Cloning and characterization of PET100, a gene required for the assembly of yeast cytochrome c oxidase

Abstract

The biogenesis of cytochrome c oxidase in Saccharomyces cerevisiae requires a protein encoded by the nuclear gene, PET100. Cells carrying a recessive mutation (pet100-1) in PET100 are respiratory deficient and have reduced levels of cytochrome c oxidase activity. The PET100 gene has been cloned by complementation of pet100-1, sequenced and disrupted. PET100 is located adjacent to the PDC2 gene on chromosome IV and contains an open reading frame of 333 base pairs. The PET100 protein contains a possible membrane-spanning segment and a putative mitochondrial import sequence at its NH2 terminus. A strain carrying a null mutation in PET100 lacks cytochrome e oxidase activity and assembled cytochromes a and a3, but the other respiratory chain carriers are present. The respiratory-deficient phenotype of this strain is not rescued by added hemin or heme A. These findings indicate that the mutation is specific for cytochrome c oxidase and does not affect the biosynthesis of heme A. In addition, mitochondria from the strain carrying a null mutation in PET100 contain each of the subunit polypeptides of cytochrome c oxidase. Together, these findings suggest that PET100p is not required for the synthesis or localization of cytochrome c oxidase subunits to mitochondria, but is required at a later step in their assembly into an active holoenzyme.