Limits...
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.

Show MeSH

Related in: MedlinePlus

Identification of the HFA1 transcription initiation site by 5′RACE: The HFA1 specific primer SP1 (Table 4) was used for the reverse transcription reaction to synthesize a first strand cDNA.a) Major 5′ RACE product (500 bp), b) Minor 5′RACE product (650 bp) and c) third band produced only occasionally.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4263661&req=5

pone-0114738-g001: Identification of the HFA1 transcription initiation site by 5′RACE: The HFA1 specific primer SP1 (Table 4) was used for the reverse transcription reaction to synthesize a first strand cDNA.a) Major 5′ RACE product (500 bp), b) Minor 5′RACE product (650 bp) and c) third band produced only occasionally.

Mentions: Amino acid sequences of the fungal acetyl-CoA carboxylases are obtained from the KEGG database (http://www.kegg.jp/). The phylogenetic tree shown in Fig. 1 was rooted by using aligned amino acid sequences by ClustalW2-Phylogeny [21] or ETH Phylogenetic Tree (http://www.cbrg.ethz.ch/services/PhylogeneticTree). The probability of the MTS of each amino acid sequences was analyzed with MitoProtII [18]. The 5′end of the nucleotide sequence was obtained from KEGG (http://www.kegg.jp/), Génolevures (http://www.genolevures.org/index.html), and NCBI (http://www.ncbi.nlm.nih.gov/).


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)

Identification of the HFA1 transcription initiation site by 5′RACE: The HFA1 specific primer SP1 (Table 4) was used for the reverse transcription reaction to synthesize a first strand cDNA.a) Major 5′ RACE product (500 bp), b) Minor 5′RACE product (650 bp) and c) third band produced only occasionally.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0114738-g001: Identification of the HFA1 transcription initiation site by 5′RACE: The HFA1 specific primer SP1 (Table 4) was used for the reverse transcription reaction to synthesize a first strand cDNA.a) Major 5′ RACE product (500 bp), b) Minor 5′RACE product (650 bp) and c) third band produced only occasionally.
Mentions: Amino acid sequences of the fungal acetyl-CoA carboxylases are obtained from the KEGG database (http://www.kegg.jp/). The phylogenetic tree shown in Fig. 1 was rooted by using aligned amino acid sequences by ClustalW2-Phylogeny [21] or ETH Phylogenetic Tree (http://www.cbrg.ethz.ch/services/PhylogeneticTree). The probability of the MTS of each amino acid sequences was analyzed with MitoProtII [18]. The 5′end of the nucleotide sequence was obtained from KEGG (http://www.kegg.jp/), Génolevures (http://www.genolevures.org/index.html), and NCBI (http://www.ncbi.nlm.nih.gov/).

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