevisiae suggest that both self-assembly and GTPase activity of fusion DRPs are required for mitochondrial fusion. The objective of our proposed research is to use Mgm1, the yeast mitochondrial inner membrane fusion DRP, to determine the molecular mechanism of fusion DRP function and mitochondrial membrane fusion. We will take advantage of the tools developed to study Mgm1 biochemistry and reconstituted membrane fusion. Thus, the following specific aims are proposed: (1) Analyze the structure of Mgm1 and nucleotide-dependent conformational changes using lipid monolayer-assisted 2D crystallization of Mgm1 wild type and mutants with nucleotide analogs that represent distinct nucleotide states. Crystals will be observed by transmission electron microscopy. A homology model for Mgm1 based on high-resolution structures of other DRPs will then be docked into the electron density calculated from the 2D crystallographic data. (2) Determine the molecular activities of Mgm1 that give rise to membrane fusion by developing in vitro assays to study distinct membrane fusion events such as membrane tethering, membrane deformation, and lipid mixing. Purified Mgm1 reconstituted into liposomes in conjunction with targeted mutagenesis will allow the analysis of the contribution of self-assembly, GTP binding and hydrolysis, and membrane binding to membrane fusion events. Mgm1-dependent changes in liposome clustering, morphology, and fusion-dependent fluorescence dequenching will be used to analyze intermediate steps of Mgm1- mediated membrane fusion. These studies will not only illuminate the basic mechanism of mitochondrial fusion, but will potentially elucidate the underlying pathology of DRP mutations linked to neurodegenerative diseases.
Mitochondrial dynamics and morphology are altered, indicating mitochondrial dysfunction, in a variety of age-related diseases such as cancer, diabetes, heart disease and neurodegenerative diseases. In fact, mutations in mitochondrial fusion dynamin-related proteins, which regulate mitochondrial dynamics and morphology, have been directly linked to two distinct neurodegenerative diseases and stroke. The research proposed here will help elucidate the mechanism of a physiological process disrupted in a variety of disease states as well as potentially explain how mutations in fusion proteins lead to disease.
