Product of Saccharomyces cerevisiae nuclear gene PET494 activates translation of a specific mitochondrial mRNA.

Abstract

The product of Saccharomyces cerevisiae nuclear gene PET494 is known to be required for a posttranscriptional step in the accumulation of one mitochondrial gene product, subunit III of cytochrome c oxidase (coxIII). Here we show that the PET494 protein probably acts in mitochondria by demonstrating that both a PET494-,-galactosidase fusion protein and unmodified PET494 are specifically associated with mitochondria. To define the PET494 site of action, we isolated mutations that suppress a pet494 deletion. These mutations were rearrangements of the mitochondrial gene oxi2 that encodes coxIII. The suppressor oxi2 genes had acquired the 5'-flanking sequences of other mitochondrial genes and gave rise to oxi2 transcripts carrying the 5'-untranslated leaders of their mRNAs. These results demonstrate that in wild-type cells PET494 specifically promotes coxIII translation, probably by interacting with the 5'-untranslated leader of the oxi2 mRNA.Mitochondrial genes are expressed within the mitochondrion by a genetic system distinct from that required for expression of nuclear genes. Only a few of the components of the mitochondrial genetic system of Saccharomyces cerevisiae are encoded on the mitochondrial genome (15); most are encoded by nuclear genes and imported posttranslationally into mitochondria. Interestingly, among the known nucleus-encoded elements of the mitochondrial genetic system, several have been found to be necessary for the accumulation of specific mitochondrial gene products. Some are required for processing of particular mitochondrial transcripts (12,20,43). Other nuclear gene products are necessary for steps subsequent to the accumulation of specific mitochondrial mRNAs (13, 42, 45; C. G. Poutre and T. D. Fox, manuscript in preparation; M. Costanzo, E. C. Seaver, and T. D. Fox, submitted for publication).The product of the nuclear gene PET494 was previously shown to be specifically required for accumulation of the mitochondrially encoded subunit III of cytochrome c oxidase (coxIII). pet494 mutant cells contain all the known mitochondrial gene products except for coxIII (7,16,17). However, they contain normal amounts of the mRNA encoding coxIII (42), a transcript of the mitochondrial oxi2 gene (51). The oxi2 gene is not interrupted by introns (51), and the oxi2 mRNA in pet494 mutant cells is indistinguishable, by Northern blot analysis or by Si nuclease protection experiments, from that in wild-type cells (42). Therefore, the PET494 protein must be necessary for some posttranscriptional step leading to the accumulation of coxIll.These previous studies left open two major questions. First, does the PET494 protein act directly in mitochondria or does it act in a more indirect fashion, for example, by promoting the expression of another nuclear gene? Second, is PET494 activity necessary for translation of the oxi2 mRNA, or is it required after translation to prevent rapid degradation of the coxIII polypeptide? In this paper we show that the product of the PET494 gene is indeed a mitochondrial protein, b...

Assembly of the Rotor Component of Yeast Mitochondrial ATP Synthase Is Enhanced When Atp9p Is Supplied by Atp9p-Cox6p Complexes

“…3, E and F). Similar experiments with mitochondria of a pet111 and pet494 mutant deficient in translation of Cox2p and Cox3p, respectively (40,41), confirmed that neither mutation interfered with formation of the Atp9p-Cox6p complex. Like the atp10 and mss51 mutants, both the pet111 and pet494 mutants showed a time-dependent decrease during the chase of radiolabel in the high molecular weight complexes seen on blue native gels (not shown).…”

Characterization of Assembly Intermediates Containing Subunit 1 of Yeast Cytochrome Oxidase

“…Direct evidence for the existence of this intermediate was obtained in the present study by analyzing Cox1p intermediates in pet111 and pet494 mutants expressing Cox1p-HAC. Both pet111 and pet494 mutants are COX-deficient due to their failure to translate Cox2p and Cox3p, respectively (31,32). Mitochondrial gene products in each stain were pulse-labeled in organello, and proteins associated with Cox1p-HAC were purified from digitonin extracts with protein C antibody beads.…”

Nucleo‐mitochondrial interactions in yeast mitochondrial biogenesis

“…The mutations characterized so far are recessive, thus defining positive-acting products. In a number of cases, it has been possible to show that these products are indeed located inside mitochondria [92,96,97]. Mitochondria1 mutations that allow bypass of the translational block in a number of these pet mutants have been isolated and shown to be deletions of mtDNA, resulting in fusion of the non-coding 5'-leader sequences of one mRNA to the body of another [98 -1011.…”