ABSTRACTTHE PROTEIN-PROTEIN INTERACTIONS OF HEME A SYNTHASEByEmily HerwaldtHeme a is an obligatory cofactor in the terminal enzyme complex of the electron transport chain, cytochrome c oxidase. The heme a molecule is synthesized from heme o within the mitochondria by a multi-spanning inner membrane protein, heme a synthase (Cox15 in yeast). The insertion of heme a is critical for cytochrome c oxidase function and assembly, but this process has not been fully elucidated. In an effort to increase our understanding of heme a insertion into cytochrome c oxidase, we investigated the protein-protein interactions that occur with Cox15 in Saccharomyces cerevisiae. Cox15 in S. cerevisiae exists in six protein complexes ranging in size from ~120 kDa - 1 MDa as observed via blue native PAGE (BN-PAGE). The two largest complexes at approximately 750 kDa and 1 MDa are reminiscent of the respiratory supercomplexes containing both complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). The large 750 kDa and 1 MDa Cox15 complexes were not observed in yeast strains in which the supercomplexes are unable to form, thus supporting the hypothesis that Cox15 is present in the respiratory supercomplexes. In addition, Cox15 was found to interact with one of the catalytic subunits of the cytochrome bc1 complex, Cyt1, and we propose that Cox15 and Cyt1 interact within the supercomplexes. No other proteins from the cytochrome bc1 complex or cytochrome c oxidase were found to interact with Cox15, although if Cox15 is present in the respiratory supercomplexes, by definition, it would seem that Cox15 must also interact (at least indirectly) with the other components of the respiratory supercomplexes. Of the lower four Cox15-containing complexes ranging from ~120 - 440 kDa, the complex at 120 kDa was the most prominent, indicating that the majority of the Cox15 observed by BN-PAGE is represented by this species. Although 120 kDa is ~1.5 times larger in molecular weight than monomeric C-terminal tagged Cox15, we were unable to identify other proteins that interact with Cox15 in this 120 kDa band. Because it is accepted that molecular weights of proteins are over-estimated via BN-PAGE due to the effect of detergent, we hypothesize that this lowest complex represents monomeric Cox15. Experiments to test the composition of the remaining Cox15-containing complexes revealed that approximately 30% of Cox15 interacts with itself in homo-oligomeric complexes. In addition, experiments to test if other proteins interacted with Cox15 revealed that cytochrome c oxidase assembly factors may exist with Cox15 in one of the Cox15-containing complexes. It does not appear, however, that assembly factors of cytochrome c oxidase represent predominant protein interactions with Cox15. Finally, Cox15 was shown to interact with the cytosolic heat shock proteins, Ssa1 and Hsc82. Deletions of Ssa1 and Hsc82, however, indicated that these proteins are not part of the Cox15-containing complexes observed via BN-PAGE. Based on previous studies implicating cytosolic heat shock proteins in mitochondrial protein uptake, we predict that Ssa1 and Hsc82 are involved in the import of Cox15 into the mitochondria.
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