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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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Schematic depiction of location of the introduced stop codons for stop –codon scanning assay and RNAse protection assay results.All nucleotide numbers are given respective to the annotated start codon at +1. Position −450 is the predicted transcribed but not translated region of HFA1. The underlined region up to position −216 region shows the putative minimum mitochondrial import sequence and upstream position −141 shows the end of the sequence similarity to ACC1. The ORFof HFA1 annotated in the Saccharomyces Genome Database starts from +1. The stop codon found to lead to a respiratory deficient phenotype in the screen performed by Kursu et al. 2013 is located at −273 and 8 more stop codons at −282, −312, −360, −363, −372 −375 −378 and −381 were introduced upstream in the promoter region of HFA1.
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pone-0114738-g002: Schematic depiction of location of the introduced stop codons for stop –codon scanning assay and RNAse protection assay results.All nucleotide numbers are given respective to the annotated start codon at +1. Position −450 is the predicted transcribed but not translated region of HFA1. The underlined region up to position −216 region shows the putative minimum mitochondrial import sequence and upstream position −141 shows the end of the sequence similarity to ACC1. The ORFof HFA1 annotated in the Saccharomyces Genome Database starts from +1. The stop codon found to lead to a respiratory deficient phenotype in the screen performed by Kursu et al. 2013 is located at −273 and 8 more stop codons at −282, −312, −360, −363, −372 −375 −378 and −381 were introduced upstream in the promoter region of HFA1.

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)

Schematic depiction of location of the introduced stop codons for stop –codon scanning assay and RNAse protection assay results.All nucleotide numbers are given respective to the annotated start codon at +1. Position −450 is the predicted transcribed but not translated region of HFA1. The underlined region up to position −216 region shows the putative minimum mitochondrial import sequence and upstream position −141 shows the end of the sequence similarity to ACC1. The ORFof HFA1 annotated in the Saccharomyces Genome Database starts from +1. The stop codon found to lead to a respiratory deficient phenotype in the screen performed by Kursu et al. 2013 is located at −273 and 8 more stop codons at −282, −312, −360, −363, −372 −375 −378 and −381 were introduced upstream in the promoter region of HFA1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114738-g002: Schematic depiction of location of the introduced stop codons for stop –codon scanning assay and RNAse protection assay results.All nucleotide numbers are given respective to the annotated start codon at +1. Position −450 is the predicted transcribed but not translated region of HFA1. The underlined region up to position −216 region shows the putative minimum mitochondrial import sequence and upstream position −141 shows the end of the sequence similarity to ACC1. The ORFof HFA1 annotated in the Saccharomyces Genome Database starts from +1. The stop codon found to lead to a respiratory deficient phenotype in the screen performed by Kursu et al. 2013 is located at −273 and 8 more stop codons at −282, −312, −360, −363, −372 −375 −378 and −381 were introduced upstream in the promoter region of HFA1.
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.

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