The research proposed constitutes a continuation of the biochemical, molecular and genetic characterization of a series of respiration-deficient mammalian cell mutants isolated by this laboratory. We are interested in the cloning, mapping and control of expression of genes which encode the multisubunit complexes of the electron transport chain in mitochondria, and the emphasis will be on succinate dehydrogenase (SDH). The SDH complex is comprised of a flavoprotein (FP) and an iron-sulfur protein (IP), which interact with two hydrophobic integral membrane proteins to make up complex II. We have cloned partial cDNAs for the IP subunit from several species including humans and yeast, and have also characterized the complete gene from S. cerevisiae. Cloning the complete human cDNA is a high priority. A mutant yeast strain has been created by gene disruption and we believe that our SDH deficient hamster mutant is also defective in this gene. The characterization of the hamster mutant and the mapping of the complementing gene on human chromosome I near the centromere will be confirmed by direct complementation tests with transfected genes, and by Southern analyses of hybrid cells. Genes with heterologous promoters and genes with alterations in their coding region will be transfected into mammalian and yeast cells to study 1) the effect of different levels of expression of the IP subunit on the assembly of complex II, and b) the effect of amino acid changes on the post-translational modifications (Fe-S clusters), interactions with the other subunits, and functions of SDH. A second major goal is to clone the cDNA (and gene) for the FP subunit of SDH. The strategy will be to clone it first from yeast, and then use it as a probe for selecting the mammalian cDNA (or gene). We have specific antisera to screen expression libraries. An alternative approach to identify this gene in yeast might be by a suppressor mutation which suppresses a specific mutation in the IP subunit. The FP subunit is subject to a particularly interesting post-translational modification: the covalent attachment of a flavin to a histidine side chain. The conservation of this reaction from prokaryotes to eukaryotes is noteworthy. The search for suppressor mutations may also yield mutations in the small integral membrane protein genes. Human patients with mitochondrial myopathies and with defects in specific complexes of the electron transport chain are being identified with increasing frequency. The defects often appear to be tissue specific. It is hoped that the proposed studies will provide further data and cloned, identified genes for a detailed genetic and biochemical analysis of mitochondrial biogenesis in eukaryotes and mammals in particular.
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