Figure 1.
Model depicting pathways involved in alanine metabolism in Saccharomyces cerevisiae.
Alanine is biosynthesized through the action of the ALT1-encoded aminotransferase (1). It is proposed that alanine is also synthesized through the irreversible action of glutamine aminotransferase (2), which belongs to the ω-amidase pathway, this enzyme catabolizes glutamine to α-ketoglutaramate leading to the formation of NH4 and α-ketoglutarate (3). Dashed lines indicate that TCA cycle enzymes are inactive when grown on glucose as the sole carbon source, except those leading to the replenishment of α-ketoglutarate.
Figure 2.
Single alt1Δ and double alt1Δ alt2Δ mutants display alanine partial auxotrophy.
Wild type, alt1Δ, alt2Δ and alt1Δ alt2Δ were grown on ammonium-glucose (A) or alanine-ammonium-glucose (B). Graphed values represent means of three independent experiments ± SD. Specific growth rate was determined during exponential phase in glucose-ammonium cultures (C). Values are presented as means ± SD from three independent experiments. Intracellular concentration of alanine in extracts obtained from glucose-ammonium-grown cells (D). Yeast cells were grown and harvested when the cultures reached the stated optical density. Cell–free extracts were prepared and alanine pools were determined as described in Material and methods.
Figure 3.
ALT1 and ALT2 display differential gene expression pattern, which is consistent with Alt1-Alt2 intracellular concentration.
Northern blot of total RNA prepared from wild type strain grown on either glucose-ammonium (A) or glucose-alanine (B). Samples were collected from various OD600 as stated. Representative results from three experiments are shown. Cell-free extracts from ALT1-TAP (C and E) or ALT2-TAP (D and F) tagged strains grown on glucose-ammonium (C and D) or glucose-alanine (E and F) were prepared and subjected to immunoblot analysis using anti-TAP polyclonal antibody. As a loading control, each nitrocellulose membrane was also subjected to immunoblot analysis using anti-Lys20/Lys21 antibody. All lanes were loaded with 100 µg of protein. An ALT1-TAP sample was included as a loading control in panel F. Results are representative of three independent experiments.
Figure 4.
Alanine determines ALT1 expression induction and ALT2 expression repression.
Northern blot of total RNA obtained from wild type strains grown on 7 mM proline with or without alanine (A) or on 3 mM or 7 mM alanine (B), as nitrogen sources.
Figure 5.
Nrg1 determines ALT1 and ALT2 repression.
Northern blot of total RNA obtained from wild type and nrg1Δ strains grown on either glucose-ammonium (A) or glucose-alanine (B). Samples were collected from various OD600 as stated. Representative results from three experiments are shown. ChIP assays were performed using anti-Myc antibody on wild type strains containing myc13 epitope-tagged NRG1. As a control, equivalent samples were treated with anti-HA antibody. Strains were grown on 2% (w/v) glucose +7 mM alanine to either 0.3 or 1.8 OD600, cells were centrifuged, collected and used to prepare samples for ChIP (C, D). PCR was performed with the deoxyoligonucleotides described in Table 3. As a negative control, PCR was also performed for LEU4 coding region. Results are representative of three independent experiments.
Figure 6.
tetO7 promoter increases ALT2 expression, which is consistent with Alt2 intracellular concentration.
Northern blot of total RNA prepared from wild type strain, alt1Δ and alt1Δ tetO7-ALT2 grown glucose-ammonium (A). Samples were collected from various OD600 as stated. Representative results from three experiments are shown. Cell-free extracts from tetO7-ALT2-yEcitrine (tetO7-ALT2-YEC) (B), and ALT2-yEcitrine (ALT2-YEC) (C) tagged strains grown on glucose-ammonium were obtained and subjected to immunoblot analysis using anti-GFP monoclonal antibody. As a loading control, nitrocellulose membranes were also immunoblotted with anti-Lys20/Lys21 antibody. All lanes were loaded with 100 μg of protein.
Table 1.
Alanine aminotransferase specific activity in extracts prepared from glucose-NH4 grown cultures.
Figure 7.
Figure. Evolutionary relationships of alanine aminotransferases present in post-WGD yeasts.
The evolutionary history was inferred using the Maximum-Likelihood method [29]. The branch lengths are measured in the number of substitutions per site. ALT1 and ALT2 are clustered in different subtrees with their corresponding post-WGD yeasts orthologues. Since ALT2 branch is longer than ALT1 branch, a higher divergence rate for ALT2 is suggested.
Table 2.
Strains used in this study.
Table 3.
Oligonucleotides used for strain construction.