Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae

Abstract

We used cells carrying plasmids causing the overproduction of Gln3p, Ure2p, or both of these proteins to elucidate the ability of Ure2p to prevent the activation of gene expression by Gln3p in cells growing in a glutamine-containing medium. Our results indicate that Ure2p probably does not interfere with the binding of the GATA factor Gln3p to GATAAG sites but acts directly on Gln3p to block its ability to activate transcription.In Saccharomyces cerevisiae, the expression of a number of genes whose products enable the organism to use a variety of compounds as sources of nitrogen is activated by the product of the GLN3 gene, Gln3p. This zinc finger protein exerts its effect by binding to GATAAG sequences located upstream of the regulated genes (1). The product of another gene, URE2, prevents the activation of the regulated genes by Gln3p in cells growing with glutamine as the source of nitrogen (5, 6).We have cloned the GLN3 and URE2 genes on plasmids, enabling us to overproduce the products of these genes (4, 7). We now present the results of experiments with cells that overproduce either one or both of the products of these genes. Our experiments suggest that Ure2p acts directly on Gln3p, converting it to a form that is unable to activate transcription.As previously reported, overproduction of Gln3p results in very slow growth, with a mass doubling time of more than 10 h in minimal media (7). We examined the effects of Ure2p on the growth inhibition exerted by Gln3p. To this end, we transformed strain PM38 (MAT␣ leu2-3,112 ura3-52) with plasmid pPM49 (2 m GAL10-GLN3 [7]). We also transformed this strain with plasmids carrying the URE2 gene fused to GAL10 as well as the LEU2 gene in order to create strains that overproduce both Gln3p and Ure2p. The high-copy-number plasmid pLE-8 was constructed by inserting a 1.7-kb StuI-SalI fragment of p8 containing the GAL10-URE2 fusion into the BamHI site of YEp13 (2, 3). The resulting plasmid has a LEU2 selectable marker and the GAL10 upstream activating sequence driving the URE2 gene. The single-copy centromere plasmid pLC-8 (YCpGAL10-URE2) was constructed by replacing the URA3 fragment of the p8 plasmid with the SalIXhoI LEU2-containing fragment of YEp13.The results presented in Fig. 1 show that overproduction of Gln3p in the strain carrying the GLN3 gene fused to GAL10 causes strong growth inhibition and that growth is restored by the overproduction of Ure2p resulting from the simultaneous presence of the plasmid carrying URE2 fused to GAL10.The effect of Ure2p is also demonstrated by the ability of Ure2p to depress the ability of overproduced Gln3p to activate the synthesis of glutamine synthetase. As shown in Table 1, overproduction of Gln3p enables the cell to produce glutamine synthetase during growth with glutamine as the source of nitrogen, but this increase in the level of the enzyme is largely prevented by the simultaneous overproduction of Ure2p.We have previously shown that Ure2p blocks neither the FIG. 1. Suppression of GLN3 lethality by overexpression of URE2...

N-Acetylglucosamine-inducible CaGAP1 encodes a general amino acid permease which co-ordinates external nitrogen source response and morphogenesis in Candida albicans

“…proline, the amino acid uptake is high (Blinder et al, 1996;Courchesne et al, 1983). In S. cerevisiae, at least five proteins (Ure2p, Dal80p, Gln3p, Nil1p and Nil2p) function co-ordinately to control the transcription of GAP1 (Blinder et al, 1996;Cunningham et al, 1993;Rowen et al, 1997;Stanbrough et al, 1995). The nitrogendependent regulation of GAP1 is complex, occurring not only at the level of GAP1 transcription but also through Gap1p sorting and degradation by ubiquitin-triggered internalization (Springael et al, 1998).…”

“…In the presence of ammonia or glutamine, the amino acid uptake is low, whereas in media containing a poor nitrogen source, e.g. proline, the amino acid uptake is high (Blinder et al, 1996;Courchesne et al, 1983). In S. cerevisiae, at least five proteins (Ure2p, Dal80p, Gln3p, Nil1p and Nil2p) function co-ordinately to control the transcription of GAP1 (Blinder et al, 1996;Cunningham et al, 1993;Rowen et al, 1997;Stanbrough et al, 1995).…”

RTG-dependent Mitochondria-to-Nucleus Signaling Is Regulated by MKS1 and Is Linked to Formation of Yeast Prion [URE3]

“…A connection between the retrograde pathway and nitrogen metabolism has recently been uncovered by the finding that the target of rapamycin (TOR) kinase pathway in yeast also involves the RTG genes (Komeili et al, 2000;Shamji et al, 2000). The TOR kinase pathway promotes the formation of a complex between Gln3p, a transcription factor that controls the expression of genes required for the utilization of poor nitrogen sources (Mitchell and Magasanik, 1984;Courchesne and Magasanik, 1988;Coschigano and Magasanik, 1991;Blinder et al, 1996), and Ure2p, a negative regulator of Gln3p (Beck and Hall, 1999). When the TOR kinase pathway is inhibited by rapamycin, or when cells are grown on a poor nitrogen source such as urea, Rtg1p and Rtg3p translocate from the cytoplasm to the nucleus in an Rtg2p-dependent manner to activate target gene expression (Komeili et al, 2000).…”

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

“…[URE3] is a selfpropagating form of the Ure2 protein, a factor that normally mediates nitrogen catabolite repression through the Gln3 transcriptional activator (Blinder et al, 1996). As is the case for [PSI ϩ ], the prion state inactivates Ure2, leading to expression of transcriptional targets including DAL5 (Brachmann et al, 2006).…”