The regulator of the yeast proline utilization pathway is differentially phosphorylated in response to the quality of the nitrogen source - PubMed (original) (raw)

The regulator of the yeast proline utilization pathway is differentially phosphorylated in response to the quality of the nitrogen source

H L Huang et al. Mol Cell Biol. 2000 Feb.

Abstract

The proline utilization pathway in Saccharomyces cerevisiae is regulated by the Put3p transcriptional activator in response to the presence of the inducer proline and the quality of the nitrogen source in the growth medium. Put3p is constitutively bound to the promoters of its target genes, PUT1 and PUT2, under all conditions studied but activates transcription to the maximum extent only in the absence of rich nitrogen sources and in the presence of proline (i.e., when proline serves as the sole source of nitrogen). Changes in target gene expression therefore occur through changes in the activity of the DNA-bound regulator. In this report, we demonstrate by phosphatase treatment of immunoprecipitates of extracts metabolically labeled with (32)P or (35)S that Put3p is a phosphoprotein. Examination of Put3p isolated from cells grown on a variety of nitrogen sources showed that it was differentially phosphorylated as a function of the quality of the nitrogen source: the poorer the nitrogen source, the slower the gel migration of the phosphoforms. The presence of the inducer does not detectably alter the phosphorylation profile. Activator-defective and activator-constitutive Put3p mutants have been analyzed. One activator-defective mutant appears to be phosphorylated in a pattern similar to that of the wild type, thus separating its ability to be phosphorylated from its ability to activate transcription. Three activator-constitutive mutant proteins from cells grown on an ammonia-containing medium have a phosphorylation profile similar to that of the wild-type protein in cells grown on proline. These results demonstrate a correlation between the phosphorylation status of Put3p and its ability to activate its target genes and suggest that there are two signals, proline induction and quality of nitrogen source, impinging on Put3p that act synergistically for maximum expression of the proline utilization pathway.

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Figures

FIG. 1

GST-Put3p and Put3p are phosphoproteins. (A) 32P labeling and IP of GST-Put3p. Extracts were made from cells of strain DB1000 carrying plasmid pHB3 (GST-PUT3) that were grown in a low-phosphate medium with ammonia (A; lane 1) or proline (P; lane 2) as the sole nitrogen source, labeled with 32P, and immunoprecipitated with mouse monoclonal anti-GST, as described in Materials and Methods. The immunoprecipitated GST-Put3p from the proline culture (lane 3) was incompletely digested with thrombin to release full-length Put3p (lane 4). After the signal decayed, the membrane was probed with anti-Put3p (lanes 5 and 6) and then reprobed with anti-GST antibodies (lanes 7 and 8) for detection of GST-Put3p and the cleaved Put3p. The proteins were resolved on a 12.5% (12.57% total acrylamide concentration [T], 0.5% cross-linker concentration [C]) polyacrylamide gel and transferred to a PVDF membrane. Circled P, phosphorylated form. (B) 32P labeling and IP of Put3p. Extracts were made from cells of strain DB1000 carrying plasmid pDB37 (PUT3) grown in a low-phosphate medium with ammonia (lane 3) or proline (lane 4) as the sole nitrogen source or carrying YEp24 grown in a low-phosphate medium with ammonia (lane 7), labeled with 32P, and immunoprecipitated with polyclonal anti-Put3p, as described in Materials and Methods. The proteins were resolved on a 7.5% (7.65% T, 2% C) polyacrylamide gel and transferred to a PVDF membrane. After the signal decayed, the membrane was probed with anti-Put3p antibody (lanes 1 and 2). The 32P-labeled immunoprecipitates from the ammonia cultures were treated with CIP in the presence (+) or absence (−) of phosphatase inhibitors (lanes 5 and 6, wild type; lane 7, _put3_Δ). (C) 35S labeling of Put3p. Extracts of 35S-labeled DB1000 carrying plasmid pDB37 grown on ammonia- or proline-containing medium were immunoprecipitated with anti-Put3p antibody (lanes 1 to 4, upper section) and treated with CIP in the presence (lanes 1 and 3) or absence (lanes 2 and 4) of phosphatase inhibitors. The proteins were resolved on a 7.5% (7.65% T, 2% C) polyacrylamide gel and transferred to a PVDF membrane. After the signal decayed, the membrane was probed with anti-Put3p antibody (lanes 1 to 4, lower section). These gels were run under conditions that do not resolve isoforms of Put3p.

FIG. 2

Put3p is differentially phosphorylated as a function of the quality of the nitrogen source. (A) Isoforms of Put3p. Extracts of strain DB1000 carrying plasmid pDB37 and grown on minimal media with different nitrogen sources were subjected to SDS–6% PAGE (6.12% T, 2% C) and immunoblotting with anti-Put3p antiserum. Lanes: 1 and 5, ammonia; 2, urea; 3, GABA; 4, proline. Below each lane is the net specific activity (Net Sp. Act.) of β-galactosidase from strain DB1000 (_put3_Δ) carrying plasmids pDB37 (PUT3) and pMB7 (PUT2-lacZ) and grown under the same conditions. (B) Addition of the inducer has little effect on the migration of Put3p isoforms. Extracts of DB1000 carrying plasmid pDB37 were treated as described for panel A. Lanes: 1 and 8, ammonia; 2, ammonia plus proline; 3, urea; 4, urea plus proline; 5, GABA; 6, GABA plus proline; 7, proline. Below each lane is the net specific activity of β-galactosidase from strain DB1000 (_put3_Δ) carrying plasmids pDB37 (PUT3) and pMB7 (PUT2-lacZ) and grown under the same conditions. (C) Isoforms of Put3p and GST-Put3p are due to differential phosphorylation. Extracts of DB1000 carrying plasmid pDB37 (PUT3) or pHB3 (GST-PUT3) were made from cells grown in minimal media with ammonia (lane 3), GABA (lane 4), or GABA plus proline (lane 5) as the sole nitrogen sources and analyzed as described for panel A. Markers: lane 1, extract from strain DB1000 carrying plasmid pHB6 (put3-75) grown on minimal GABA medium; lane 2, extract from strain DB1000 carrying plasmid pDB37 (PUT3) grown on minimal proline medium. Immunoprecipitates from the same extracts were treated with CIP in the presence (lanes 6, 8, and 10) or absence (lanes 7, 9, and 11) of phosphatase inhibitors.

FIG. 3

Phosphorylation is altered in regulatory Put3p mutants. (A) Activator-constitutive mutants have unique phosphorylation profiles of Put3p. Extracts of strain DB1000 carrying plasmids pMB3 (_PUT3_c-903, upper section), pMB4 (_PUT3_c-914, middle section), or pMB5 (_PUT3_c-683, lower section) grown on minimal medium with ammonia (lane 3), GABA (lane 4), or GABA plus proline (lane 5) were subjected to SDS–6% PAGE (6.12% T, 2% C) and immunoblotting with anti-Put3p antiserum. Extracts from GABA-plus-proline-grown cultures were immunoprecipitated with anti-Put3p antiserum and treated with CIP in the presence (lane 6) or absence (lane 7) of phosphatase inhibitors. Markers: lane 1, extract from strain DB1000 carrying plasmid pHB6 (put3-75) grown on minimal GABA medium; lane 2, extract from strain DB1000 carrying plasmid pDB37 (PUT3) grown on minimal proline medium. The net specific activities (Net Sp. Act.) of β-galactosidase from a PUT2-lacZ gene in each strain are given below lanes 3 to 5. (B) Two activator-defective Put3p mutants differ from each other in their phosphorylation profiles. Extracts of strain DB1000 carrying plasmids pHB6 (put3-316; upper section) or pDB193 (put3-75; lower section) grown on minimal medium with ammonia (lane 3), GABA (lane 4), or GABA plus proline (lane 5) were subjected to SDS-PAGE and immunoblotting with anti-Put3p antiserum. Extracts from GABA-plus-proline-grown cultures were immunoprecipitated with anti-Put3p antiserum and treated with CIP in the presence (lane 6) or absence (lane 7) of phosphatase inhibitors. Markers are as described for panel A. Full specific activities of β-galactosidase from a PUT2-lacZ gene in each strain are given below lanes 3 to 5.

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The regulator of the yeast proline utilization pathway is differentially phosphorylated in response to the quality of the nitrogen source - PubMed (original) (raw)