Isabelle Georis

The yeast GATA factor Gat1 occupies a central position in nitrogen catabolite repression-sensitive gene activation.

ABSTRACT Saccharomyces cerevisiae cells are able to adapt their metabolism according to the quality of the nitrogen sources available in the environment. Nitrogen catabolite repression (NCR) restrains the yeasts capacity to use poor nitrogen sources when rich ones are available. NCR-sensitive expression is modulated by the synchronized action of four DNA-binding GATA factors. Although the first identified GATA factor, Gln3, was considered the major activator of NCR-sensitive gene expression, our work positions Gat1 as a key factor for the integrated control of NCR in yeast for the following reasons: (i) Gat1 appeared to be the limiting factor for NCR gene expression, (ii) GAT1 expression was regulated by the four GATA factors in response to nitrogen availability, (iii) the two negative GATA factors Dal80 and Gzf3 interfered with Gat1 binding to DNA, and (iv) Gln3 binding to some NCR promoters required Gat1. Our study also provides mechanistic insights into the mode of action of the two negative GATA factors. Gzf3 interfered with Gat1 by nuclear sequestration and by competition at its own promoter. Dal80-dependent repression of NCR-sensitive gene expression occurred at three possible levels: Dal80 represses GAT1 expression, it competes with Gat1 for binding, and it directly represses NCR gene transcription.

Tor Pathway Control of the Nitrogen-responsive DAL5 Gene Bifurcates at the Level of Gln3 and Gat1 Regulation in Saccharomyces cerevisiae

The Tor1,2 protein kinases globally influence many cellular processes including nitrogen-responsive gene expression that correlates with intracellular localization of GATA transcription activators Gln3 and Gat1/Nil1. Gln3-Myc13 and Gat1-Myc13 are restricted to the cytoplasm of cells provided with good nitrogen sources, e.g. glutamine. Following the addition of the Tor1,2 inhibitor, rapamycin, both transcription factors relocate to the nucleus. Gln3-Myc13 localization is highly dependent upon Ure2 and type 2A-related phosphatase, Sit4. Ure2 is required for Gln3 to be restricted to the cytoplasm of cells provided with good nitrogen sources, and Sit4 is required for its location to the nucleus following rapamycin treatment. The paucity of analogous information concerning Gat1 regulation prompted us to investigate the effects of deleting SIT4 and URE2 on Gat1-Myc13 localization, DNA binding, and NCR-sensitive transcription. Our data demonstrate that Tor pathway control of NCR-responsive transcription bifurcates at the regulation of Gln3 and Gat1. Gat1-Myc13 localization is not strongly influenced by deleting URE2, nor is its nuclear targeting following rapamycin treatment strongly dependent on Sit4. ChIP experiments demonstrated that Gat1-Myc13 can bind to the DAL5 promoter in the absence of Gln3. Gln3-Myc13, on the other hand, cannot bind to DAL5 in the absence of Gat1. We conclude that: (i) Tor pathway regulation of Gat1 differs markedly from that of Gln3, (ii) nuclear targeting of Gln3-Myc13 is alone insufficient for its recruitment to the DAL5 promoter, and (iii) the Tor pathway continues to play an important regulatory role in NCR-sensitive transcription even after Gln3-Myc13 is localized to the nucleus.

Rapamycin-induced Gln3 Dephosphorylation Is Insufficient for Nuclear Localization Sit4 AND PP2A PHOSPHATASES ARE REGULATED AND FUNCTION DIFFERENTLY

Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Δ22Δ, tpd3Δ, or cdc55Δ cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.

Nitrogen catabolite repression-sensitive transcription as a readout of Tor pathway regulation: the genetic background, reporter gene and GATA factor assayed determine the outcomes.

Nitrogen catabolite repression (NCR)-sensitive genes, whose expression is highly repressed when provided with excess nitrogen and derepressed when nitrogen is limited or cells are treated with rapamycin, are routinely used as reporters in mechanistic studies of the Tor signal transduction pathway in Saccharomyces cerevisiae. Two GATA factors, Gln3 and Gat1, are responsible for NCR-sensitive transcription, but recent evidence demonstrates that Tor pathway regulation of NCR-sensitive transcription bifurcates at the level of GATA factor localization. Gln3 requires Sit4 phosphatase for nuclear localization and NCR-sensitive transcription while Gat1 does not. In this article, we demonstrate that the extent to which Sit4 plays a role in NCR-sensitive transcription depends upon whether or not (i) Gzf3, a GATA repressor homologous to Dal80, is active in the genetic background assayed; (ii) Gat1 is able to activate transcription of the assayed gene in the absence of Gln3 in that genetic background; and (iii) the gene chosen as a reporter is able to be transcribed by Gln3 or Gat1 in the absence of the other GATA factor. Together, the data indicate that in the absence of these three pieces of information, overall NCR-sensitive gene transcription data are unreliable as Tor pathway readouts.

Distinct phosphatase requirements and GATA factor responses to nitrogen catabolite repression and rapamycin treatment in Saccharomyces cerevisiae.

In yeast, rapamycin (Rap)-inhibited TorC1, and the phosphatases it regulates (Sit4 and PP2A) are components of a conserved pathway regulating the response of eukaryotic cells to nutrient availability. TorC1 and intracellular nitrogen levels regulate the localization of Gln3 and Gat1, the activators of nitrogen catabolite repression (NCR)-sensitive genes whose products are required to utilize poor nitrogen sources. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and NCR-sensitive transcription is repressed. During nitrogen limitation or Rap treatment, Gln3 and Gat1 are nuclear, and transcription is derepressed. We previously demonstrated that the Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for nuclear Gln3 localization differ. We now show that Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for NCR-sensitive and Rap-induced nuclear Gat1 localization markedly differ from those of Gln3. Our data suggest that Gln3 and Gat1 localizations are controlled by two different regulatory pathways. Gln3 localization predominantly responds to intracellular nitrogen levels, as reflected by its stronger NCR-sensitivity, weaker response to Rap treatment, and strong response to methionine sulfoximine (Msx, a glutamine synthetase inhibitor). In contrast, Gat1 localization predominantly responds to TorC1 regulation as reflected by its weaker NCR sensitivity, stronger response to Rap, and immunity to the effects of Msx. Nuclear Gln3 localization in proline-grown (nitrogen limited) cells exhibits no requirement for Pph21/22-Tpd3/Cdc55, whereas nuclear Gat1 localization under these conditions is absolutely dependent on Pph21/22-Tpd3/Cdc55. Furthermore, the extent to which Pph21/22-Tpd3-Cdc55 is required for the TorC1 pathway (Rap) to induce nuclear Gat1 localization is regulated in parallel with Pph21/22-Tpd3-Cdc55-dependent Gln3 dephosphorylation and NCR-sensitive transcription, being highest in limiting nitrogen and lowest when nitrogen is in excess.

Nitrogen-responsive Regulation of GATA Protein Family Activators Gln3 and Gat1 Occurs by Two Distinct Pathways, One Inhibited by Rapamycin and the Other by Methionine Sulfoximine

Background: Nitrogen availability and TorC1 regulate the localization and function of transcription factors Gln3 and Gat1. Results: Gln3 and Gat1 responses to rapamycin and methionine sulfoximine differ markedly and exhibit different phosphatase requirements. Conclusion: Gln3 and Gat1 localization and function are regulated by two distinct pathways, one inhibited by rapamycin and the other by methionine sulfoximine. Significance: Glutamine starvation regulates Gln3 via a TorC1-independent pathway. Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.

Intranuclear function for protein phosphatase 2A: Pph21 and Pph22 are required for rapamycin-induced GATA factor binding to the DAL5 promoter in yeast.

ABSTRACT Protein phosphatase 2A (PP2A), a central Tor pathway phosphatase consisting of a catalytic subunit (Pph21 or Pph22), a scaffold subunit (Tpd3), and one of two regulatory subunits (Cdc55 or Rts1), has been repeatedly shown to play important roles in cytoplasmically localized signal transduction activities. In contrast, its involvement in intranuclear control of mRNA production has heretofore not been reported. Here, we demonstrate for the first time that binding of the nitrogen catabolite repression-responsive GATA transcription activators (Gln3 and Gat1) to the DAL5 promoter and DAL5 expression require Pph21/22-Tpd3-Cdc55/Rts1 in rapamycin-treated glutamine-grown cells. This conclusion is supported by the following observations. (i) Rapamycin-induced DAL5 expression along with Gln3 and Gat1 binding to the DAL5 promoter fails to occur in pph21Δ pph22Δ, tpd3Δ, and cdc55Δ rts1Δ mutants. (ii) The Pph21/22 requirement persists even when Gat1 and Gln3 are rendered constitutively nuclear, thus dissociating the intranuclear requirement of PP2A from its partial requirement for rapamycin-induced nuclear Gat1 localization. (iii) Pph21-Myc13 (Ppp21 tagged at the C terminus with 13 copies of the Myc epitope) weakly associates with the DAL5 promoter in a Gat1-dependent manner, whereas a similar Pph22-Myc13 association requires both Gln3 and Gat1. Finally, we demonstrate that a pph21Δ pph22Δ double mutant is epistatic to ure2Δ for nuclear Gat1 localization in untreated glutamine-grown cells, whereas for Gln3, just the opposite occurs: i.e., ure2Δ is epistatic to pph21Δ pph22Δ. This final observation adds additional support to our previous conclusion that the Gln3 and Gat1 GATA factor localizations are predominantly controlled by different regulatory pathways.

Alterations in the Ure2 αCap Domain Elicit Different GATA Factor Responses to Rapamycin Treatment and Nitrogen Limitation

Background: TorC1, excess nitrogen, and Ure2 negatively regulate Gln3 and Gat1. Results: Nitrogen catabolite repression-sensitive control of Gln3/Gat1 is normal in ure2 αcap mutants that no longer respond to TorC1 inhibitor, rapamycin. Conclusion: Different regions of Ure2 are associated with the Gln3/Gat1 response to TorC1 inhibition and nitrogen availability. Significance: Gln3, Gat1, and Ure2 respond to TorC1 and nitrogen availability via distinct regulatory pathways. Ure2 is a phosphoprotein and central negative regulator of nitrogen-responsive Gln3/Gat1 localization and their ability to activate transcription. This negative regulation is achieved by the formation of Ure2-Gln3 and -Gat1 complexes that are thought to sequester these GATA factors in the cytoplasm of cells cultured in excess nitrogen. Ure2 itself is a dimer the monomer of which consists of two core domains and a flexible protruding αcap. Here, we show that alterations in this αcap abolish rapamycin-elicited nuclear Gln3 and, to a more limited extent, Gat1 localization. In contrast, these alterations have little demonstrable effect on the Gln3 and Gat1 responses to nitrogen limitation. Using two-dimensional PAGE we resolved eight rather than the two previously reported Ure2 isoforms and demonstrated Ure2 dephosphorylation to be stimulus-specific, occurring after rapamycin treatment but only minimally if at all in nitrogen-limited cells. Alteration of the αcap significantly diminished the response of Ure2 dephosphorylation to the TorC1 inhibitor, rapamycin. Furthermore, in contrast to Gln3, rapamycin-elicited Ure2 dephosphorylation occurred independently of Sit4 and Pph21/22 (PP2A) as well as Siw14, Ptc1, and Ppz1. Together, our data suggest that distinct regions of Ure2 are associated with the receipt and/or implementation of signals calling for cessation of GATA factor sequestration in the cytoplasm. This in turn is more consistent with the existence of distinct pathways for TorC1- and nitrogen limitation-dependent control than it is with these stimuli representing sequential steps in a single regulatory pathway.

Yeast nitrogen catabolite repression is sustained by signals distinct from glutamine and glutamate reservoirs.

Nitrogen catabolite repression (NCR) is a wide transcriptional regulation program enabling bakers yeast to downregulate genes involved in the utilization of poor nitrogen sources when preferred ones are available. Nowadays, glutamine and glutamate, the major nitrogen donors for biosyntheses, are assumed to be key metabolic signals regulating NCR. NCR is controlled by the conserved TORC1 complex, which integrates nitrogen signals among others to regulate cell growth. However, accumulating evidence indicate that the TORC1‐mediated control of NCR is only partial, arguing for the existence of supplementary regulatory processes to be discovered. In this work, we developed a genetic screen to search for new players involved in NCR signaling. Our data reveal that the NADP‐glutamate dehydrogenase activity of Gdh1 negatively regulates NCR‐sensitive gene transcription. By determining the total, cytoplasmic and vacuolar pools of amino acids, we show that there is no positive correlation between glutamine/glutamate reservoirs and the extent of NCR. While our data indicate that glutamine could serve as initial trigger of NCR, they show that it is not a sufficient signal to sustain repression and point to the existence of yet unknown signals. Providing additional evidence uncoupling TORC1 activity and NCR, our work revisits the dogmas underlying NCR regulation.

GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation

The TorC1 protein kinase complex is a central component in a eukaryotic cell’s response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g., proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor−dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3-Myc13 localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells’ nitrogen environment.