The W1536 8B Δ<i>hfa1</i> strain carrying plasmids with inserted stop codon mutation at position downstream of −372 resulted in unchanged lactate deficiency.

<p>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 Δ<i>hfa1</i> or W1536 8B Δ<i>htd2</i> (respiratory deficient control) and the plasmids carried by the strains are indicated at the left side of the panels. YCp33: empty plasmid; HFA1: YCp33 <i>HFA1</i>; −381: YCp33 <i>HFA1</i> −381; −372: YCp33 <i>HFA1</i> −372; −363: YCp33<i>HFA1</i> −363. Only stop codon mutations relevant to define the putative translation initiation site and controls are shown. The results for other mutants shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114738#pone-0114738-g002" target="_blank">Fig. 2</a> can be found as supplementary data.</p

Plasmids used for the acetyl-CoA carboxylases study in yeasts.

<p>*these plasmids are unstable in <i>E. coli</i> and have to be maintained in <i>S. cerevisiae</i> frozen stocks</p><p>Plasmids used for the acetyl-CoA carboxylases study in yeasts.</p

Expression and Evolution of the Non-Canonically Translated Yeast Mitochondrial Acetyl-CoA Carboxylase Hfa1p

<div><p>The <i>Saccharomyces cerevisiae</i> 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 <i>HFA1</i> gene and its evolutionary origin have remained unclear. Here, we determined the <i>HFA1</i> transcription initiation sites by 5′ RACE analysis. Using a novel “Stop codon scanning” approach, we mapped the location of the <i>HFA1</i> 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. <i>In silico</i> analyses of fungal <i>ACC</i> genes revealed conserved “cryptic” upstream mitochondrial targeting sequences in yeast species that have not undergone a whole genome duplication. Our Δ<i>hfa1</i> baker's yeast mutant phenotype rescue studies using the protoploid <i>Kluyveromyces lactis ACC</i> 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 <i>S. cerevisiae</i> 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.</p></div

Identification of the <i>HFA1</i> transcription initiation site by 5′RACE: The <i>HFA1</i> specific primer SP1 (Table 4) was used for the reverse transcription reaction to synthesize a first strand cDNA.

<p>a) Major 5′ RACE product (500 bp), b) Minor 5′RACE product (650 bp) and c) third band produced only occasionally.</p

Oligonucleotides used for the acetyl-CoA carboxylases study in yeasts.

<p>Oligonucleotides used for the acetyl-CoA carboxylases study in yeasts.</p

Mitochondrial targeting prediction of selected proteins in <i>S. cerevisiae.</i>

<p>The whole amino acid sequence and the whole amino acid sequence with the 5′ upstream region of the ORF of each protein was analyzed up to the first in-frame stop codon with MitoProt II. In case of the Hfa1p, the identified upstream start codon with the addition of the methionine for the convenience of the calculation with MitoProt II was used. The whole amino acid sequence with the 5′ upstream region of the ORF of each protein was analyzed up to the first in-frame stop codon with Target P. In case of Acp1p, the whole amino acid sequence was used for the calculation with Target P. <b>mTP, SP</b>: Final NN scores on which the final prediction is based. Note that the scores are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class, see below) may be an indication of how certain the prediction is. <b>Loc: S</b>: Secretory pathway, i.e. the sequence contains SP, a signal peptide <b>M</b>: Mitochondrion, i.e. the sequence contains mTP, a mitochondrial targeting peptide; -: Any other location; <b>RC</b>: Reliability class, from 1 to 5, where 1 indicates the strongest prediction. RC is a measure of the size of the difference ('diff') between the highest (winning) and the second highest output scores. There are 5 reliability classes, defined as follows: 1: diff>0.800, 2: 0.800>diff>0.600, 3: 0.600> diff>0.400, 4: 0.400> diff>0.200, 5: 0.200> diff HFA1 and ACP1 are listed as controls for the prediction calculation of MitoProt II and target P.</p><p>Mitochondrial targeting prediction of selected proteins in <i>S. cerevisiae.</i></p

Schematic depiction of location of the introduced stop codons for stop –codon scanning assay and RNAse protection assay results.

<p>All nucleotide numbers are given respective to the annotated start codon at +1. Position −450 is the predicted transcribed but not translated region of <i>HFA1</i>. 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 <i>ACC1</i>. The ORFof <i>HFA1</i> annotated in the <i>Saccharomyces</i> Genome Database starts from +1. The stop codon found to lead to a respiratory deficient phenotype in the screen performed by Kursu <i>et al</i>. 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 <i>HFA1</i>.</p

Acetyl-CoA carboxylases from yeast split into two subgroups with the exception of the <i>S. cerevisiae HFA1</i>.

<p>The phylogenetic tree of representative members of the acetyl-CoA carboxylases from fungi species was constructed using Clustal W2 Phylogeny and NJPlot as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114738#s2" target="_blank">Material and methods</a>. Numbers at branch points are bootstrap values and were calculated with Clustal X2.</p

<i>K.lactis ACC</i> complemented the respiratory deficiency when appended with the putative 5′ non-AUG initiated MTS, but not without this sequence.

<p>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 Δ<i>hfa1</i> +YCp33 <i>HFA1.</i> 2) W1536 8B Δ<i>hfa1</i>+YCp33 3) W1536 8B Δ<i>hfa1</i>. 4) W1536 8B (wild type). 5) W1536 8B Δ<i>hfa1</i>+<i>K.lactis ACC</i> without 5′- encoded putative cryptic MTS sequence. 6) W1536 8B Δ<i>hfa1</i>+<i>K.lactis ACC</i> with 5′ -encoded putative cryptic 5′ MTS.</p