Ongoing resolution of duplicate gene functions shapes the diversification of a metabolic network
- University of Wisconsin-Madison, United States
- University of Wisconsin-Madison, Madison, United States
- Morgridge Institute for Research, United States
Figures
Figure 1
The S. uvarum GAL network.
(A) The GAL regulatory network. (B) The GAL or Leloir metabolic pathway. The colors show the amino acid identity of each component compared to their S. cerevisiae homologs (full data in Figure …
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Figure 1—source data 1
Amino acid identity and GAL gene composition between S. uvarum and S. cerevisiae GAL network.
Quantitative data underlying Figure 1.
- https://doi.org/10.7554/eLife.19027.004
Figure 2 with 1 supplement
SuvaGAL1 and SuvaGAL3 are not as subfunctionalized as ScerGAL1 and ScerGAL3.
(A) S. uvarum GAL3 likely encodes a functional galactokinase. The error bars represent standard deviations of three biological replicates. A Wilcoxon rank sum test comparing the average times to …
Figure 2—figure supplement 1
S. uvarum and S. cerevisiae have qualitatively similar gal1 null phenotypes.
‘+’ indicates growth after 7 days, while ‘−’ indicates no growth after 7 days when compared to the negative control (minimal media without a carbon source) (Materials and methods). Note that driving …
Figure 3 with 1 supplement
SuvaGAL80 and SuvaGAL80B encode co-repressors with partially overlapping functions.
(A) Expression divergence between SuvaGAL80 and SuvaGAL80B. The bar graph on the left shows the mRNA levels (in log2 of Reads Per Kilobase of transcript per Million mapped reads or RPKM) of SuvaGAL80…
Figure 3—figure supplement 1
In SC + 2% galactose, S. uvarum gal80∆ and gal80b∆ had GAL1 expression levels similar to the wild-type at mid-log phase.
Flow cytometry histogram of PGAL1-EGFP fluorescence.
Figure 4 with 6 supplements
The galactose-dependent temporary growth arrest phenotype of S.uvarum gal80∆ gal80b∆.
(A) The Temporary Growth Arrest (TGA) phenotype in SC + 2% galactose. The averages of the log2 of the ratios between absorbances at each time point (ODt) and initial absorbances (OD0) for three …
Figure 4—figure supplement 1
The TGA phenotype of S. uvarum gal80∆ gal80b∆ can be rescued by S. cerevisiae GAL80 or by re-introducing SuvaGAL80.
The bar graphs show the average times to first doubling time of three biological replicates. Strains were cultured in SC + 2% galactose.
Figure 4—figure supplement 2
Galactose-specific global differential expression of S. uvarum gal80∆ gal80b∆.
(A) S. uvarum GAL network comprises similar targets as the S. cerevisiae GAL network. The bar graph shows the log2 of the RPKM ratio between S. uvarum gal80∆ gal80b∆ and wild-type in SC + 5% …
Figure 4—figure supplement 3
High performance liquid chromatography measurements of key metabolites in SC + 2% galactose in S. uvarum gal80∆ gal80b∆ and wild-type during the TGA phase and after the growth resumed.
Statistically significant data points are marked by asterisks (*p<0.05, **p<0.01, one-tailed Student’s t-test). Red corresponds to ethanol, and blue corresponds to galactose; ethanol was produced by …
Figure 4—figure supplement 4
GAL1 expression was higher at the early stages of growth in SC + 2% galactose in the S. uvarum gal80∆ gal80b∆ background but gradually decreased.
Fluorescence levels were obtained by flow cytometry, normalized to forward scatter, and plotted as histograms. 4 hr was during the TGA phase, whereas 8 hr was approaching the end of the TGA phase.
Figure 4—figure supplement 5
Fructose, mannose, or glucose alone did not lead to a TGA phenotype or other growth defects.
All experiments were performed in SC media with the carbon sources indicated.
Figure 4—figure supplement 6
The regulation of PGM1 by galactose was inferred as the ancestral state.
(A) mRNA levels of S. uvarum PGM1 and PGM2 during mid-log phase in SC + 2% galactose, SC + 5% glycerol, and SC + 2% glucose. Note that PGM2, which encodes the major isoform of phosphoglucomutase, …
Figure 5 with 1 supplement
Overly rapid galactose catabolism leads to metabolic overload and bottlenecks.
The graph shows the metabolite levels and transcript expression for the Leloir pathway, glycolysis, trehalose cycle, glycerol biosynthesis, TCA cycle, and electron transport chain. Purple steps cost …
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Figure 5—source data 1
Differential gene expression between S. uvarum gal80∆ gal80b∆ and wild-type during the TGA phase and at mid-log phase.
- https://doi.org/10.7554/eLife.19027.017
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Figure 5—source data 2
Mass spectrometry metabolomic results comparing S. uvarum gal80∆ gal80b∆ to wild-type during the TGA phase and mid-log phase.
- https://doi.org/10.7554/eLife.19027.018
Figure 5—figure supplement 1
Galactose-1-phosphate accumulation of S. cerevisiae gal7∆ and gal10∆.
Galactose-1-phosphate levels were quantified by mass spectrometry. Samples were harvested after 4.5 hr of growth in 2% galactose. ‘LOQ’ stands for “Limit of Quantification”.
Figure 6
The addition of sugars downstream of the trehalose cycle exacerbated metabolic overload.
(A) Fructose and mannose exacerbated the TGA phenotype in the S. uvarum gal80∆ gal80b∆ background, whereas glucose partially rescued the TGA phenotype. (B) The S. uvarum TGA phenotype in galactose …
Figure 7
The less active S. cerevisiae GAL1 gene is partially responsible for a subtle temporary growth arrest.
(A) Elevated accumulation of ROS in S. cerevisiae gal80∆ in SC + 1% galactose +1% mannose. S. cerevisiae gal80∆ had significantly higher ROS than wild-type (p=2.3e-6, n = 12, Wilcoxon rank sum …
Figure 8
Ongoing diversification of the functions of the GAL1-GAL3 and GAL80-GAL80B duplicate gene pairs in Saccharomyces.
Important evolutionary events are shown on the cladogram. WGD, the whole genome duplication that created the two pairs of paralogs. The inferred duplicate divergence fates are shown at the bottom of …
Additional files
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Supplementary file 1
Gene Ontology Term enrichment results of mis-regulated processes in Suva gal80∆ gal80b∆ during the TGA phase and mid-log phase.
- https://doi.org/10.7554/eLife.19027.023
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Supplementary file 2
List of strains used in this study.
- https://doi.org/10.7554/eLife.19027.024
