Rajendra Rai

Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae.

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

Saccharomyces cerevisiae GATA sequences function as TATA elements during nitrogen catabolite repression and when Gln3p is excluded from the nucleus by overproduction of Ure2p

Saccharomyces cerevisiae selectively uses good nitrogen sources (glutamine) in preference to poor ones (proline) by repressing GATA factor-dependent transcription of the genes needed to transport and catabolize poor nitrogen sources, a physiological process designated nitrogen catabolite repression (NCR). We show that some NCR-sensitive genes (CAN1,DAL5, DUR1,2, and DUR3) produce two transcripts of slightly different sizes. Synthesis of the shorter transcript is NCR-sensitive and that of the longer transcript is not. The longer transcript also predominates in gln3Δ mutants irrespective of the nitrogen source provided. We demonstrate that the longer mRNA species arises through the use of an alternative transcription start site generated by Gln3p-binding sites (GATAAs) being able to act as surrogate TATA elements. The ability of GATAAs to serve as surrogate TATAs, i.e. when synthesis of the shorter, NCR-sensitive transcripts are inhibited, correlates with sequestration of enhanced green fluorescent protein (EGFP)-Gln3p in the cytoplasm in a way that is indistinguishable from that seen with EGFP-Ure2p. However, when the shorter, NCR-sensitive DAL5transcript predominates, EGFP-Gln3p is nuclear. These data suggest that the mechanism underlying NCR involves the cytoplasmic association of Ure2p with Gln3p, an interaction that prevents Gln3p from reaching it is binding sites upstream of NCR-sensitive genes.

Mks1p Is Required for Negative Regulation of Retrograde Gene Expression in Saccharomyces cerevisiae but Does Not Affect Nitrogen Catabolite Repression-sensitive Gene Expression

The Tor1/2p signal transduction pathway regulates nitrogen catabolite repression (NCR)-sensitive (GAP1,GAT1, DAL5) and retrograde (CIT2,DLD3, IDH1/2) gene expression by controlling intracellular localization of the transcription activators, Gln3p and Gat1p, and Rtg1p and Rtg3p, respectively. The accepted pathway for this regulation is NH3 or excess nitrogen ⊣ Mks1p ⊣ Ure2p ⊣ Gln3p → DAL5, and rapamycin or limiting nitrogen ⊣ Torp → Tap42 ⊣ Mks1p → Rtg1/3p → CIT2, respectively. In current models, Mks1p positively regulates both Gln3p (and DAL5 expression) and Rtg1/3p (and CIT2expression). Here, in contrast, we show the following. (i) Mks1p is a strong negative regulator of CIT2 expression and does not effect NCR-sensitive expression of DAL5 orGAP1. (ii) Retrograde carbon and NCR-sensitive nitrogen metabolism are not linked by the quality of the nitrogen source,i.e. its ability to elicit NCR, but by the product of its catabolism, i.e. glutamate or ammonia. (iii) In some instances, we can dissociate rapamycin-induced CIT2expression from Mks1p function, i.e. rapamycin does not suppress Mks1p-mediated down-regulation of CIT2 expression. These findings suggest that currently accepted models of Tor1/2p signal transduction pathway regulation require revision.

The Level of DAL80 Expression Down-Regulates GATA Factor-Mediated Transcription in Saccharomyces cerevisiae

Nitrogen-catabolic gene expression in Saccharomyces cerevisiae is regulated by the action of four GATA family transcription factors: Gln3p and Gat1p/Nil1p are transcriptional activators, and Dal80 and Deh1p/Gzf3p are repressors. In addition to the GATA sequences situated upstream of all nitrogen catabolite repression-sensitive genes that encode enzyme and transport proteins, the promoters of the GAT1, DAL80, and DEH1 genes all contain multiple GATA sequences as well. These GATA sequences are the binding sites of the GATA family transcription factors and are hypothesized to mediate their autogenous and cross regulation. Here we show, using DAL80 fused to the carbon-regulated GAL1,10 or copper-regulated CUP1 promoter, that GAT1 expression is inversely regulated by the level of DAL80 expression, i.e., as DAL80 expression increases, GAT1 expression decreases. The amount of DAL80 expression also dictates the level at which DAL3, a gene activated almost exclusively by Gln3p, is transcribed. Gat1p was found to partially substitute for Gln3p in transcription. These data support the contention that regulation of GATA-factor gene expression is tightly and dynamically coupled. Finally, we suggest that the complicated regulatory circuit in which the GATA family transcription factors participate is probably most beneficial as cells make the transition from excess to limited nitrogen availability.

Ammonia-specific regulation of Gln3 localization in Saccharomyces cerevisiae by protein kinase Npr1.

Events directly regulating Gln3 intracellular localization and nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae are interconnected with many cellular processes that influence the utilization of environmental metabolites. Among them are intracellular trafficking of the permeases that transport nitrogenous compounds and their control by the Tor1,2 signal transduction pathway. Npr1 is a kinase that phosphorylates and thereby stabilizes NCR-sensitive permeases, e.g. Gap1 and Mep2. It is also a phosphoprotein for which phosphorylation and kinase activity are regulated by Tor1,2 via Tap42 and Sit4. Npr1 has been reported to negatively regulate nuclear localization of Gln3 in SD (ammonia)-grown cells. Thus we sought to distinguish whether Npr1: (i) functions directly as a component of NCR control; or (ii) influences Gln3 localization indirectly, possibly as a consequence of participating in protein trafficking. If Npr1 functions directly, then the ability of all good nitrogen sources to restrict Gln3 to the cytoplasm should be lost in an npr1Δ just as occurs when URE2 (encoding this well studied negative Gln3 regulator) is deleted. We show that nuclear localization of Gln3-Myc13 in an npr1Δ occurred only with ammonia as the nitrogen source. Other good nitrogen sources, e.g. glutamine, serine, or asparagine, restricted Gln3-Myc13 to the cytoplasm of both wild type and npr1Δ cells. In other words, the npr1Δ did not possess the uniform phenotype for all repressive nitrogen sources characteristic of ure2Δ. This suggests that the connection between Gln3 localization and Npr1 is indirect, arising from the influence of Npr1 on the ability of cells to utilize ammonia as a repressive nitrogen source.

gln3 Mutations Dissociate Responses to Nitrogen Limitation (Nitrogen Catabolite Repression) and Rapamycin Inhibition of TorC1

Background: Gln3 localization is hypothesized to be co-regulated by TorC1 and nitrogen limitation. Results: gln3 mutations abrogating Gln3-Tor1 interaction abolish the Gln3 response to rapamycin without adversely affecting its response to nitrogen limitation. Conclusion: Different Gln3 regions mediate responses to rapamycin and nitrogen limitation. Significance: Controlled Gln3 localization occurs via two separable regulatory pathways, both of which are required for overall WT Gln3 control. The GATA family transcription activator, Gln3 responds to the nitrogen requirements and environmental resources of the cell. When rapidly utilized, “good” nitrogen sources, e.g., glutamine, are plentiful, Gln3 is completely sequestered in the cytoplasm, and the transcription it mediates is minimal. In contrast, during nitrogen-limiting conditions, Gln3 quickly relocates to the nucleus and activates transcription of genes required to scavenge alternative, “poor” nitrogen sources, e.g., proline. This physiological response has been designated nitrogen catabolite repression (NCR). Because rapamycin treatment also elicits nuclear Gln3 localization, TorC1 has been thought to be responsible for NCR-sensitive Gln3 regulation. However, accumulating evidence now suggests that GATA factor regulation may occur by two separate pathways, one TorC1-dependent and the other NCR-sensitive. Therefore, the present experiments were initiated to identify Gln3 amino acid substitutions capable of dissecting the individual contributions of these pathways to overall Gln3 regulation. The rationale was that different regulatory pathways might be expected to operate through distinct Gln3 sensor residues. We found that C-terminal truncations or amino acid substitutions in a 17-amino acid Gln3 peptide with a predicted propensity to fold into an α-helix partially abolished the ability of the cell to sequester Gln3 in the cytoplasm of glutamine-grown cells and eliminated the rapamycin response of Gln3 localization, but did not adversely affect its response to limiting nitrogen. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. We also found that Gln3 possesses at least one Tor1-interacting site in addition to the one previously reported.

DNA binding site specificity of the Neurospora global nitrogen regulatory protein NIT2: analysis with mutated binding sites.

NIT2, a positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes that encode nitrogen catabolic enzymes. NIT2 binding sites in the promoter regions of nit3, alc and lao have at least two GATA sequence elements. We have examined the binding affinity of the NIT2 protein for the yeast DAL5 wild-type upstream activation sequence UASNTR, which contains two GATA elements, and for a series of mutated binding sites, each differing from the wild-type site by a single base. Substitution for individual nucleotides within 5′ or 3′ sequences that flank the GATA elements had only modest effects upon NIT2 binding. In contrast, nearly all substitutions within the GATA elements almost completely eliminated NIT2 binding, demonstrating the importance of the GATA sequence for NIT2 binding. Four high-affinity binding sites for the NIT2 protein were found within a central region of the nit-2 gene itself.

In vivo specificity of Ure2 protection from heavy metal ion and oxidative cellular damage in Saccharomyces cerevisiae

The S. cerevisiae Ure2 protein is a prion precursor able to form large homopolymers with the characteristics of amyloid particles, a function largely restricted to its 90 N‐terminal amino acids. The remaining C‐terminal domain of Ure2 plays two important roles in cellular metabolism. First, it regulates nitrogen catabolic gene expression by forming a complex with the GATA transcription factor Gln3. This complex formation correlates with Gln3 being sequestered in the cytoplasm under conditions of excess nitrogen, where Gln3/Gat1‐mediated transcription is minimal. Second, Ure2, which possesses structural homology with glutathione S‐transferases and binds to xenobiotics and glutathione, has been recently shown to be required for Cd(II) and hydrogen peroxide detoxification. Present experiments demonstrate that Ure2 possesses a far broader protection specificity, being required to avoid the toxic effects of As(III), As(V), Cr(III), Cr(VI), Se(IV), as well as Cd(II) and Ni(II), and to varying lesser degrees Co(II), Cu(II), Fe(II), Ag(I), Hg(II), cumene and t‐butyl hydroperoxides. In contrast, deletion of URE2 greatly enhances a cells ability to withstand toxic concentrations of Zn(II) and Mo(VI). In the case of Cd(II), Ure2 does not function to decrease intracellular Cd(II) levels or influence glutathione availability for glutathionation. In fact, ure2 hypersensitivity to Cd(II) remains the same, even when glutathione is used as sole source of nitrogen for cell growth. These data suggest that Ure2 possesses a central role in metal ion detoxification, a role not demonstrably shared by either of the two known S. cerevisiae glutathione S‐transferases, Gtt1 and Gtt2, or the two glutaredoxins, Grx1 and Grx2, that also possess glutathione S‐transferase activity. Copyright

Overlapping positive and negative GATA factor binding sites mediate inducible DAL7 gene expression in Saccharomyces cerevisiae.

Allantoin pathway gene expression inSaccharomyces cerevisiae responds to two different environmental stimuli. The expression of these genes is induced in the presence of allantoin or its degradative metabolites and repressed when a good nitrogen source (e.g. asparagine or glutamine) is provided. Three types of cis-acting sites and trans-acting factors are required for allantoin pathway gene transcription as follows: (i)UAS NTR element associated with the transcriptional activators Gln3p and Gat1p, (ii)URS GATA element associated with the repressor Dal80p, and (iii) UIS ALL element associated with the Dal82 and Dal81 proteins required for inducer-dependent transcription. Most of the work leading to the above conclusions has employed inducer-independent allantoin pathway genes (e.g. DAL5 and DAL3). The purpose of this work is to extend our understanding of these elements and their roles to inducible allantoin pathway genes using theDAL7 (encoding malate synthase) as a model. We show that eight distinct cis-acting sites participate in the process as follows: a newly identified GC-rich element, two UAS NTR, two UIS ALL, and threeURS GATA elements. The two GATA-containingUAS NTR elements are coincident with two of the three GATA sequences that make up the URS GATAelements. The remaining URS GATA GATA sequence, however, is not a UAS NTR element but appears to function only in repression. The data provide insights into how these cis- and trans-acting factors function together to accomplish the regulated expression of the DAL7 gene that is observedin vivo.

General amino acid control and 14-3-3 proteins Bmh1/2 are required for nitrogen catabolite repression-sensitive regulation of Gln3 and Gat1 localization

Nitrogen catabolite repression (NCR), the ability of Saccharomyces cerevisiae to use good nitrogen sources in preference to poor ones, derives from nitrogen-responsive regulation of the GATA family transcription activators Gln3 and Gat1. In nitrogen-replete conditions, the GATA factors are cytoplasmic and NCR-sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear, dramatically increasing GATA factor-mediated transcription. This regulation was originally attributed to mechanistic Tor protein kinase complex 1 (mTorC1)-mediated control of Gln3. However, we recently showed that two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration, only one of which is mTorC1. Present experiments demonstrate that the other previously elusive component is uncharged transfer RNA-activated, Gcn2 protein kinase-mediated general amino acid control (GAAC). Gcn2 and Gcn4 are required for NCR-sensitive nuclear Gln3-Myc13 localization, and from epistasis experiments Gcn2 appears to function upstream of Ure2. Bmh1/2 are also required for nuclear Gln3-Myc13 localization and appear to function downstream of Ure2. Overall, Gln3 phosphorylation levels decrease upon loss of Gcn2, Gcn4, or Bmh1/2. Our results add a new dimension to nitrogen-responsive GATA-factor regulation and demonstrate the cumulative participation of the mTorC1 and GAAC pathways, which respond oppositely to nitrogen availability, in the nitrogen-responsive control of catabolic gene expression in yeast.