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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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K.lactis ACC complemented the respiratory deficiency when appended with the putative 5′ non-AUG initiated MTS, but not without this sequence.Strains were grown on media containing glucose (SCD-URA), glycerol (SCG, non-fermentable) or lactate (SCL, non-fermentable) as the sole carbon source at 30°C and 33°C. 1) W1536 8B Δhfa1 +YCp33 HFA1. 2) W1536 8B Δhfa1+YCp33 3) W1536 8B Δhfa1. 4) W1536 8B (wild type). 5) W1536 8B Δhfa1+K.lactis ACC without 5′- encoded putative cryptic MTS sequence. 6) W1536 8B Δhfa1+K.lactis ACC with 5′ -encoded putative cryptic 5′ MTS.
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pone-0114738-g004: K.lactis ACC complemented the respiratory deficiency when appended with the putative 5′ non-AUG initiated MTS, but not without this sequence.Strains were grown on media containing glucose (SCD-URA), glycerol (SCG, non-fermentable) or lactate (SCL, non-fermentable) as the sole carbon source at 30°C and 33°C. 1) W1536 8B Δhfa1 +YCp33 HFA1. 2) W1536 8B Δhfa1+YCp33 3) W1536 8B Δhfa1. 4) W1536 8B (wild type). 5) W1536 8B Δhfa1+K.lactis ACC without 5′- encoded putative cryptic MTS sequence. 6) W1536 8B Δhfa1+K.lactis ACC with 5′ -encoded putative cryptic 5′ MTS.

Mentions: Turunen et al. have suggested that HFA1 and ACC1 originated following a whole genome duplication event from a single ACC gene encoding a dually localized ACC [13]. Our database research confirms that K.lactis is predicted to have only one ACC (KLLA0F06072g). We analyzed the translation of the 5′-upstream region of the K. lactis ACC with two different subcellular localization prediction programs, MitoProtII and Target P [18], [19]. MitoProtII predicted that the region upstream to the canonical translation initiation codon encodes an in-frame protein sequence with a probability of 0.9999 to be a mitochondrial import signal, while Target P produces a mTP (mitochondrial targeting peptide) score of 0.586 for mitochondrial targeting potential. In order to add experimental evidence to the hypothesis that the dually localized, one-gene encoded variant of ACC is the original state of ACC in yeasts, we cloned and expressed the K. lactis ACC with and without the 5′ upstream region predicted to encode a MTS, in the S. cerevisiae W1536 8B Δhfa1 strain. Because K. lactis ACC was apparently toxic to E. coli even when expressed in low plasmid copy number, we resorted to a gap repair cloning approach in yeast (see material and methods). Functional mitochondrial localization of the K. lactis ACC carrying the extra 5′ sequence was successfully demonstrated by the complementation of the respiratory deficiency of the mutant strain (12 out of 18 Ura+ candidates were respiratory competent) at 33°C. All 18 isolates from the W1536 8B Δhfa1 strain transformed with the K. lactis ACC without MTS were tested and failed to grown on the lactate plate at 33°C, suggesting that the hypothetical MTS is required for the complementation (Fig. 4).


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)

K.lactis ACC complemented the respiratory deficiency when appended with the putative 5′ non-AUG initiated MTS, but not without this sequence.Strains were grown on media containing glucose (SCD-URA), glycerol (SCG, non-fermentable) or lactate (SCL, non-fermentable) as the sole carbon source at 30°C and 33°C. 1) W1536 8B Δhfa1 +YCp33 HFA1. 2) W1536 8B Δhfa1+YCp33 3) W1536 8B Δhfa1. 4) W1536 8B (wild type). 5) W1536 8B Δhfa1+K.lactis ACC without 5′- encoded putative cryptic MTS sequence. 6) W1536 8B Δhfa1+K.lactis ACC with 5′ -encoded putative cryptic 5′ MTS.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263661&req=5

pone-0114738-g004: K.lactis ACC complemented the respiratory deficiency when appended with the putative 5′ non-AUG initiated MTS, but not without this sequence.Strains were grown on media containing glucose (SCD-URA), glycerol (SCG, non-fermentable) or lactate (SCL, non-fermentable) as the sole carbon source at 30°C and 33°C. 1) W1536 8B Δhfa1 +YCp33 HFA1. 2) W1536 8B Δhfa1+YCp33 3) W1536 8B Δhfa1. 4) W1536 8B (wild type). 5) W1536 8B Δhfa1+K.lactis ACC without 5′- encoded putative cryptic MTS sequence. 6) W1536 8B Δhfa1+K.lactis ACC with 5′ -encoded putative cryptic 5′ MTS.
Mentions: Turunen et al. have suggested that HFA1 and ACC1 originated following a whole genome duplication event from a single ACC gene encoding a dually localized ACC [13]. Our database research confirms that K.lactis is predicted to have only one ACC (KLLA0F06072g). We analyzed the translation of the 5′-upstream region of the K. lactis ACC with two different subcellular localization prediction programs, MitoProtII and Target P [18], [19]. MitoProtII predicted that the region upstream to the canonical translation initiation codon encodes an in-frame protein sequence with a probability of 0.9999 to be a mitochondrial import signal, while Target P produces a mTP (mitochondrial targeting peptide) score of 0.586 for mitochondrial targeting potential. In order to add experimental evidence to the hypothesis that the dually localized, one-gene encoded variant of ACC is the original state of ACC in yeasts, we cloned and expressed the K. lactis ACC with and without the 5′ upstream region predicted to encode a MTS, in the S. cerevisiae W1536 8B Δhfa1 strain. Because K. lactis ACC was apparently toxic to E. coli even when expressed in low plasmid copy number, we resorted to a gap repair cloning approach in yeast (see material and methods). Functional mitochondrial localization of the K. lactis ACC carrying the extra 5′ sequence was successfully demonstrated by the complementation of the respiratory deficiency of the mutant strain (12 out of 18 Ura+ candidates were respiratory competent) at 33°C. All 18 isolates from the W1536 8B Δhfa1 strain transformed with the K. lactis ACC without MTS were tested and failed to grown on the lactate plate at 33°C, suggesting that the hypothetical MTS is required for the complementation (Fig. 4).

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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