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.     

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.     

Improved anaerobic use of arginine by Saccharomyces cerevisiae

Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Delta(1)-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.     

Improved Anaerobic Use of Arginine by Saccharomyces cerevisiae

Anaerobic arginine catabolism in Saccharomyces cerevisiae was genetically modified to allow assimilation of all four rather than just three of the nitrogen atoms in arginine. This was accomplished by bypassing normal formation of proline, an unusable nitrogen source in the absence of oxygen, and causing formation of glutamate instead. A pro3 ure2 strain expressing a PGK1 promoter-driven PUT2 allele encoding Δ¹-pyrroline-5-carboxylate dehydrogenase lacking a mitochondrial targeting sequence produced significant cytoplasmic activity, accumulated twice as much intracellular glutamate, and produced twice as much cell mass as the parent when grown anaerobically on limiting arginine as sole nitrogen source.     

The Saccharomyces Cerevisiae Rtg2 Gene Is a Regulator of Aconitase Expression under Catabolite Repression Conditions

The ACO1 gene, encoding mitochondrial aconitase of Saccharomyces cerevisiae, is required both for oxidative metabolism and for glutamate prototrophy. This gene is subject to catabolite repression; the ACO1 mRNA level is further reduced when glutamate is supplied with glucose. To further explore regulation of ACO1 expression, we have screened for mutations that reduce expression of an ACO1-lacZ fusion borne on a multicopy vector. We identified a gene required for wild-type expression of ACO1 only under catabolite repression conditions. Sequencing of the corresponding cloned gene revealed that it is identical to RTG2 previously cloned as a pivotal gene in controlling interorganelle retrograde communication. Cells containing either the original rtg2-2 mutation or a null rtg2 allele are not petite but show a residual growth on minimum glucose medium with ammonium sulfate as the sole nitrogen source. This growth defect is partially restored by supplying aspartate or threonine, and fully with glutamate or proline supplement. Surprisingly, this phenotype is not observed on complete medium lacking either of these amino acids. In addition, a genetic analysis revealed an interaction between RTG2 and ASP5 (encoding aspartate amino transferase), thus supporting our hypothesis that RTG2 may be involved in the control of several anaplerotic pathways.     

Proline accumulation by mutation or disruption of the proline oxidase gene improves resistance to freezing and desiccation stresses in Saccharomyces cerevisiae

We examined the role of intracellular proline under freezing and desiccation stress conditions in Saccharomyces cerevisiae. When cultured in liquid minimal medium, the proline-nonutilizing mutant containing the put1 mutation (proline oxidase-deficient) produced more intracellular proline, and increased the cell survival rate as compared to the wild-type strain after freezing and desiccation. We also constructed two PUT1 gene disruptants. PUT1-disrupted mutants in minimal medium supplemented with external proline at 0.1% accumulated higher proline levels than those of the control strains (17-22-fold). These disruptants also had a 2-5-fold increase in cell viability compared to the control strains after freezing and desiccation stresses. These results indicate that proline has a stress-protective function in yeast.     

Activation of a new proline transport system in Salmonella typhimurium.

Proline uptake can be mediated by three different transport systems in wild-type Salmonella typhimurium: a high-affinity proline transport system encoded by the putP gene and two glycine-betaine transport systems with a low affinity for proline encoded by the proP and proU genes. However, only the PutP permease transports proline well enough t allow growth on proline as a sole carbon or nitrogen source. By selecting for mutations that allow a putP mutant to grow on proline as a sole nitrogen source, we isolated mutants (designated proZ) that appeared to activate a cryptic proline transport system. These mutants enhanced the transport of proline and proline analogs but did not require the function of any of the known proline transport genes. The mutations mapped between 75 and 77.5 min on the S. typhimurium linkage map. Proline transport by the proZ mutants was competitively inhibited by isoleucine and leucine, which suggests that the ProZ phenotype may be due to unusual mutations that alter the substrate specificity of the branched-chain amino acid transport system encoded by the liv genes.     

Molecular characterization of yeast regulatory gene CAT3 necessary for glucose derepression and nuclear localization of its product

The yeast regulatory gene CAT3 has an essential function for the depression of several glucose-repressible enzymes. Therefore, cat3 mutants are unable to grow on maltose or on non-fermentable carbon sources. Unlike the point mutants isolated previously, cat3 null allele strains also failed to utilize raffinose or galactose as sole carbon sources. Sequencing of an 1.6-kb HindIII-BglII fragment complementing cat3 mutations revealed an open reading frame of 322 codons, size of which is in good agreement with the 1.3-kb size of mRNA. No significant similarities with previously sequenced genes could be detected. CAT3-lacZ fusions confirmed the proposed reading frame. A CAT3-lacZ fusion encoding 307 amino acids of CAT3 was able to complement the growth defects of cat3 point mutants and null allele strains. Assay of beta-galactosidase activity under different growth conditions indicated a constitutive expression of the CAT3 gene product. Cellular fractionation studies showed the nuclear localization of the CAT3 protein.     

Genetic control of amino acid transport inAspergillus nidulans: Evidence for polymeric amino acid permease

On a medium containing either acetate as the sole source of carbon or arginine as the sole source of nitrogen and the two amino acid analogs,p-fluorophenylalanine (FPA) and ethionine, eight FPA-resistant mutants were selected. Dominance tests in heterozygous diploids showed that 3 out of 8 are recessive, 1 semidominant, and 4 dominant to their wild-type alleles. Mutants were characterized by the nature of amino acid transport detected on the basis of amino acid utilization patterns. Six new loci identified after genetic analysis were located on two linkage groups: three each on linkage groups I and II. Recombinants between pairs of locifpaD andfpaQ, andfpaK andfpaP, were found to be sensitive to FPA. The patterns of segregation of resistant markers and amino acid utilization were considered to characterize the specificity of transport mutants.     

Genetics and physiology of proline utilization in Saccharomyces cerevisiae: enzyme induction by proline.

Proline is converted to glutamate in the yeast Saccharomyces cerevisiae by the sequential action of two enzymes, proline oxidase and delta 1-pyrroline-5-carboxylate (P5C) dehydrogenase. The levels of these enzymes appear to be controlled by the amount of proline in the cell. The capacity to transport proline is greatest when the cell is grown on poor nitrogen sources, such as proline or urea. Mutants have been isolated which can no longer utilize proline as the sole source of nitrogen. Mutants in put1 are deficient in proline oxidase, and those in put2 lack P5C dehydrogenase. The put1 and put2 mutations are recessive, segregate 2:2 in tetrads, and appear to be unlinked to one another. Proline induces both proline oxidase and P5C dehydrogenase. The arginine-degradative pathway intersects the proline-degradative pathway at P5C. The P5C formed from the breakdown of arginine or ornithine can induce both proline-degradative enzymes by virtue of its conversion to proline.     

Self-cloning baker's yeasts that accumulate proline enhance freeze tolerance in doughs

We constructed self-cloning diploid baker's yeast strains by disrupting PUT1, encoding proline oxidase, and replacing the wild-type PRO1, encoding gamma-glutamyl kinase, with a pro1(D154N) or pro1(I150T) allele. The resultant strains accumulated intracellular proline and retained higher-level fermentation abilities in the frozen doughs than the wild-type strain. These results suggest that proline-accumulating baker's yeast is suitable for frozen-dough baking.