The Regulator of the Yeast Proline Utilization Pathway Is Differentially Phosphorylated in Response to the Quality of the Nitrogen Source

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 andPUT2, 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 with³²P or ³⁵S 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.

ACKNOWLEDGMENTS

We thank C. Michels and J. Thorner for gifts of strains and plasmids and D. Barber for the construction of plasmids pDNB109 and pDNB118 and strains DB1000 and DB8-5C. We are grateful to members of the laboratory for helpful discussions and to S. Garrett and S. A. des Etages for critical reading of the manuscript.

This work was supported by Public Health Service grant 5 R01 GM40751 from the National Institutes of Health and grant 21-98 from the Foundation of UMDNJ.