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The Evolutionary Potential of Phenotypic Mutations.

Yanagida H, Gispan A, Kadouri N, Rozen S, Sharon M, Barkai N, Tawfik DS - PLoS Genet. (2015)

Bottom Line: Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals.Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location.Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT
Errors in protein synthesis, so-called phenotypic mutations, are orders-of-magnitude more frequent than genetic mutations. Here, we provide direct evidence that alternative protein forms and phenotypic variability derived from translational errors paved the path to genetic, evolutionary adaptations via gene duplication. We explored the evolutionary origins of Saccharomyces cerevisiae IDP3 - an NADP-dependent isocitrate dehydrogenase mediating fatty acids ß-oxidation in the peroxisome. Following the yeast whole genome duplication, IDP3 diverged from a cytosolic ancestral gene by acquisition of a C-terminal peroxisomal targeting signal. We discovered that the pre-duplicated cytosolic IDPs are partially localized to the peroxisome owing to +1 translational frameshifts that bypass the stop codon and unveil cryptic peroxisomal targeting signals within the 3'-UTR. Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals. Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location. Further, as exemplified here, the sequences that promote translational frameshifts are also more prone to genetic deletions. Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

No MeSH data available.


Related in: MedlinePlus

The potential peroxisomal localization of a pathway supplying NADPH for β-oxidation.(A) The putative pathway follows the TCA reaction cycle, beginning with pyruvate and comprising four enzymes: PYC, pyruvate carboxylase; CIT, citrate synthase; ACO, aconitase; and finally IDP, isocirate dehydrogenase. Two enzymes have peroxisomal S. cerevisiae paralogous:—CIT3 and IDP3; the other two, PYC and ACO, are thought to act in the cytosol and mitochondria, respectively. Hypothetical transporters permitting the export and import of intermediate metabolites are depicted as white circles. (B) The PTS1-like motif of S.cer PYC1 (last 11 amino acids plus the 3’UTR ending with SHL*) was tested by fusion to the C-terminus of S.cer IDP2 and complementation of ΔIdp3 growth on petroselinate. Shown is growth with the original motif (orange line), and a version obtained by a single base deletion upstream the stop codon (green line) that puts the putative PTS1 in-frame (PYC1ΔG; the deleted G is highlighted in grey). (C) The PTS1-like motif of S.cer ACO2 (last 10 amino acids plus the 3’UTR ending with NKF*) was tested as described in part (B) above. An in-frame version obtained via a deletion of a single base upstream the stop codon (ACO2ΔA) was also tested. Whilst the initial growth was very slow, upon serial transfers to fresh petrosalinate media (5-fold dilutions) a marked increase in growth rate was observed. Single colonies were randomly isolated from the 2nd transferred culture (at 900 h) and sequenced. All 7 sequenced clones possessed a single mutation in the PTS1-like motif converting it to NKL (S4C Fig).
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pgen.1005445.g003: The potential peroxisomal localization of a pathway supplying NADPH for β-oxidation.(A) The putative pathway follows the TCA reaction cycle, beginning with pyruvate and comprising four enzymes: PYC, pyruvate carboxylase; CIT, citrate synthase; ACO, aconitase; and finally IDP, isocirate dehydrogenase. Two enzymes have peroxisomal S. cerevisiae paralogous:—CIT3 and IDP3; the other two, PYC and ACO, are thought to act in the cytosol and mitochondria, respectively. Hypothetical transporters permitting the export and import of intermediate metabolites are depicted as white circles. (B) The PTS1-like motif of S.cer PYC1 (last 11 amino acids plus the 3’UTR ending with SHL*) was tested by fusion to the C-terminus of S.cer IDP2 and complementation of ΔIdp3 growth on petroselinate. Shown is growth with the original motif (orange line), and a version obtained by a single base deletion upstream the stop codon (green line) that puts the putative PTS1 in-frame (PYC1ΔG; the deleted G is highlighted in grey). (C) The PTS1-like motif of S.cer ACO2 (last 10 amino acids plus the 3’UTR ending with NKF*) was tested as described in part (B) above. An in-frame version obtained via a deletion of a single base upstream the stop codon (ACO2ΔA) was also tested. Whilst the initial growth was very slow, upon serial transfers to fresh petrosalinate media (5-fold dilutions) a marked increase in growth rate was observed. Single colonies were randomly isolated from the 2nd transferred culture (at 900 h) and sequenced. All 7 sequenced clones possessed a single mutation in the PTS1-like motif converting it to NKL (S4C Fig).

Mentions: Following the above findings, and a report that appeared while this work was ongoing on cryptic peroxisomal targeting of two cytosolic enzymes [30], we performed a systematic computational search of the 3’-UTR regions of four closely related post-WGD Saccharomyces genomes: S. cerevisiae, S. paradoxus, S. bayanus and S. mikatae (S1 and S3 Tables). The search was based on PTS1 motifs containing all possible variation of amino acids ((S/A/C/E/I/H/Q)-(K/R/H)-(L/F)-stop) from 20 peroxisomal proteins in the Saccaromyces genome database. We searched for the motif starting up to 30 bp downstream the stop codon in all frames. PTS1-like motifs were found in around 1% of total genes of the genomes. However, about 40% of these were interrupted by another stop codon (S1 Table). Further, only a small number of these potentially cryptic motifs were found in more than one species, suggesting that these motifs are under functional selection. We thus focused on few interesting candidates that are conserved among the post-WGD species, and foremost on pyruvate carboxylase 1 (PYC1)—a cytosolic enzyme converting pyruvate to oxaloacetate. The NADPH used for peroxisomal β-oxidation could be produced from pyruvate by a putative pathway that includes four enzymes: PYC: pyruvate carboxylase; CIT: citrate synthase; ACO: aconitase; and finally IDP: isocirate dehydrogenase. Although not established as a peroxisomal NADPH providing pathway, this reaction sequence comprises part of the TCA cycle. Among these, two enzymes have known peroxisomal paralogues in S. cerevisiae: CIT2 and IDP3. The other two, PYC and ACO, are thought to act in the cytosol and mitochondria, respectively, thus demanding the shuttle of their substrates and products to and from the peroxisome (Fig 3A) [44]. We identified, however, a cryptic PTS1-like motif (SHL*) in the PYC1 genes of all four Saccharomyces species. The motif of S. cerevisiae PYC1 is located at 11 bp downstream from the stop codon, in a +1-shifted frame, and predicted as a weak motif by the PTS1 predictor [45] (S3A Fig).


The Evolutionary Potential of Phenotypic Mutations.

Yanagida H, Gispan A, Kadouri N, Rozen S, Sharon M, Barkai N, Tawfik DS - PLoS Genet. (2015)

The potential peroxisomal localization of a pathway supplying NADPH for β-oxidation.(A) The putative pathway follows the TCA reaction cycle, beginning with pyruvate and comprising four enzymes: PYC, pyruvate carboxylase; CIT, citrate synthase; ACO, aconitase; and finally IDP, isocirate dehydrogenase. Two enzymes have peroxisomal S. cerevisiae paralogous:—CIT3 and IDP3; the other two, PYC and ACO, are thought to act in the cytosol and mitochondria, respectively. Hypothetical transporters permitting the export and import of intermediate metabolites are depicted as white circles. (B) The PTS1-like motif of S.cer PYC1 (last 11 amino acids plus the 3’UTR ending with SHL*) was tested by fusion to the C-terminus of S.cer IDP2 and complementation of ΔIdp3 growth on petroselinate. Shown is growth with the original motif (orange line), and a version obtained by a single base deletion upstream the stop codon (green line) that puts the putative PTS1 in-frame (PYC1ΔG; the deleted G is highlighted in grey). (C) The PTS1-like motif of S.cer ACO2 (last 10 amino acids plus the 3’UTR ending with NKF*) was tested as described in part (B) above. An in-frame version obtained via a deletion of a single base upstream the stop codon (ACO2ΔA) was also tested. Whilst the initial growth was very slow, upon serial transfers to fresh petrosalinate media (5-fold dilutions) a marked increase in growth rate was observed. Single colonies were randomly isolated from the 2nd transferred culture (at 900 h) and sequenced. All 7 sequenced clones possessed a single mutation in the PTS1-like motif converting it to NKL (S4C Fig).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526572&req=5

pgen.1005445.g003: The potential peroxisomal localization of a pathway supplying NADPH for β-oxidation.(A) The putative pathway follows the TCA reaction cycle, beginning with pyruvate and comprising four enzymes: PYC, pyruvate carboxylase; CIT, citrate synthase; ACO, aconitase; and finally IDP, isocirate dehydrogenase. Two enzymes have peroxisomal S. cerevisiae paralogous:—CIT3 and IDP3; the other two, PYC and ACO, are thought to act in the cytosol and mitochondria, respectively. Hypothetical transporters permitting the export and import of intermediate metabolites are depicted as white circles. (B) The PTS1-like motif of S.cer PYC1 (last 11 amino acids plus the 3’UTR ending with SHL*) was tested by fusion to the C-terminus of S.cer IDP2 and complementation of ΔIdp3 growth on petroselinate. Shown is growth with the original motif (orange line), and a version obtained by a single base deletion upstream the stop codon (green line) that puts the putative PTS1 in-frame (PYC1ΔG; the deleted G is highlighted in grey). (C) The PTS1-like motif of S.cer ACO2 (last 10 amino acids plus the 3’UTR ending with NKF*) was tested as described in part (B) above. An in-frame version obtained via a deletion of a single base upstream the stop codon (ACO2ΔA) was also tested. Whilst the initial growth was very slow, upon serial transfers to fresh petrosalinate media (5-fold dilutions) a marked increase in growth rate was observed. Single colonies were randomly isolated from the 2nd transferred culture (at 900 h) and sequenced. All 7 sequenced clones possessed a single mutation in the PTS1-like motif converting it to NKL (S4C Fig).
Mentions: Following the above findings, and a report that appeared while this work was ongoing on cryptic peroxisomal targeting of two cytosolic enzymes [30], we performed a systematic computational search of the 3’-UTR regions of four closely related post-WGD Saccharomyces genomes: S. cerevisiae, S. paradoxus, S. bayanus and S. mikatae (S1 and S3 Tables). The search was based on PTS1 motifs containing all possible variation of amino acids ((S/A/C/E/I/H/Q)-(K/R/H)-(L/F)-stop) from 20 peroxisomal proteins in the Saccaromyces genome database. We searched for the motif starting up to 30 bp downstream the stop codon in all frames. PTS1-like motifs were found in around 1% of total genes of the genomes. However, about 40% of these were interrupted by another stop codon (S1 Table). Further, only a small number of these potentially cryptic motifs were found in more than one species, suggesting that these motifs are under functional selection. We thus focused on few interesting candidates that are conserved among the post-WGD species, and foremost on pyruvate carboxylase 1 (PYC1)—a cytosolic enzyme converting pyruvate to oxaloacetate. The NADPH used for peroxisomal β-oxidation could be produced from pyruvate by a putative pathway that includes four enzymes: PYC: pyruvate carboxylase; CIT: citrate synthase; ACO: aconitase; and finally IDP: isocirate dehydrogenase. Although not established as a peroxisomal NADPH providing pathway, this reaction sequence comprises part of the TCA cycle. Among these, two enzymes have known peroxisomal paralogues in S. cerevisiae: CIT2 and IDP3. The other two, PYC and ACO, are thought to act in the cytosol and mitochondria, respectively, thus demanding the shuttle of their substrates and products to and from the peroxisome (Fig 3A) [44]. We identified, however, a cryptic PTS1-like motif (SHL*) in the PYC1 genes of all four Saccharomyces species. The motif of S. cerevisiae PYC1 is located at 11 bp downstream from the stop codon, in a +1-shifted frame, and predicted as a weak motif by the PTS1 predictor [45] (S3A Fig).

Bottom Line: Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals.Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location.Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, Israel.

ABSTRACT
Errors in protein synthesis, so-called phenotypic mutations, are orders-of-magnitude more frequent than genetic mutations. Here, we provide direct evidence that alternative protein forms and phenotypic variability derived from translational errors paved the path to genetic, evolutionary adaptations via gene duplication. We explored the evolutionary origins of Saccharomyces cerevisiae IDP3 - an NADP-dependent isocitrate dehydrogenase mediating fatty acids ß-oxidation in the peroxisome. Following the yeast whole genome duplication, IDP3 diverged from a cytosolic ancestral gene by acquisition of a C-terminal peroxisomal targeting signal. We discovered that the pre-duplicated cytosolic IDPs are partially localized to the peroxisome owing to +1 translational frameshifts that bypass the stop codon and unveil cryptic peroxisomal targeting signals within the 3'-UTR. Exploring putative cryptic signals in all 3'-UTRs of yeast genomes, we found that other enzymes related to NADPH production such as pyruvate carboxylase 1 (PYC1) might be prone to peroxisomal localization via cryptic signals. Using laboratory evolution we found that these translational frameshifts are rapidly imprinted via genetic single base deletions occurring within the very same gene location. Further, as exemplified here, the sequences that promote translational frameshifts are also more prone to genetic deletions. Thus, genotypes conferring higher phenotypic variability not only meet immediate challenges by unveiling cryptic 3'-UTR sequences, but also boost the potential for future genetic adaptations.

No MeSH data available.


Related in: MedlinePlus