Pet111p, an Inner Membrane-bound Translational Activator That Limits Expression of the Saccharomyces cerevisiaeMitochondrial Gene COX2

Abstract

The protein specified by the Saccharomyces cerevisiae nuclear gene PET111 specifically activates translation of the mitochondrially coded mRNA for cytochrome c oxidase subunit II (Cox2p). We found Pet111p specifically in mitochondria of both wild-type cells and cells expressing a chromosomal gene for a functional epitopetagged form of Pet111p. Pet111p was associated with mitochondrial membranes and was highly resistant to extraction with alkaline carbonate. Pet111p was protected from proteolytic digestion by the mitochondrial inner membrane. Thus, it is exposed only on the matrix side, where it could participate directly in organellar translation and localize Cox2p synthesis by virtue of its functional interaction with the COX2 mRNA 5-untranslated leader. We also found that Pet111p is present at levels limiting the synthesis of Cox2p by examining the effect of altered PET111 gene dosage in the nucleus on expression of a reporter gene, cox2::ARG8 m , that was inserted into mitochondrial DNA. The level of the reporter protein, Arg8p, was one-half that of wild type in a diploid strain heterozygous for a pet111 deletion mutation, whereas it was increased 2.8-fold in a strain bearing extra copies of PET111 on a high-copy plasmid. Thus, Pet111p could play dual roles in both membrane localization and regulation of Cox2p synthesis within mitochondria.

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

“…Consistent with this hypothesis, topological information required for efficient assembly of two mitochondrially coded subunits of the cytochrome c oxidase complex, Cox2p and Cox3p, has been shown to reside in untranslated portions of their mRNAs (Sanchirico et al, 1998). In addition to their apparent role in localizing translation on the matrix side of the inner membrane, the COX2 and COX3 mRNA-specific translational activators are also present at levels that limit expression of their target mitochondrial genes (Steele et al, 1996;Green-Willms et al, 2001).…”

“…Pet309p, detected as an overproduced epitope-tagged protein, is an integral inner membrane protein partially exposed on the intermembrane space side (outside) (Man-they et al, 1998). Pet111p, detected as an epitope-tagged protein at wild-type levels, is an integral inner membrane protein facing the matrix (Green-Willms et al, 2001). Pet54p is a peripheral inner membrane protein, whereas Pet122p and Pet494p, detected as overproduced proteins, behaved like integral inner membrane proteins (McMullin and Fox, 1993).…”

Characterization of Gtf1p, the Connector Subunit of Yeast Mitochondrial tRNA-dependent Amidotransferase

“…The absence of the novel Cox2p* band in a pet111 and gtf1 double mutant (Fig. 2D) defective in translation of the COX2 mRNA (34,35), confirmed that this translation product is related to Cox2p. That the novel Cox2p* and the Atp8p-deficient pheno- ATPase activity was measured at 37°C in the absence and presence of 10 g/ml oligomycin as described (30).…”

“…Pet111p is required for Cox2p translation initiation (34). The absence of the novel Cox2p* band in a pet111 and gtf1 double mutant (Fig.…”

Nascent RNA structure modulates the transcriptional dynamics of RNA polymerases

“…This effect may reflect an evolutionary pressure to maximize expression rates through the attenuation of polymerase pausing; however, it has been proposed that there also exists a selective pressure for endosymbiont genomes to use the energetically cheaper ATP and TTP nucleotides (42). Interestingly, the mitochondrial organelle is one system in which gene expression is limited not by transcription, but by mRNA-specific translational activation (43,44). Consequently, polymerase pausing may not be as important for the control of gene expression in mitochondria as it is in the nucleus.…”