André Feller

Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activation.

The product of the LYS14 gene of Saccharomyces cerevisiae activates the transcription of at least four genes involved in lysine biosynthesis. Physiological and genetic studies indicate that this activation is dependent on the inducer alpha-aminoadipate semialdehyde, an intermediate of the pathway. The gene LYS14 was sequenced and, from its nucleotide sequence, predicted to encode a 790-amino-acid protein carrying a cysteine-rich DNA-binding motif of the Zn(II)2Cys6 type in its N-terminal portion. Deletion of this N-terminal portion including the cysteine-rich domain resulted in the loss of LYS14 function. To test the function of Lys14 as a transcriptional activator, this protein without its DNA-binding motif was fused to the DNA-binding domain of the Escherichia coli LexA protein. The resulting LexA-Lys14 hybrid protein was capable of activating transcription from a promoter containing a lexA operator, thus confirming the transcriptional activation function of Lys14. Furthermore, evidence that this function, which is dependent on the presence of alpha-aminoadipate semialdehyde, is antagonized by lysine was obtained. Such findings suggest that activation by alpha-aminoadipate semialdehyde and the apparent repression by lysine are related mechanisms. Lysine possibly acts by limiting the supply of the coinducer, alpha-aminoadipate semialdehyde.

Saccharomyces cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided, and Gln3 Phosphorylation Levels Become Nitrogen Source-responsive in a sit4-deleted Strain

Tor1,2 control of type 2A-related phosphatase activities in Saccharomyces cerevisiae has been reported to be responsible for the regulation of Gln3 phosphorylation and intracellular localization in response to the nature of the nitrogen source available. According to the model, excess nitrogen stimulates Tor1,2 to phosphorylate Tip41 and/or Tap42. Tap42 then complexes with and inactivates Sit4 phosphatase, thereby preventing it from dephosphorylating Gln3. Phosphorylated Gln3 complexes with Ure2 and is sequestered in the cytoplasm. When Tor1,2 kinase activities are inhibited by limiting nitrogen, or rapamycin-treatment, Tap42 can no longer complex with Sit4. Active Sit4 dephosphorylates Gln3, which can then localize to the nucleus and activate transcription. The paucity of experimental data directly correlating active Sit4 and Pph3 with Gln3 regulation prompted us to assay Gln3-Myc13 phosphorylation and intracellular localization in isogenic wild type, sit4, pph3, and sit4pph3 deletion strains. We found that Sit4 actively brought about Gln3-Myc13 dephosphorylation in both good (glutamine or ammonia) and poor (proline) nitrogen sources. This Sit4 activity masked nitrogen source-dependent changes in Gln3-Myc13 phosphorylation which were clearly visible when SIT4 was deleted. The extent of Sit4 requirement for Gln3 nuclear localization was both nitrogen source- and strain-dependent. In some strains, Sit4 was not even required for Gln3 nuclear localization in untreated or rapamycin-treated, proline-grown cells or Msx-treated, ammonia-grown cells.

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.

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.

Lys80p of Saccharomyces cerevisiae, previously proposed as a specific repressor of LYS genes, is a pleiotropic regulatory factor identical to Mks1p.

In Saccharomyces cerevisiae, an intermediate of the lysine pathway, α‐aminoadipate semialdehyde (αAASA), acts as a coinducer for the transcriptional activation of LYS genes by Lys14p. The limitation of the production of this intermediate through feedback inhibition of the first step of the pathway results in apparent repression by lysine. Previously, the lys80 mutations, reducing the lysine repression and increasing the production of lysine, were interpreted as impairing a repressor of LYS genes expression. In order to understand the role of Lys80p in the control of the lysine pathway, we have analysed the effects of mutations epistatic to lys80 mutations. The effects of lys80 mutations on LYS genes expression were dependent on the integrity of the activation system (Lys14p and αAASA). The increased production of lysine in lys80 mutants appeared to result from an improvement of the metabolic flux through the pathway and was correlated to an increase of the α‐ketoglutarate pool and of the level of several enzymes of the tricarboxylic acid cycle. The LYS80 genes has been cloned and sequenced; it turned out to be identical to gene MKS1 cloned as a gene encoding a negative regulator of the RAS‐cAMP pathway. We conclude that Lys80p is a pleiotropic regulatory factor rather than a specific repressor of LYS genes.

Transduction of the nitrogen signal activating Gln3-mediated transcription is independent of Npr1 kinase and Rsp5-Bul1/2 ubiquitin ligase in Saccharomyces cerevisiae.

Nitrogen Catabolite Repression (NCR) allows the adaptation of yeast cells to the quality of nitrogen supply by inhibiting the transcription of genes encoding proteins involved in transport and degradation of nonpreferred nitrogen sources. In cells using ammonium or glutamine, the GATA transcription factor Gln3 is sequestered in the cytoplasm by Ure2 whereas it enters the nucleus after a shift to a nonpreferred nitrogen source like proline or upon addition of rapamycin, the TOR complex inhibitor. Recently, the Npr1 kinase and the Rsp5, Bul1/2 ubiquitin ligase complex were reported to have antagonistic roles in the nuclear import and Gln3-mediated activation. The Npr1 kinase controls the activity of various permeases including transporters for nitrogen sources that stimulate NCR such as the Mep ammonium transport systems. Combining data from growth tests, Northern blot analysis and Gln3 immunolocalization, we show that the Npr1 kinase is not a direct negative regulator of Gln3-dependent transcription. The derepression of Gln3-activated genes in ammonium-grown npr1 cells results from the reduced uptake of the nitrogen-repressing compound because NCR could be restored in npr1 cells by repairing ammonium-uptake defects through different means. Finally, we show that the impairment of the ubiquitin ligase complex does not prevent induction of NCR genes under nonpreferred nitrogen conditions. The apparent Rsp5-, Bul1/2-dependent Gln3 activation keeps to the cellular status, as it is only observed in cells having left the balanced phase of exponential growth.

Control-mechanisms acting at the transcriptional and post-transcriptional levels are involved in the synthesis of the arginine pathway carbamoylphosphate synthase of yeast.

In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synthase (CPSase A) is subject to two control mechanisms. One mechanism, the general control of amino acid biosynthesis, influences the expression of both CPA1 and CPA2 genes, the structural genes for the two subunits of the enzyme. The second mechanism, the specific control of arginine biosynthesis, only affects the expression of CPA1. To study these mechanisms in more detail, we have cloned the CPA1 and CPA2 genes and used their DNA to measure the CPA1 and CPA2 mRNA content of cells grown under various conditions. A close coordination was observed in the variation of the levels of CPA1 and CPA2 mRNAs and polypeptide products under conditions where the general control of amino acid biosynthesis operates. In contrast, little correlation was found between the levels of CPA1 mRNA and the corresponding protein for conditions affecting repression by arginine: the total amplitude of variation was 6‐fold higher for the CPA1 protein than for the CPA1 messenger transcript. Such findings are consistent with the conclusion that the general control operates at the transcriptional level and that the specific arginine control acts primarily at a post‐transcriptional level.

A nonameric core sequence is required upstream of the LYS genes of Saccharomyces cerevisiae for Lys14p-mediated activation and apparent repression by lysine

The expression of the structural genes for lysine (LYS) biosynthesis is controlled by a pathway‐specific regulation mediated by the transcriptional activator Lys14 in the presence of α‐aminoadipate semialdehyde, an intermediate of the pathway acting as a co‐inducer. Owing to end product inhibition of the first step of the pathway, excess lysine reduces the production of the co‐inducer and causes apparent repression of the LYS genes. Analysis of LYS promoters and insertions within an heterologous reporter gene have allowed the characterization of an upstream activating element (UASLYS) able to confer Lys14‐ and α‐aminoadipate semialdehyde‐dependent activation as well as apparent repression by lysine to another yeast gene. This DNA motif is present as one or several copies in the promoters of at least six LYS genes. The consensus sequence derived from the comparison of the UASLYS showing the highest activation capacities comprises the nonameric core sequence TCCRNYGGA. The RNY sequence of the 3 bp spacer as well as the presence of flanking AT‐rich regions on both sides of the core sequence appear essential for optimal activation. Further evidence that this element is the target of Lys14p was provided by the demonstration that Lys14p binds to UASLYSin vitro. The binding is independent of the presence of the co‐inducer and is not affected by lysine. It depends on the integrity of the putative Zn(II)2Cys6 binuclear cluster contained in the Lys14p.

Identification of a gene encoding a homocitrate synthase isoenzyme of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, most of the LYS structural genes have been identified except the genes encoding homocitrate synthase and α‐aminoadipate aminotransferase. Expression of several LYS genes responds to an induction mechanism mediated by the product of LYS14 and an intermediate of the pathway, α‐aminoadipate semialdehyde (αAASA) as an inducer. This activation is modulated by the presence of lysine in the growth medium leading to an apparent repression. Since the first enzyme of the pathway, homocitrate synthase, is feedback inhibited by lysine, it could be a major element in the control of αAASA supply.