Interactions among COX1, COX2, and COX3 mRNA-specific Translational Activator Proteins on the Inner Surface of the Mitochondrial Inner Membrane of Saccharomyces cerevisiae

The core of the cytochrome c oxidase complex is composed of its three largest subunits, Cox1p, Cox2p, and Cox3p, which are encoded in mitochondrial DNA of Saccharomyces cerevisiae and inserted into the inner membrane from the inside. Mitochondrial translation of the COX1, COX2, and COX3 mRNAs is activated mRNA specifically by the nuclearly coded proteins Pet309p, Pet111p, and the concerted action of Pet54p, Pet122p, and Pet494p, respectively. Because the translational activators recognize sites in the 5′-untranslated leaders of these mRNAs and because untranslated mRNA sequences contain information for targeting their protein products, the activators are likely to play a role in localizing translation. Herein, we report physical associations among the mRNA-specific translational activator proteins, located on the matrix side of the inner membrane. These interactions, detected by coimmune precipitation and by two-hybrid experiments, suggest that the translational activator proteins could be organized on the surface of the inner membrane such that synthesis of Cox1p, Cox2p, and Cox3p would be colocalized in a way that facilitates assembly of the core of the cytochrome c oxidase complex. In addition, we found interactions between Nam1p/Mtf2p and the translational activators, suggesting an organized delivery of mitochondrial mRNAs to the translation system

Cox18p Is Required for Export of the Mitochondrially Encoded Saccharomyces cerevisiae Cox2p C-Tail and Interacts with Pnt1p and Mss2p in the Inner Membrane

The amino- and carboxy-terminal domains of mitochondrially encoded cytochrome c oxidase subunit II (Cox2p) are translocated out of the matrix to the intermembrane space. We have carried out a genetic screen to identify components required to export the biosynthetic enzyme Arg8p, tethered to the Cox2p C terminus by a translational gene fusion inserted into mtDNA. We obtained multiple alleles of COX18, PNT1, and MSS2, as well as mutations in CBP1 and PET309. Focusing on Cox18p, we found that its activity is required to export the C-tail of Cox2p bearing a short C-terminal epitope tag. This is not a consequence of reduced membrane potential due to loss of cytochrome oxidase activity because Cox2p C-tail export was not blocked in mitochondria lacking Cox4p. Cox18p is not required to export the Cox2p N-tail, indicating that these two domains of Cox2p are translocated by genetically distinct mechanisms. Cox18p is a mitochondrial integral inner membrane protein. The inner membrane proteins Mss2p and Pnt1p both coimmunoprecipitate with Cox18p, suggesting that they work together in translocation of Cox2p domains, an inference supported by functional interactions among the three genes