Characterization of redox-mediated protein in mitochondria

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DESCRIPTION (provided by applicant): The overall goal of this project is to further our understanding of the role of redox chemistry in mitochondrial biogenesis. Previous studies, including ours, have shown that the mitochondrial intermembrane space contains a novel oxidative folding pathway. A redox-regulated import pathway consisting of Mia40 was identified to mediate the import of small Tim proteins and cysteine-rich proteins in the intermembrane space. The sulfhydryl oxidase Erv1 also functions in this pathway as a putative oxidant for Mia40. Both Mia40 and Erv1 contain sets of highly conserved cysteine pairs that play an important role in the thiol/disulfide exchange required for import of a subset of cysteine-rich proteins in the intermembrane space. In addition, we have shown that cytochrome c and oxygen act as a terminal electron acceptors. However, our genetic studies suggest that other acceptors, including anaerobic acceptors, may also function in this pathway. The goal of this proposal is to use biochemical and genetic approaches to characterize this import pathway. In contrast to Erv1, the redox state of the 6 cysteine residues of Mia40 and the identity and function of its disulfide bonds in regulating import has not been determined. Thus, the first aim is to reconstitute the disulfide exchange reaction with Mia40, Erv1 and potential substrates, including biochemical characterization of the redox properties of Mia40, Erv1 and substrates. A battery of tests including monobromobimane titration, intrinsic tryptophan fluorescence, and AMS thiol-trapping will be utilized on wildtype and mutants of Erv1, Mia40, and substrates to dilineate the role that each cysteine residues play in the thiol/disulfide exchange mechanism. In addition, the second aim is to use a genetic approach to identify potential substrates and interacting factors of Erv1. In this case, a multi-copy suppressor screen using the temperature-sensitive en/1 mutants will be used for the identification of possible substrates. In addition, candidate interacting proteins will be investigated in genetic and biochemical approaches to determine how they function with Erv1. In all, characterization of this pathway will provide insight into in the redox environment of the mitochondrion. Characterization of this pathway is important for public health because mitochondrial dysfunction has been linked to a broad range of neurodegenerative and muscular diseases such as cardiac ischemia and perfusion injury, mitochondrial myopathies and neuropathies and general neurodegenerative diseases such as Parkinson's and Alzheimer's. This proposal will provide insight into fundamental pathways in mitochondrial assembly that are impaired in the disease state.