Regulation of the urea active transporter gene (DUR3) in Saccharomyces cerevisiae.

The DUR3 gene, which encodes a component required for active transport of urea in Saccharomyces cerevisiae, has been isolated, and its sequence has been determined. The deduced DUR3 protein profile possesses alternating hydrophobic and hydrophilic regions characteristics of integral membrane proteins. Strong negative complementation observed during genetic analysis of the DUR3 locus suggests that the DUR3 product may polymerize to carry out its physiological function. Expression of DUR3 is regulated in a manner similar to that of other genes in the allantoin pathway. High-level expression is inducer dependent, requiring functional DAL81 and DAL82 genes. Maintenance of DUR3 mRNA at uninduced, nonrepressed basal levels requires the negatively acting DAL80 gene product. DUR3 expression is highly sensitive to nitrogen catabolite repression and also has a partial requirement for the GLN3 product.     

Regulation of glutamine-repressible gene products by the GLN3 function in Saccharomyces cerevisiae.

Mutants of the yeast Saccharomyces cerevisiae have been isolated which fail to derepress glutamine synthetase upon glutamine limitation. The mutations define a single nuclear gene, GLN3, which is located on chromosome 5 near HOM3 and HIS1 and is unlinked to the structural gene for glutamine synthetase, GLN1. The three gln3 mutations are recessive, and one is amber suppressible, indicating that the GLN3 product is a positive regulator of glutamine synthetase expression. Four polypeptides, in addition to the glutamine synthetase subunit are synthesized at elevated rates when GLN3+ cultures are shifted from glutamine to glutamate media as determined by pulse-labeling and one- and two-dimensional gel electrophoresis. The response of all four proteins is blocked by gln3 mutations. In addition, the elevated NAD-dependent glutamate dehydrogenase activity normally found in glutamate-grown cells is not found in gln3 mutants. Glutamine limitation of gln1 structural mutants has the opposite effect, causing elevated levels of NAD-dependent glutamate dehydrogenase even in the presence of ammonia. We suggest that there is a regulatory circuit that responds to glutamine availability through the GLN3 product.     

GLN3 encodes a global regulator of nitrogen metabolism and virulence of C. albicans

The function of GLN3, a GATA factor encoding gene, in nitrogen metabolism of Candida albicans was examined. GLN3 null mutants had reduced growth rates on multiple nitrogen sources. More severe growth defects were observed in mutants lacking both GLN3 and GAT1, a second GATA factor gene. GLN3 was an activator of two genes involved in ammonium assimilation, GDH3, encoding NADP-dependent glutamate dehydrogenase, and MEP2, which encodes an ammonium permease. GAT1 contributed to MEP2 expression, but not that of GDH3. A putative general amino acid permease gene, GAP2, was also activated by both GLN3 and GAT1, but activation by GLN3 was nitrogen source dependent. GLN3 was constitutively expressed, but GAT1 expression varied with nitrogen source and was reduced 2- to 3-fold in gln3 mutants. Both gln3 and gat1 mutants exhibited reduced sensitivity to rapamycin, suggesting they function downstream of TOR kinase. Hyphae formation by gln3 and gat1 mutants differed in relation to nitrogen source. The gln3 mutants formed hyphae on several nitrogen sources, but not ammonium or urea, suggesting a defect in ammonium assimilation. Virulence of gln3 mutants was reduced in a murine model of disseminated disease. We conclude that GLN3 has a broad role in nitrogen metabolism, partially overlapping, but distinct from that of GAT1, and that its function is important for the ability of C. albicans to survive within the host environment.     

GLN3 encodes a global regulator of nitrogen metabolism and virulence of C. albicans.

The function of GLN3, a GATA factor encoding gene, in nitrogen metabolism of Candida albicans was examined. GLN3 null mutants had reduced growth rates on multiple nitrogen sources. More severe growth defects were observed in mutants lacking both GLN3 and GAT1, a second GATA factor gene. GLN3 was an activator of two genes involved in ammonium assimilation, GDH3, encoding NADP-dependent glutamate dehydrogenase, and MEP2, which encodes an ammonium permease. GAT1 contributed to MEP2 expression, but not that of GDH3. A putative general amino acid permease gene, GAP2, was also activated by both GLN3 and GAT1, but activation by GLN3 was nitrogen source dependent. GLN3 was constitutively expressed, but GAT1 expression varied with nitrogen source and was reduced 2- to 3-fold in gln3 mutants. Both gln3 and gat1 mutants exhibited reduced sensitivity to rapamycin, suggesting they function downstream of TOR kinase. Hyphae formation by gln3 and gat1 mutants differed in relation to nitrogen source. The gln3 mutants formed hyphae on several nitrogen sources, but not ammonium or urea, suggesting a defect in ammonium assimilation. Virulence of gln3 mutants was reduced in a murine model of disseminated disease. We conclude that GLN3 has a broad role in nitrogen metabolism, partially overlapping, but distinct from that of GAT1, and that its function is important for the ability of C. albicans to survive within the host environment.     

Regulatory circuit for responses of nitrogen catabolic gene expression to the GLN3 and DAL80 proteins and nitrogen catabolite repression in Saccharomyces cerevisiae.

We demonstrate that expression of the UGA1, CAN1, GAP1, PUT1, PUT2, PUT4, and DAL4 genes is sensitive to nitrogen catabolite repression. The expression of all these genes, with the exception of UGA1 and PUT2, also required a functional GLN3 protein. In addition, GLN3 protein was required for expression of the DAL1, DAL2, DAL7, GDH1, and GDH2 genes. The UGA1, CAN1, GAP1, and DAL4 genes markedly increased their expression when the DAL80 locus, encoding a negative regulatory element, was disrupted. Expression of the GDH1, PUT1, PUT2, and PUT4 genes also responded to DAL80 disruption, but much more modestly. Expression of GLN1 and GDH2 exhibited parallel responses to the provision of asparagine and glutamine as nitrogen sources but did not follow the regulatory responses noted above for the nitrogen catabolic genes such as DAL5. Steady-state mRNA levels of both genes did not significantly decrease when glutamine was provided as nitrogen source but were lowered by the provision of asparagine. They also did not respond to disruption of DAL80.     

Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes.

Mutations in the GLN3 gene prevented a normal increase in the NAD-glutamate dehydrogenase and glutamine synthetase levels in glutamate-grown Saccharomyces cerevisiae cells, whereas mutations in the URE2 gene resulted in high levels of these enzymes in glumate- and glutamine-grown cells. A ure2 gln3 double mutant had low levels of glutamate dehydrogenase and glutamine synthetase in cells grown on glutamate and glutamine; thus, gln3 mutations were epistatic to the ure2 mutations. The results suggest that the GLN3 product is capable of promoting increases in enzyme levels in the absence of a functional URE2 product and that the URE2 product antagonizes the GLN3 product. The URE2 and GLN3 genes were also found to regulate the level of arginase activity. This regulation is completely independent of the regulation of arginase by substrate induction. The activities of glutamate dehydrogenase, glutamine synthetase, and arginase were higher in cells grown on glutamate as the nitrogen source than they were in cells grown under a nitrogen-limiting condition. It had previously been shown that the levels of these enzymes can be increased by glutamine deprivation. We propose that the URE2-GLN3 system regulates enzyme synthesis, in response to glutamine and glutamate, to adjust the intracellular concentration of ammonia so as to maintain glutamine at the level required for optimal growth.     

Transcriptional regulation of the DAL5 gene in Saccharomyces cerevisiae

We demonstrate that the DAL5 gene, encoding a necessary component of the allantoate transport system, is constitutively expressed in Saccharomyces cerevisiae. Its relatively high basal level of expression did not increase further upon addition of allantoin pathway intermediates. However, steady-state DAL5 mRNA levels dropped precipitously when a repressive nitrogen source was provided. These control characteristics of DAL5 expression make this gene a good model with which to unravel the mechanism of nitrogen catabolite repression. Its particular advantage relative to other potentially useful genes derives from its lack of control by induction and hence the complicating effects of inducer exclusion.     

Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae.

This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae. During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene. Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have previously been shown to regulate the allantoin degradation system. Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions. When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression. Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.     

Identification of a glutaminyl-tRNA synthetase mutation Saccharomyces cerevisiae

Saccharomyces cerevisiae glutaminyl-tRNA synthetase mutants were isolated through systematic screening of tight Gln- derivatives of a leaky glutamine auxotroph. These mutations define a single nuclear gene, GLN4. The gln4-1 mutation is specific for Gln-tRNA synthetase and shows a dosage effect in heterozygous diploids. The wild-type Gln-tRNA synthetase exhibits a Km for glutamine of 25 microM; the gln4-1 mutation increases this value 20-fold. These observations strongly suggest that GLN4 encodes the Gln-tRNA synthetase.