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Expression and evolution of the non-canonically translated yeast mitochondrial acetyl-CoA carboxylase Hfa1p.

Suomi F, Menger KE, Monteuuis G, Naumann U, Kursu VA, Shvetsova A, Kastaniotis AJ - PLoS ONE (2014)

Bottom Line: Our Δhfa1 baker's yeast mutant phenotype rescue studies using the protoploid Kluyveromyces lactis ACC confirmed functionality of the cryptic upstream mitochondrial targeting signal.These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in S. cerevisiae have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms.Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5' upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland.

ABSTRACT
The Saccharomyces cerevisiae genome encodes two sequence related acetyl-CoA carboxylases, the cytosolic Acc1p and the mitochondrial Hfa1p, required for respiratory function. Several aspects of expression of the HFA1 gene and its evolutionary origin have remained unclear. Here, we determined the HFA1 transcription initiation sites by 5' RACE analysis. Using a novel "Stop codon scanning" approach, we mapped the location of the HFA1 translation initiation site to an upstream AUU codon at position -372 relative to the annotated start codon. This upstream initiation leads to production of a mitochondrial targeting sequence preceding the ACC domains of the protein. In silico analyses of fungal ACC genes revealed conserved "cryptic" upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δhfa1 baker's yeast mutant phenotype rescue studies using the protoploid Kluyveromyces lactis ACC confirmed functionality of the cryptic upstream mitochondrial targeting signal. These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in S. cerevisiae have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms. Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5' upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.

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The W1536 8B Δhfa1 strain carrying plasmids with inserted stop codon mutation at position downstream of −372 resulted in unchanged lactate deficiency.Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The yeast cells were grown on media containing Glucose (SCD) or lactate (Lactate) as the sole carbon source at 33°C. Strains used for this study are W1536 8B, W1536 8B Δhfa1 or W1536 8B Δhtd2 (respiratory deficient control) and the plasmids carried by the strains are indicated at the left side of the panels. YCp33: empty plasmid; HFA1: YCp33 HFA1; −381: YCp33 HFA1 −381; −372: YCp33 HFA1 −372; −363: YCp33HFA1 −363. Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The results for other mutants shown in Fig. 2 can be found as supplementary data.
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pone-0114738-g003: The W1536 8B Δhfa1 strain carrying plasmids with inserted stop codon mutation at position downstream of −372 resulted in unchanged lactate deficiency.Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The yeast cells were grown on media containing Glucose (SCD) or lactate (Lactate) as the sole carbon source at 33°C. Strains used for this study are W1536 8B, W1536 8B Δhfa1 or W1536 8B Δhtd2 (respiratory deficient control) and the plasmids carried by the strains are indicated at the left side of the panels. YCp33: empty plasmid; HFA1: YCp33 HFA1; −381: YCp33 HFA1 −381; −372: YCp33 HFA1 −372; −363: YCp33HFA1 −363. Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The results for other mutants shown in Fig. 2 can be found as supplementary data.

Mentions: A mutation located upstream of the annotated start codon of HFA1 was obtained during our study on the mtFAS in yeast seeking for respiratory deficient synthetic petite mutants [3]. This mutation is located upstream at −273 in frame with the designated AUG (Fig. 2). This finding further supported the hypothesis that the translation of HFA1 mRNA does not start at the designated codon but further upstream. W1536 8B Δhfa1 proved to be respiratory deficient when grown on lactate as the only carbon source at a growth temperature of 33°C. None of the transformants carrying plasmids with stop codon mutations downstream of the −372 site were able to grow like the wild type strain or the Δhfa1 strains carrying a wild type copy of HFA1 on the YCp33 HFA1 plasmid. However, stop codon mutations inserted upstream of the −375 position did not impede the rescue of the respiratory deficiency of the W1536 8B Δhfa1 strain. The only triplet in between the mutated codons that matches the sequence of previously reported non-AUG initiation codons is ATT at position −372. The sequence context is a good match for a Kozak consensus sequence [22] (Fig. 3). The mutation of the codon in at location −372 resulted in unchanged lactate deficiency of Δhfa1 strains transformed with the mutagenized rescue plasmid (Fig. 3). This is not the case when the preceding likely non-AUG translation initiation codon is (−381, ATA encoding Ile) is mutated to a stop codon (Fig. 3). We therefore suggest that the native translation initiation codon of HFA1 is ATT at −372, encoding isoleucine.


Expression and evolution of the non-canonically translated yeast mitochondrial acetyl-CoA carboxylase Hfa1p.

Suomi F, Menger KE, Monteuuis G, Naumann U, Kursu VA, Shvetsova A, Kastaniotis AJ - PLoS ONE (2014)

The W1536 8B Δhfa1 strain carrying plasmids with inserted stop codon mutation at position downstream of −372 resulted in unchanged lactate deficiency.Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The yeast cells were grown on media containing Glucose (SCD) or lactate (Lactate) as the sole carbon source at 33°C. Strains used for this study are W1536 8B, W1536 8B Δhfa1 or W1536 8B Δhtd2 (respiratory deficient control) and the plasmids carried by the strains are indicated at the left side of the panels. YCp33: empty plasmid; HFA1: YCp33 HFA1; −381: YCp33 HFA1 −381; −372: YCp33 HFA1 −372; −363: YCp33HFA1 −363. Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The results for other mutants shown in Fig. 2 can be found as supplementary data.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4263661&req=5

pone-0114738-g003: The W1536 8B Δhfa1 strain carrying plasmids with inserted stop codon mutation at position downstream of −372 resulted in unchanged lactate deficiency.Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The yeast cells were grown on media containing Glucose (SCD) or lactate (Lactate) as the sole carbon source at 33°C. Strains used for this study are W1536 8B, W1536 8B Δhfa1 or W1536 8B Δhtd2 (respiratory deficient control) and the plasmids carried by the strains are indicated at the left side of the panels. YCp33: empty plasmid; HFA1: YCp33 HFA1; −381: YCp33 HFA1 −381; −372: YCp33 HFA1 −372; −363: YCp33HFA1 −363. Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The results for other mutants shown in Fig. 2 can be found as supplementary data.
Mentions: A mutation located upstream of the annotated start codon of HFA1 was obtained during our study on the mtFAS in yeast seeking for respiratory deficient synthetic petite mutants [3]. This mutation is located upstream at −273 in frame with the designated AUG (Fig. 2). This finding further supported the hypothesis that the translation of HFA1 mRNA does not start at the designated codon but further upstream. W1536 8B Δhfa1 proved to be respiratory deficient when grown on lactate as the only carbon source at a growth temperature of 33°C. None of the transformants carrying plasmids with stop codon mutations downstream of the −372 site were able to grow like the wild type strain or the Δhfa1 strains carrying a wild type copy of HFA1 on the YCp33 HFA1 plasmid. However, stop codon mutations inserted upstream of the −375 position did not impede the rescue of the respiratory deficiency of the W1536 8B Δhfa1 strain. The only triplet in between the mutated codons that matches the sequence of previously reported non-AUG initiation codons is ATT at position −372. The sequence context is a good match for a Kozak consensus sequence [22] (Fig. 3). The mutation of the codon in at location −372 resulted in unchanged lactate deficiency of Δhfa1 strains transformed with the mutagenized rescue plasmid (Fig. 3). This is not the case when the preceding likely non-AUG translation initiation codon is (−381, ATA encoding Ile) is mutated to a stop codon (Fig. 3). We therefore suggest that the native translation initiation codon of HFA1 is ATT at −372, encoding isoleucine.

Bottom Line: Our Δhfa1 baker's yeast mutant phenotype rescue studies using the protoploid Kluyveromyces lactis ACC confirmed functionality of the cryptic upstream mitochondrial targeting signal.These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in S. cerevisiae have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms.Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5' upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Biochemistry and Molecular Medicine and Biocenter Oulu, University of Oulu, Oulu, Finland.

ABSTRACT
The Saccharomyces cerevisiae genome encodes two sequence related acetyl-CoA carboxylases, the cytosolic Acc1p and the mitochondrial Hfa1p, required for respiratory function. Several aspects of expression of the HFA1 gene and its evolutionary origin have remained unclear. Here, we determined the HFA1 transcription initiation sites by 5' RACE analysis. Using a novel "Stop codon scanning" approach, we mapped the location of the HFA1 translation initiation site to an upstream AUU codon at position -372 relative to the annotated start codon. This upstream initiation leads to production of a mitochondrial targeting sequence preceding the ACC domains of the protein. In silico analyses of fungal ACC genes revealed conserved "cryptic" upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δhfa1 baker's yeast mutant phenotype rescue studies using the protoploid Kluyveromyces lactis ACC confirmed functionality of the cryptic upstream mitochondrial targeting signal. These results lend strong experimental support to the hypothesis that the mitochondrial and cytosolic acetyl-CoA carboxylases in S. cerevisiae have evolved from a single gene encoding both the mitochondrial and cytosolic isoforms. Leaning on a cursory survey of a group of genes of our interest, we propose that cryptic 5' upstream mitochondrial targeting sequences may be more abundant in eukaryotes than anticipated thus far.

Show MeSH
Related in: MedlinePlus