The PUT) gene of Saccharomyces cerevisiae, believed to encode proline oxidase, has been completely sequenced and contains an open reading frame capable of encoding a polypeptide of 476 amino acids in length. The amino terminus of the protein deduced from the DNA sequence has a characteristic mitochondrial import signal; two PUT)-lacZ gene fusions were constructed that produced mitochondrially localized ,(-galactosidase in vivo. The transcription initiation and termination sites of the PUT) mRNA were determined. By using a PUT)-lacZ gene fusion that makes a cytoplasmic jI-galactosidase, the regulation of the PUT) gene was studied. PUT) is inducible by proline, responds only slightly to carbon catabolite repression, and is not regulated by the cytochrome activator proteins HAPI and HAP2.

The PUT) and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT) promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT) UASs showed that they were functionally independent and additive in producing maximal levels of gene expression.

The PUT2 gene was isolated on a 6.5-kilobase insert of a recombinant DNA plasmid by functional complementation of a put2 (A -pyrroline-5-carboxylate dehydrogenase-deficient) mutation in Saccharomyces cerevisiae. Its identity was confirmed by a gene disruption technique in which the chromosomal PUT2+ gene was replaced by plasmid DNA carrying the pit2 gene into which the S. cerevisiae HIS3+ gene had been inserted. The cloned PUT2 gene was used to probe specific mRNA levels: full induction of the PUT2 gene resulted in a 15-fold increase over the uninduced level. The PUT2-specific mRNA was approximately 2 kilobases in length and was used in Si nuclease protection experiments to locate the gene to a 3-kilobase HindIll fragment. When Al-pyrroline-5-carboxylate dehydrogenase activity levels were measured in strains carrying the original plasmid, as well as in subclones, similar induction ratios were found as compared with enzyme levels in haploid yeast strains. Effects due to increased copy number or position were also seen. The cloned gene on a 2i1-containing vector was used to map the PUT2 gene to chromosome VIII.

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.Saccharomyces cerevisiae cells can sense the quality of the nitrogen source in their environment, enabling them to utilize preferred nitrogen-containing compounds over nonpreferred ones or to express pathways for the utilization of alternative nitrogen sources when the preferred ones have been consumed. Although very little is known about the sensing mechanism itself, work over the last decade has led to the discovery of a set of regulatory proteins, the GATA factors, whose role is to regulate, in both positive and negative directions, the expression of pathways of nitrogen assimilation in yeast. These proteins, Gln3p (26), Nil1p/Gat1p (10, 44), Dal80p/Uga43p (12, 13), and Nil2p/Gzf3p/Deh1p (11,34,42), are involved in a complex set of regulatory loops, competition for GATA binding sites, and possibly even some autoregulation. Recently, the coactivator Ada1p, isolated as Gan1p, was identified as a link between the GATA binding proteins and the basal transcriptional machinery (41). Global nitrogen repressor Ure2p is believed to interact with Gln3p to obtain appropriate expression of a variety of nitrogen assimilator...

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...