Conformational changes play a role in regulating the activity of the proline utilization pathway-specific regulator in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA-bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the 'on' conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non-inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.

Functional analysis of the PUT3 transcriptional activator of the proline utilization pathway in Saccharomyces cerevisiae.

Proline can serve as a nitrogen source for the yeast Saccharomyces cerevisiae when preferred sources of nitrogen are absent from the growth medium. PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate. PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence to the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen. To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken. Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain. A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding protein stability. Intragenic suppressors of the central domain mutation have been isolated and analyzed.

Roles of URE2 and GLN3 in the proline utilization pathway in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, gamma-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment. Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes. The products of the GLN3 and URE2 genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways. GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed. The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study. Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect on PUT2 is comparatively small. Recessive mutations in URE2 elevated expression of the PUT1 and PUT2 genes 5- to 10-fold when cells were grown on a nitrogen-repressing medium. Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the sole nitrogen source, a put3 ure2 strain had somewhat elevated PUT gene expression, suggesting an effect of the ure2 mutation in the absence of the PUT3 product. PUT1 and PUT2 gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions. Therefore, regulation of the PUT genes by URE2 does not require a functional GLN3 protein. The effect of the ure2 mutation on the PUT genes is not due to increased internal proline levels. URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation.

Proline biosynthesis in Saccharomyces cerevisiae: analysis of the PRO3 gene, which encodes delta 1-pyrroline-5-carboxylate reductase.

The PRO3 gene of Saccharomyces cerevisiae encodes the 286-amino-acid protein delta 1-pyrroline-5-carboxylate reductase [L-proline:NAD(P+) 5-oxidoreductase; EC 1.5.1.2], which catalyzes the final step in proline biosynthesis. The protein has substantial similarity to the pyrroline carboxylate reductases of diverse bacterial species, soybean, and humans. Using RNA hybridization and measurements of enzyme activity, we have determined that the expression of the PRO3 gene appears to be constitutive. It is not repressed by the pathway end product (proline), induced by the initial substrate (glutamate), or regulated by the general control system. Its expression is not detectably altered when cells are grown in a wide range of nitrogen sources or when glycerol and ethanol replace glucose as the carbon source. The possibility that this enzyme has other functions in addition to proline biosynthesis is discussed.