Abstract
Cox17 is an essential protein in the assembly of cytochrome c oxidase within the mitochondrion. Cox17 is implicated in providing copper ions for formation of CuA and CuB sites in the oxidase complex. To address whether Cox17 is functional in shuttling copper ions to the mitochondrion, Cox17 was tethered to the mitochondrial inner membrane by a fusion to the transmembrane domain of the inner membrane protein, Sco2. The copper-binding domain of Sco2 that projects into the inter-mitochondrial membrane space was replaced with Cox17. The Sco2/Cox17 fusion protein containing the mitochondrial import sequence and transmembrane segment of Sco2 is exclusively localized within the mitochondrion. The Sco2/Cox17 protein restores respiratory growth and normal cytochrome oxidase activity in cox17Delta cells. These studies suggest that the function of Cox17 is confined to the mitochondrial intermembrane space. Domain mapping of yeast Cox17 reveals that the carboxyl-terminal segment of the protein has a function within the intermembrane space that is independent of copper ion binding. The essential C-terminal function of Cox17 maps to a candidate amphipathic helix that is important for mitochondrial uptake and retention of the Cox17 protein. This motif can be spatially separated from the N-terminal copper-binding functional motif. Possible roles of the C-terminal motif are discussed.
Highlights
Copper plays an essential role in the biochemistry of all aerobic organisms [1]
The truncated mutants of COX17 under the control of the MET25 promoter were transformed into cox17⌬ cells, and the transformants were tested for respiratory growth and cytochrome c oxidase activity
Cox17 has been implicated as the mitochondrial copper metallochaperone responsible for shuttling Cu(I) ions to the mitochondrion for assembly of cytochrome c oxidase
Summary
Copper plays an essential role in the biochemistry of all aerobic organisms [1] This metal functions as a cofactor permitting the facile transfer of electrons in key enzymes including Cu/Zn superoxide dismutase for antioxidant defense, tyrosinase for melanin synthesis, and cytochrome c oxidase for electron transport in the mitochondrial respiratory chain. Assembly of functional CcO requires transport of nuclear-encoded subunits across both mitochondrial membranes. It is unclear whether the assembly of newly synthesized subunits occurs within the cristae or on the peripheral surface of the IM. Based on the necessity of Lys and Atx for shuttling copper ions to sites of utilization, the prediction is that CcO would require a specific protein involved in copper shuttling to the mitochondrion. Three cysteinyl residues present in a Cys-CysXaa-Cys sequence motif are critical for in vivo Cox function and Cu(I) ion binding [21]
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