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      Physical interaction and functional studies of human SCO1, a mitochondrial metallochaperone

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      Boulet, Aren_M Sc_thesis_April_20141.pdf (1.567Mb)
      Date
      2014-04-24
      Author
      Boulet, Aren
      Type
      Thesis
      Degree Level
      Masters
      Metadata
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      Abstract
      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.
      Degree
      Master of Science (M.Sc.)
      Department
      Biochemistry
      Program
      Biochemistry
      Committee
      Leary, Scot; Lee, Jeremy; Moore, Stan
      Copyright Date
      April 2014
      URI
      http://hdl.handle.net/10388/ETD-2014-04-1468
      http://hdl.handle.net/10388/7282
      Subject
      SCO1
      SCO2
      COX
      Cytochrome c oxidase
      Copper
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      • Graduate Theses and Dissertations
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