Physical interaction and functional studies of human SCO1, a mitochondrial metallochaperone
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Cytochrome c oxidase (COX) is a multimeric protein complex embedded in the inner mitochondrial membrane that contributes to the electrochemical potential ultimately required for adenosine triphosphate (ATP) synthesis. Synthesis of Cytochrome c Oxidase 1 (SCO1) and SCO2 are two of many accessory proteins that facilitate the assembly of individual COX structural subunits into a functional holoenzyme complex. SCO1 and SCO2 also function to regulate cellular copper homeostasis. Both of these functions require that SCO proteins collaborate with several interacting partners. ¬With few exceptions, however, their protein partners have yet to be identified and, as a consequence, we lack mechanistic insight into SCO protein function. To address this gap in our knowledge, I used physical methods to identify interacting partners of SCO1. Physical interactions between potential interacting partners of endogenous SCO1 or overexpressed SCO1-FLAG were stabilized with a chemical crosslinker, and the protein complexes were purified using the appropriate primary antibody. Mass spectrometric analysis of the eluates provided two lists of potential interacting partners that were subsequently filtered by gene ontology function and mitochondrial localization. The final list was comprised of 85 proteins, which were rank ordered based on total peptide counts per protein. Three of these candidate interacting partners were then further characterized; COX20, tricarboxylate transport protein (SLC25A1) and mitochondrial 2-oxoglutarate/malate carrier (SLC25A11). I first confirmed the authenticity of the observed interactions by conducting the reciprocal co-immunoprecipitations in the absence of a chemical crosslinker. I then investigated the effect of transient knockdown of each protein on the abundance of COX II, a direct proxy of total COX content. Knockdown of COX20 and SLC25A1 reduced COX II levels in SCO1 and SCO2 patient fibroblasts, but did not affect COX II abundance in control cells. SLC25A11 knockdown lowered COX II abundance in both control and SCO patient cells. These data suggest that the ability of SCO1 to interact with each of these proteins somehow facilitates the maturation of the CuA site of COX II.
DegreeMaster of Science (M.Sc.)
CommitteeLeary, Scot; Lee, Jeremy; Moore, Stan
Copyright DateApril 2014
Cytochrome c oxidase