Identification of direct and indirect targets of the Gln3 and Gat1 activators by transcriptional profiling in response to nitrogen availability in the short and long term.

Nitrogen catabolite repression (NCR) consists in the specific inhibition of transcriptional activation of genes encoding the permeases and catabolic enzymes needed to degrade poor nitrogen sources. Under nitrogen limitation or rapamycin treatment, NCR genes are activated by Gln3 or Gat1, or by both factors. To compare the sets of genes responding to rapamycin or to nitrogen limitation, we used DNA microarrays to establishing the expression profiles of a wild type strain, and of a double gln3Delta-gat1Delta strain, grown on glutamine, after addition of rapamycin, on proline, or after a shift from glutamine to proline. Analysis of microarray data revealed 392 genes whose expression was dependent on the nitrogen source quality. 91 genes were activated in a GATA factor-dependent manner in all growth conditions, suggesting a direct role of Gln3 and Gat1 in their expression. Other genes were only transiently up-regulated (stress-responsive genes) or down-regulated (genes encoding ribosomal proteins and translational factors) upon nitrogen limitation, and this regulation was delayed in a gln3Delta-gat1Delta strain. Repression of amino acid and nucleotide biosynthetic genes after a nitrogen shift did not depend on Gcn4. Several transporter genes were repressed as a consequence of enhanced levels of NCR-responsive permeases present at the plasma membrane.

Role of MADS box proteins and their cofactors in combinatorial control of gene expression and cell development.

In all organisms, correct development, growth and function depends on the precise and integrated control of the expression of their genes. Often, gene regulation depends upon the cooperative binding of proteins to DNA and upon protein-protein interactions. Eukaryotes have widely exploited combinatorial strategies to create gene regulatory networks. MADS box proteins constitute the perfect example of cellular coordinators. These proteins belong to a large family of transcription factors present in most eukaryotic organisms and are involved in diverse and important biological functions. MADS box proteins are combinatorial transcription factors in that they often derive their regulatory specificity from other DNA binding or accessory factors. This review is aimed at analyzing how MADS box proteins combine with a variety of cofactors to achieve functional diversity.

Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast.

In Saccharomyces cerevisiae, the synthesis of inositol pyrophosphates is essential for vacuole biogenesis and the cell's response to certain environmental stresses. The kinase activity of Arg82p and Kcs1p is required for the production of soluble inositol phosphates. To define physiologically relevant targets of the catalytic products of Arg82p and Kcs1p, we used DNA microarray technology. In arg82delta or kcs1delta cells, we observed a derepressed expression of genes regulated by phosphate (PHO) on high phosphate medium and a strong decrease in the expression of genes regulated by the quality of nitrogen source (NCR). Arg82p and Kcs1p are required for activation of NCR-regulated genes in response to nitrogen availability, mainly through Nil1p, and for repression of PHO genes by phosphate. Only the catalytic activity of both kinases was required for PHO gene repression by phosphate and for NCR gene activation in response to nitrogen availability, indicating a role for inositol pyrophosphates in these controls. Arg82p also controls expression of arginine-responsive genes by interacting with Arg80p and Mcm1p, and expression of Mcm1-dependent genes by interacting with Mcm1p. We show here that Mcm1p and Arg80p chaperoning by Arg82p does not involve the inositol polyphosphate kinase activity of Arg82p, but requires its polyaspartate domain. Our results indicate that Arg82p is a bifunctional protein whose inositol kinase activity plays a role in multiple signalling cascades, and whose acidic domain protects two MADS-box proteins against degradation.

Yeast epiarginase regulation, an enzyme-enzyme activity control: identification of residues of ornithine carbamoyltransferase and arginase responsible for enzyme catalytic and regulatory activities.

In the presence of ornithine and arginine, ornithine carbamoyltransferase (OTCase) and arginase form a one-to-one enzyme complex in which the activity of OTCase is inhibited whereas arginase remains catalytically active. The mechanism by which these nonallosteric enzymes form a stable complex triggered by the binding of their respective substrates raises the question of how such a cooperative association is induced. Analyses of mutations in both enzymes identify residues that are required for their association, some of them being important for catalysis. In arginase, two cysteines at the C terminus of the protein are crucial for its epiarginase function but not for its catalytic activity and trimeric structure. In OTCase, mutations of putative ornithine binding residues, Asp-182, Asn-184, Asn-185, Cys-289, and Glu-256 greatly reduced the affinity for ornithine and impaired the interaction with arginase. The four lysine residues located in the SMG loop, Lys-260, Lys-263, Lys-265, and Lys-268, also play an important role in mediating the sensitivity of OTCase to ornithine and to arginase and appear to be involved in transducing and enhancing the signal given by ornithine for the closure of the catalytic domain.

Swapping functional specificity of a MADS box protein: residues required for Arg80 regulation of arginine metabolism.

Arg80 and Mcm1, two members of the MADS box family of DNA-binding proteins, regulate the metabolism of arginine in association with Arg81, the arginine sensor. In spite of the high degree of sequence conservation between the MADS box domains of the Arg80 and Mcm1 proteins (56 of 81 amino acids), these domains are not interchangeable. To determine which amino acids define the specificity of Arg80, we swapped the amino acids in each secondary-structure element of the Arg80 MADS box domain with the corresponding amino acids of Mcm1 and assayed the ability of these chimeras to regulate arginine-metabolic genes in place of the wild-type Arg80. Also performed was the converse experiment in which each variant residue in the Mcm1 MADS box domain was swapped with the corresponding residue of Arg80 in the context of an Arg80-Mcm1 fusion protein. We show that multiple regions of Arg80 are important for its function. Interestingly, the residues which have important roles in determining the specificity of Arg80 are not those which could contact the DNA but are residues that are likely to be involved in protein interactions. Many of these residues are clustered on one side of the protein, which could serve as an interface for interaction with Arg81 or Mcm1. This interface is distinct from the region used by the Mcm1 and human serum response factor MADS box proteins to interact with their cofactors. It is possible that this alternative interface is used by other MADS box proteins to interact with their cofactors.

In Saccharomyces cerevisiae, the inositol polyphosphate kinase activity of Kcs1p is required for resistance to salt stress, cell wall integrity, and vacuolar morphogenesis.

A problem for inositol signaling is to understand the significance of the kinases that convert inositol hexakisphosphate to diphosphoinositol polyphosphates. This kinase activity is catalyzed by Kcs1p in the yeast Saccharomyces cerevisiae. A kcs1Delta yeast strain that was transformed with a specifically "kinase-dead" kcs1p mutant did not synthesize diphosphoinositol polyphosphates, and the cells contained a fragmented vacuolar compartment. Biogenesis of the yeast vacuole also required another functional domain in Kcs1p, which contains two leucine heptad repeats. The kinase activity of Kcs1p was also found to sustain cell growth and integrity of the cell wall and to promote adaptive responses to salt stress. Thus, the synthesis of diphosphoinositol polyphosphates has wide ranging physiological significance. Furthermore, we showed that these phenotypic responses to Kcs1p deletion also arise when synthesis of precursor material for the diphosphoinositol polyphosphates is blocked in arg82Delta cells. This metabolic block was partially bypassed, and the phenotype was partially rescued, when Kcs1p was overexpressed in the arg82Delta cells. This was due, in part, to the ability of Kcs1p to phosphorylate a wider range of substrates than previously appreciated. Our results show that diphosphoinositol polyphosphate synthase activity is essential for biogenesis of the yeast vacuole and the cell's responses to certain environmental stresses.