Rapamycin treatment results in GATA factor-independent hyperphosphorylation of the proline utilization pathway activator in Saccharomyces cerevisiae

Abstract

Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.

FIG. 1.

Phosphorylation profiles of Put3p in wild-type and GATA factor-deficient yeast strains. (A) Hyperphosphorylation of Put3p after rapamycin treatment in a wild-type strain. Protease-deficient strain DB1000 carrying high-copy PUT3-containing plasmid pMDB4 was grown in minimal glucose-glutamine medium supplemented with tryptophan. Rapamycin (lanes +; 200 ng/ml) or the drug vehicle alone (lanes −; 90% ethanol-10% Tween 20) was added, and the cells were harvested 60 mins later. Lanes:1 and 2, extracts were prepared and separated by SDS-PAGE followed by immunoblotting with anti-Put3p antibody; 3 and 4, rapamycin-treated extracts were incubated with calf intestinal phosphatase (CIP) in the presence or absence of phosphatase inhibitors (CIP Inh.), as described in Materials and Methods. (B) Hyperphosphorylation of Put3p after rapamycin treatment in GATA factor-deficient yeast strains. Strains BJ2168 (wild type [WT]) and BJ2168 nil1Δgln3Δ (nil1::hisG gln3::LEU2) carrying high-copy plasmid pMDB9 (PUT3) were grown in a minimal glucose-glutamine medium supplemented with tryptophan and leucine and treated with rapamycin (lanes 2 and 4) or drug vehicle (lanes 1 and 3) as in panel A. Extracts were prepared and separated by SDS-PAGE followed by immunoblotting with anti-Put3p antibody as in panel A. (C) Hyperphosphorylation of Put3p in wild-type and nil1 strains grown in nitrogen-derepressing media. Strains BJ2168 and BJ2168 nil1Δ (nil1::hisG) carrying plasmid pMDB9 were grown in a minimal glucose medium supplemented with tryptophan and leucine and containing either glutamine (Q; lanes 1 and 3) or GABA (G; lanes 2 and 4) as the sole nitrogen source. Extracts were prepared and separated by SDS-PAGE followed by immunoblotting with anti-Put3p antibody as in panel A. (D) Hyperphosphorylation of Put3p in a ure2Δ strain. Extracts of strains DB1000 or BJ2168 ure2Δ (ure2::LEU2) carrying plasmid pMDB4 (PUT3) grown in minimal media with either glutamine (Q; lanes 1, 2, 5, and 6) or GABA (G; lanes 3, 4, 7, and 8) as the nitrogen source were subjected to modified SDS-PAGE and immunoblotting with anti-Put3p antiserum. In lanes 5 to 8, the same extracts were incubated with calf intestinal phosphatase in the presence or absence of phosphatase inhibitors followed by SDS-PAGE and immunoblotting.

FIG. 2.

PUT gene expression after treatment with rapamycin. Protease-deficient strain BJ2168 carrying plasmids pYEp13 (LEU2) and either pWB36 (PUT1-lacZ) or pABC4 (PUT2-lacZ) was grown in minimal glucose-glutamine medium supplemented with tryptophan. Rapamycin (+; 200 ng/ml) or the drug vehicle alone (−; 90% ethanol-10% Tween 20) was added, and the cells were harvested 60 min later and assayed for β-galactosidase activity. Results are the average of three independent experiments with standard deviation as indicated.

FIG. 3.

Roles of Put3p, Gln3p, and Nil1p in rapamycin induction of PUT1 expression. Strains DB26-3A (wild type [WT]), DB1200 (gln3Δ), DB2000 (nil1Δ), DB2200 (gln3Δnil1Δ), DB26-2B (put3Δ), DB1201 (put3Δgln3Δ), DB2201 (put3Δnil1Δ), and DB2300 (put3Δgln3Δnil1Δ) carrying a genomic copy of PUT1-lacZ were grown in minimal glucose-glutamine medium supplemented with adenine, uracil, and leucine and treated as described in the legend to Fig. 2. Rapamycin induced PUT1 expression in wild-type, gln3Δ, and put3Δ strains by 4-, 3.6-, and 3.5-fold, respectively.

FIG. 4.

Effects of removal of Put3p, Gln3p, and Nil1p on nitrogen regulation of PUT1. (A) Nitrogen regulation of PUT1 under noninducing conditions. (B) Expression of PUT1 with proline as the sole source of nitrogen. The strains listed in Fig. 3 were grown in minimal medium containing glutamine (Gln, 0.1%), ammonia (Amm, 0.2%), glutamate (Glt, 0.1%), GABA (0.1%), or proline (Pro, 0.1%) as the nitrogen source. Extracts were made and assayed for β-galactosidase activity. Results are the average of two to five independent measurements with variations as indicated. WT, wild type.

FIG. 5.

Working model for the regulation of PUT1 transcription by nitrogen derepression and proline induction. See the text for details. Put3p binding to proline, binding of negatively acting GATA factors, DNA bending, and Put3p-GATA factor interactions are speculative at present. Abbreviations: UAS_PUT, binding sites for Put3p (bp −307 to −293 and −280 to −265 from the ATG of PUT1); GATAA, binding sites for GATA factors (bp −235 and −204); TATA, TATA box (bp −118). Symbols: →, extent and levels of PUT1 transcription relative to level on Gln; *, phosphorylation sites; , Put3p dimers; , proline; , positive GATA factors; , negative GATA factors.