Mutation of a phosphorylatable residue in Put3p affects the magnitude of rapamycin-induced PUT1 activation in a Gat1p-dependent manner

Abstract

Saccharomyces cerevisiae can utilize high quality (e.g. glutamine and ammonia) as well as low quality (e.g. gamma-amino butyric acid and proline) nitrogen sources. The transcriptional activator Put3p allows yeast cells to utilize proline as a nitrogen source through expression of the PUT1 and PUT2 genes. Put3p activates high level transcription of these genes by binding proline directly. However, Put3p also responds to other lower quality nitrogen sources. As nitrogen quality decreases, Put3p exhibits an increase in phosphorylation concurrent with an increase in PUT gene expression. The proline-independent activation of the PUT genes requires both Put3p and the positively acting GATA factors, Gln3p and Gat1p. Conversely, the phosphorylation of Put3p is not dependent on GATA factor activity. Here, we find that the mutation of Put3p at amino acid Tyr-788 modulates the proline-independent activation of PUT1 through Gat1p. The phosphorylation of Put3p appears to influence the association of Gat1p, but not Gln3p, to the PUT1 promoter. Combined, our findings suggest that this may represent a mechanism through which yeast cells rapidly adapt to use proline as a nitrogen source under nitrogen limiting conditions.

FIGURE 1.

Detection of the phosphorylation state of Put3p. a, Put3p purified from insect and S. cerevisiae cells. Protein samples were incubated at 37 °C for 1 h with either calf intestinal phosphastase (+)or buffer (−). The difference in the apparent molecular masses of the two protein preparations is because of the size of the tags used, His (4.3 kDa) or MORF (19 kDa). The asterisk indicates the location of the lower mobility-phosphorylated version of Put3p. b, identified phosphopeptides from the proteolytic digestion of Put3p purified from yeast. The phosphorylated amino acid is each peptide is highlighted in red. Peptides were generated using either trypsin (cleavage sites indicated by the backslash), chymotrypsin or AspN, or a combination of these proteases. c, a schematic representation of Put3p illustrating the location of phosphorylation sites and subdomains. MHR, middle homology region; DBD, DNA binding domain; AD, activation domain.

FIGURE 2.

Effects of N-terminal phosphorylation sites on PUT1:lacZ activation. Relative β-galactosidase activity of put3Δ, PUT1-lacZ cells carrying pRS415, a pRS415 plasmid expressing wild-type (WT) Put3p, or a version of Put3p bearing the annotated mutation(s). Cells were grown in media containing glutamate to an A₆₀₀ ≈ 0.6 and treated with either rapamycin (gray bars) or the drug vehicle (open bars) for 1 h. Values represent the mean of three separate cultures subjected to the same treatment, and error bars indicate S.D.

FIGURE 3.

Effects of C-terminal phosphorylation sites on PUT1:lacZ activation. a, relative β-galactosidase activity of C-terminal phosphorylation-incompetent mutants. b, relative β-galactosidase activity of C-terminal mock-phosphorylation mutants. put3Δ, PUT1-lacZ cells carrying pRS415 (39), the pRS415 plasmid expressing wild-type (WT) Put3p, or a version of Put3p bearing the annotated mutation(s) was grown in media containing glutamate to an A₆₀₀ ≈ 0.6 and treated with either rapamycin (gray bars) or the drug vehicle (open bars) for 1 h. Values represent the mean of three separate cultures subjected the same treatment, and error bars indicate S.D.

FIGURE 4.

Relative β-galactosidase activity of Put3p.Y788F grown in either glutamine or proline +/− rapamycin (Rap). put3Δ cells carrying either pRS415 (open bars), the pRS415 plasmid expressing wild-type (WT) Put3p (gray bars), or a version of Put3p bearing Y788F mutation (black bars) were grown in media containing either glutamate or proline to an A₆₀₀ ≈ 0.6 and treated with either rapamycin or the drug vehicle for 1 h. Values represent the mean of three separate cultures subjected the same treatment, and error bars indicate S.D.

FIGURE 5.

Relative β-galactosidase activity of Put3p.Y788F in yeast cell GATA factor-deleted backgrounds. a, put3Δ, PUT1-lacZ and put3Δ, ure2Δ, PUT1-lacZ cells carrying either pRS415, the pRS415 plasmid expressing wild-type (WT) Put3p, or a version of Put3p bearing Y788F mutation in the presence and absence of rapamycin. b, put3Δ, PUT1-lacZ, and put3Δ, gatlΔ, PUT1-lacZ cells carrying either pRS415, the pRS415 plasmid expressing wild-type Put3p, or a version of Put3p bearing Y788F mutation in the presence and absence of rapamycin. c, put3Δ, PUT1-lacZ and put3Δ, gln3Δ PUT1-lacZ cells carrying either pRS415, the pRS415 plasmid expressing wild-type Put3p, or a version of Put3p bearing Y788F mutation in the presence and absence of rapamycin. Cells were grown in media containing glutamine to an A₆₀₀ ≈ 0.6 and treated with either rapamycin (gray bars) or the drug vehicle (open bars) for 1 h. Values represent the mean of three separate cultures subjected the same treatment, and error bars indicate S.D.

FIGURE 6.

The interaction of Gat1p and Gln3p with the PUT1 promoter in the presence of wild type and Y788F versions of Put3p. a, cells expressing Rpb3p-cAVI were treated with rapamycin, and ChIP-quantitative PCR was performed for either the PUT1 start site (−98 to +52) or the PUT1 upstream region (−303 to −178, as indicated). ChIP-quantitative PCR data are expressed as IP:Input ratio of PUT1 to HMR. Data were normalized to the wild-type rapamycin control. Error bars represent S.D. for at least three biological replicates. An asterisk indicates a p value of less than 0.05 in comparison to the wild-type control. b, as in a, using cells expressing Gat1p-cAVI and either the wild-type or Y788F versions of Put3p were treated with rapamycin, and ChIP-quantitative PCR was performed for the PUT1 upstream region (−303 to −178). c, as in b, using cells expressing Gln3p-cAVI.