Cytochrome c oxidase biosynthesis and assembly in Candida utilis yeast cells

The data show that copper plays a key role in the assembly of active cytochrome c oxidase, and the insertion of copper is not necessary for the integration of the apoprotein into the membrane, which necessitates a cytoplasmic protein which is sensitive to cycloheximide.

Abstract

1. Cytochrome c oxidase from Candida utilis yeast cells grown in copper deficient and copper sufficient media has been studied by labeling the apoprotein with radioactive leucine (33H- and 14C-labeled leucine). The six subunits of cytochrome c oxidase were found to be present in copper deficient cells. 2. The kinetics of reconstitution of cytochrome c oxidase when copper deficient cells are grown in copper supplemented medium suggest that an active process is involved in the transfer and/or integration of copper into cytochrome c oxidase. 3. When copper deficient cells are grown in copper supplemented media containing cycloheximide so that no cytoplasmic protein synthesis occurs, cytochrome c oxidase reconstitution is totally inhibited. 4. When copper deficient cells are grown in copper supplemented media containing either chloramphenicol which blocks mitochondrial translation, or ethidium bromide which inhibits mitochondrial transcription, cytochrome c oxidase reconstitution is 20–25% of the control value. 5. The data show that: (a) the apoprotein of cytochrome c oxidase is synthesized in copper deficient cells; (b) the insertion of copper is not necessary for the integration of the apoprotein into the membrane; (c) the transfer of copper and/or its integration into cytochrome c oxidase necessitates a cytoplasmic protein which is sensitive to cycloheximide. Thus copper plays a key role in the assembly of active cytochrome c oxidase.