THE SACCHAROMYCES-CEREVISIAE PET309 PROTEIN IS EMBEDDED IN THE MITOCHONDRIAL INNER MEMBRANE

The nuclear PET309 gene of Saccharomyces cerevisiae is necessary for e xpression of the mitochondrial COX1 gene, which encodes subunit I of c ytochrome c oxidase. In a pet309 null mutant, there is a defect both i n accumulation of COX1 pre-RNA, if it contains introns, and in transla tion of COX1 RNAs [Manthey, G. M. & McEwen, J. E. (1995) EMBO J. 14, 4 031-4043]. To facilitate identification and intracellular localization of the protein Pet309p, that is encoded by the PET309 gene, Pet309p w as tagged at the carboxy terminus with an epitope from the human c-myc protein. A monoclonal antibody against the c-myc epitope detected a 9 8-kDa protein in mitochondria of yeast cells that expressed the PET309 -c-myc fusion protein from a high copy number plasmid. This protein wa s not detectable in cells that did not express the fusion protein, or that expressed it from a single copy centromeric vector. Additional an alyses of mitochondrial subfractions demonstrated that the PET309-c-my c fusion protein is localized specifically in the inner mitochondrial membrane. It could not be extracted by alkaline sodium carbonate, yet it was susceptible to proteinase K digestion in mitoplasts (mitochondr ia with a disrupted outer membrane). These results indicate that Pet30 9p spans the inner membrane, with domain(s) exposed to the intermembra ne space side of the membrane. How Pet309p may function in concert wit h ether gene products necessary for COX1 RNA translation or accumulati on, such as Mss51p or Nam1p, respectively, is discussed.