The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, ␥-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.The proline utilization pathway in Saccharomyces cerevisiae enables cells to use proline as the sole source of nitrogen when preferred nitrogen sources are not available in the environment. The proline utilization enzymes proline oxidase and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase, encoded by the nuclear genes PUT1 and PUT2, respectively, convert proline to glutamate in mitochondria (9-11). The expression of the PUT genes is regulated by the PUT3 activator protein, which responds to the presence of proline in the medium and increases the transcription of the PUT1 and PUT2 genes (7,8,67). The PUT3 protein constitutively binds to the upstream activation sequences in the promoters of both PUT1 and PUT2 genes in vitro and in vivo but activates transcription only in the presence of proline (2,44,45,58).Early studies on the proline utilization pathway concluded that the structural genes PUT1 and PUT2 were not regulated by nitrogen repression (10). We speculated that the proline transpor...

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as allantoin, urea, γ-aminobutyrate, or proline when preferred nitrogen sources such as asparagine, glutamine, or ammonium ions are unavailable in the environment. To use proline as the sole nitrogen source, cells must activate the expression of the proline transporters and the genes that encode the catabolic enzymes proline oxidase (PUT1) and Δ1-pyrroline-5-carboxylate dehydrogenase (PUT2). Transcriptional activation of the PUT genes requires the PUT3 regulatory protein, proline, and relief from nitrogen repression. PUT3 is a 979 amino acid protein that binds a short DNA sequence in the promoters of PUT1 and PUT2, independent of the presence of proline. The functional domains of PUT3 have been studied by biochemical and molecular tests and analysis of activator-constitutive and activator-defective mutant proteins. Mutations in the URE2 gene relieve nitrogen repression, permitting inducer-independent transcription of the PUT genes in the presence of repressing nitrogen sources. The GLN3 protein that activates the expression of many genes in alternative nitrogen source pathways is not required for the expression of the PUT genes under inducing, derepressing conditions (proline) or noninducing, repressing conditions (ammonia). Although it has been speculated that the URE2 protein antagonizes the action of GLN3 in the regulation of many nitrogen assimilatory pathways, URE2 appears to act independently of GLN3 in the proline-utilization pathway. Key words: Saccharomyces cerevisiae, proline utilization, nitrogen repression.