A novel small-subunit ribosomal protein of yeast mitochondria that interacts functionally with an mRNA-specific translational activator.

Abstract

Mitochondrial translation of the mRNA encoding cytochrome c oxidase subunit III (coxIII) specifically requires the action of three position activator proteins encoded in the nucleus of Saccharomyces cerevisiae. Some mutations affecting one of these activators, PET122, can be suppressed by mutations in an unlinked nuclear gene termed PET123. PET123 function was previously demonstrated to be required for translation of all mitochondrial gene products. We have now generated an antibody against the PET123 protein and have used it to demonstrate that PET123 is a mitochondrial ribosomal protein of the small subunit. PET123 appears to be present at levels comparable to those of other mitochondrial ribosomal proteins, and its accumulation is dependent on the presence of the 15S rRNA gene in mitochondria. Taken together with the previous genetic data, these results strongly support a model in which the mRNA-specific translational activator PET122 works by directly interacting with the small ribosomal subunit to promote translation initiation on the coxll mRNA.Protein synthesis in mitochondria of Saccharomyces cerevisiae depends on nuclear genes that encode components of the general translation machinery such as mitochondrial ribosomal proteins, translation factors, and tRNA synthetases (1, 21; M. C. Costanzo and T. D. Fox, Annu. Rev. Genet., in press). In addition to these general components, other proteins are required specifically to promote the translation of particular mitochondrial mRNA species (1, 18, 21; Costanzo and Fox, in press). For example, the proteins coded by the nuclear genes PET122, PET54, and PET494 have been shown to be required specifically for mitochondrial translation of cytochrome c oxidase subunit III (coxIII) (2,5,8,10,26,27,31). All three of these coxIII-specific translational activators have been shown to accumulate in mitochondria (5, 9, 35; unpublished results), and their site (or sites) of action has been mapped genetically to the 5'-untranslated leader of the coxIII mRNA (6). A possible explanation for these findings is that one or more of the three translational activator proteins interact directly both with the coxIII 5' leader and with some component of the general mitochondrial translation apparatus to activate coxIII translation.To understand better the mechanism of action of the coxIII-specific translational activators, we have been isolating and analyzing allele-specific nuclear suppressors of mutations in PET122, PET54, and PET494. Such allele-specific suppressors can frequently identify genes encoding proteins that directly interact (23,25

Interactions amongCOX1,COX2, andCOX3mRNA-specific Translational Activator Proteins on the Inner Surface of the Mitochondrial Inner Membrane ofSaccharomyces cerevisiae

“…Strains were CAB30 and SN33. (McMullin et al, 1990;Haffter et al, 1991;Haffter and Fox, 1992) and interactions between of Nam1p and Rpo41p (mitochondrial RNA polymerase) (Rodeheffer et al, 2001) were reported previously. Two-hybrid and genetic interactions among Pet54p, Pet122p, and Pet494p were reported previously (Brown et al, 1994;Wiesenberger et al, 1995). without eliminating respiratory complex formation, by in vivo expression of chimeric mRNAs containing 5Ј-UTLs and coding sequences derived from different respiratory complex genes (Mü ller et al, 1984;Fox, 1986, 1988;Poutre and Fox, 1987;Rö del and Fox, 1987;Mulero and Fox, 1993b;Manthey and McEwen, 1995).…”

The Nucleus-Encodedtrans-Acting Factor MCA1 Plays a Critical Role in the Regulation of CytochromefSynthesis inChlamydomonasChloroplasts

“…Some T factors may act by unmasking the initiation codon of their target mRNA, sequestered into a secondary structure (Stampacchia et al, 1997;Klinkert et al, 2006;Schwarz et al, 2007). Alternatively, T factors may recruit the translation machinery, but their affinity for components of this machinery remains to be documented in most cases (however, see McMullin et al, 1990;Haffter et al, 1991;Haffter and Fox, 1992).…”

Protein synthesis in mitochondria

“…It has been speculated that the yeast mitoribosome consists of a core structure of homologous components, responsible for carrying out central steps of protein synthesis common to both eubacteria and mitochondria, and an additional set of proteins with a more specialized function in mitochondrial protein synthesis [19]. In this respect it is worth noting that none of the three mitoribosomal proteins (PET123, MRP1, MRP17) found to functionally interact with the unique mRNA-specific translational activator PET122 (see below) displays homology to other known ribosomal proteins [20][21][22].…”

“…Three of these suppressors have turned out to be mutations in genes encoding proteins of the small mitoribosomal subunit [20][21][22]80], thus strongly suggesting that PET122 activates translation of the COX3 mRNA via an interaction with this ribosomal subunit. The fourth nuclear gene isolated in this screen, PET12 7, differs from the previous three in that its function is not absolutely required for mitochondrial gene expression [21].…”