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

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.     

Evidence for Positive Regulation of the Proline Utilization Pathway in Saccharomyces cerevisiae

A mutation has been identified that prevents Saccharomyces cerevisiae cells from growing on proline as the sole source of nitrogen, causes noninducible expression of the PUT1 and PUT2 genes, and is completely recessive. In the put3-75 mutant, the basal level of expression (ammonia as nitrogen source) of PUT1-lacZ and PUT2-lacZ gene fusions as measured by beta-galactosidase activity is reduced 4- and 7-fold, respectively, compared with the wild-type strain. Normal regulation is not restored when the cells are grown on arginine as the sole nitrogen source and put3-75 cells remain sensitive to the proline analog, L-azetidine-2-carboxylic acid, indicating that the block is not at the level of transport of the inducer, proline. In a cross between the put3-75 strain and the semidominant, constitutive mutation PUT3c-68, only parental ditype tetrads were found, indicating allelism of the two mutations. Further support for allelism derives from the comparison of enzyme levels in heteroallelic and heterozygous diploid strains. The constitutive allele appears to be fully dominant to the noninducible allele but only partially dominant to the wild type, suggesting an interaction between the wild-type and PUT3c-68 gene products. The PUT3 gene maps on chromosome XI, about 5.7 cM from the centromere. The phenotypes of alleles of the PUT3 gene, either recessive and noninducible (the put3-75 phenotype) or semidominant and constitutive (the PUT3c-68 phenotype), and their pleiotropy suggest that the PUT3 gene product is a positive activator of the proline utilization pathway.     

Amino-terminal fragments of delta 1-pyrroline-5-carboxylate dehydrogenase direct beta-galactosidase to the mitochondrial matrix in Saccharomyces cerevisiae.

delta 1-Pyrroline-5-carboxylate (P5C) dehydrogenase, the second enzyme in the proline utilization (Put) pathway of Saccharomyces cerevisiae and the product of the PUT2 gene, was localized to the matrix compartment by a mitochondrial fractionation procedure. This result was confirmed by demonstrating that the enzyme had limited activity toward an externally added substrate that could not penetrate the inner mitochondrial membrane (latency). To learn more about the nature of the import of this enzyme, three gene fusions were constructed that carried 5'-regulatory sequences through codons 14, 124, or 366 of the PUT2 gene ligated to the lacZ gene of Escherichia coli. When these fusions were introduced into S. cerevisiae either on multicopy plasmids or stably integrated into the genome, proline-inducible beta-galactosidase was made. The shortest gene fusion, PUT2-lacZ14, caused the production of a high level of beta-galactosidase that was found exclusively in the cytoplasm. The PUT2-lacZ124 and PUT2-lacZ366 fusions made lower levels of beta-galactosidases that were mitochondrially localized. Mitochondrial fractionation and protease-protection experiments showed that the PUT2-lacZ124 hybrid protein was located exclusively in the matrix, while the PUT2-lacZ366 hybrid was found in the matrix as well as the inner membrane. Thus, the amino-terminal 124 amino acids of P5C dehydrogenase carries sufficient information to target and deliver beta-galactosidase to the matrix compartment. The expression of the longer hybrids had deleterious effects on cell growth; PUT2-lacZ366-containing strains failed to grow on proline as the sole source of nitrogen. In the presence of the longest hybrid beta-galactosidase, the wild-type P5C dehydrogenase was still properly localized in the matrix compartment, but its activity was reduced. The nature of the effects of these hybrid proteins on cell growth is discussed.     

Purification and characterization of Put1p from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH) enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A K_m value of 36 mM proline and a k_cat = 27 s⁻¹ were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ₁) as an electron acceptor with a k_cat = 9.6 s⁻¹ and a K_m of 33 μM for CoQ₁. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast.     

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

The PUT1 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 PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.     

Proline: an essential intermediate in arginine degradation in Saccharomyces cerevisiae.

Results of studies on proline-nonutilizing (Put-) mutants of the yeast Saccharomyces cerevisiae indicate that proline is an essential intermediate in the degradation of arginine. Put- mutants excreted proline when grown on arginine or ornithine as the sole nitrogen source. Yeast cells contained a single enzyme, delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase, which is essential for the complete degradation of both proline and arginine. The sole inducer of this enzyme was found to be proline. P5C dehydrogenase converted P5C to glutamate, but only when the P5C was derived directly from proline. When the P5C was derived from ornithine, it was first converted to proline by the enzyme P5C reductase. Proline was then converted back to P5C and finally to glutamate by the Put enzymes proline oxidase and P5C dehydrogenase.     

Role of L-proline in the biosynthesis of prodigiosin.

Nonproliferating cells of Serratia marcescens, wild-type strain Nima, synthesized the pigment, prodigiosin, when saline suspensions were incubated with aeration at 27 degrees C in the presence of proline or alanine. Mutants PutS1 and PutS2 derived from strain Nima formed prodigiosin from alanine, but not from proline, unless alanine also was added. Strain Nima utilized proline as a sole source of carbon and of nitrogen for growth, whereas Put mutants did not. Investigation of enzymes degrading proline showed that the wild-type strain contained proline oxidase, which was absent in Put mutants. The wild type, as well as the mutants, utilized alanine as the sole source of carbon and nitrogen for growth. Although nonproliferating cells of Put mutants failed to synthesize prodigiosin from proline, addition of L-[U-14C]proline to suspensions metabolizing and synthesizing the pigment because of addition of alanine resulted in the incorporation of radioactive label into prodigiosin, as well as into cellular protein. Since Put mutants could not catabolize proline, the incorporation of [14C]proline into the prodigiosin molecule indicated that proline was incorporated directly into the pigment.     

Elimination of cdc2 phosphorylation sites in the cdc25 phosphatase blocks initiation of M-phase.

The cdc25 phosphatase is a mitotic inducer that activates p34cdc2 at the G2/M transition by dephosphorylation of Tyr15 in p34cdc2. cdc25 itself is also regulated through periodic changes in its phosphorylation state. To elucidate the mechanism for induction of mitosis, phosphorylation of cdc25 has been investigated using recombinant proteins. cdc25 is phosphorylated by both cyclin A/p34cdc2 and cyclin B/p34cdc2 at similar sets of multiple sites in vitro. This phosphorylation retards its electrophoretical mobility and activates its ability to increase cyclin B/p34cdc2 kinase activity three- to fourfold in vitro, as found for endogenous Xenopus cdc25 in M-phase extracts. The threonine and serine residues followed by proline that are conserved between Xenopus and human cdc25 have been mutated. Both the triple mutation of Thr48, Thr67, and Thr138 and the quintuple mutation of these three threonine residues plus Ser205 and Ser285, almost completely abolish the shift in electrophoretic mobility of cdc25 after incubation with M-phase extracts or phosphorylation by p34cdc2. These mutations inhibit the activation of cdc25 by phosphorylation with p34cdc2 by 70 and 90%, respectively. At physiological concentrations these mutants cannot activate cyclin B/p34cdc2 in cdc25-immunodepleted oocyte extracts, suggesting that a positive feed-back loop between cdc2 and cdc25 is necessary for the full activation of cyclin B/p34cdc2 that induces abrupt entry into mitosis in vivo.