COX3 mRNA-specific translational activator proteins are associated with the inner mitochondrial membrane in...

Three yeast proteins coded by nuclear genes, PET54, PET122, and PET494, are specifically required to activate translation of the mitochondrially coded COX3 mRNA, and one of them, PET122, has been shown to interact functionally with both the mitochondrial ribosomal small subunit and the COX3 mRNA leader. To determine the location of these proteins within mitochondria, we have used antisera directed against them to assay for their presence in submitochondrial fractions. Approximately half of the PET54 protein present in mitochondria from wild-type cells pelleted with membranes, while half was released from disrupted mitochondria in soluble form. The membrane-bound PET54 floated with the inner membrane during buoyant density gradient centrifugation, but was efficiently solubilized by extraction with alkaline carbonate. The PET122 and PET494 proteins could only be detected in cells overproducing these proteins. PET122 was completely membrane-bound and could not be extracted with alkaline carbonate. Most of the PET494 protein pelleted with membranes and very little could be solubilized by alkaline carbonate. Both PET122 and PET494 floated with membranes during buoyant density gradient centrifugation. These data suggest the possibility that synthesis of the highly hydrophobic subunit III of cytochrome c oxidase is activated by PET54, PET122, and PET494 at the inner mitochondrial membrane. Amino-terminal sequencing of proteins isolated from mitochondria revealed that PET54 is not processed during import to mitochondria while the PET122 precursor is cleaved between the 8th and 9th residues.