CLONING AND CHARACTERIZATION OF PET100, A GENE REQUIRED FOR THE ASSEMBLY OF YEAST CYTOCHROME-C-OXIDASE

The biogenesis of cytochrome c oxidase in Saccharomyces cerevisiae req uires 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 disrupte d. PET100 is located adjacent to the PDC2 gene on chromosome IV and co ntains an open reading frame of 333 base pairs. The PET100 protein con tains a possible membrane-spanning segment and a putative mitochondria l import sequence at its NH2 terminus. A strain carrying a null mutati on in PET100 lacks cytochrome c oxidase activity and assembled cytochr omes a and a(3), 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 sp ecific for cytochrome c oxidase and does not affect the biosynthesis o f heme A. In addition, mitochondria from the strain carrying a null mu tation in PET100 contain each of the subunit polypeptides of cytochrom e c oxidase. Together, these findings suggest that PET100p is not requ ired for the synthesis or localization of cytochrome c oxidase subunit s to mitochondria, but is required at a later step in their assembly i nto an active holoenzyme.