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 kcs1⌬ 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 arg82⌬ cells. This metabolic block was partially bypassed, and the phenotype was partially rescued, when Kcs1p was overexpressed in the arg82⌬ 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.

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 yeast's 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.

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.

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The products of three genes named CARGRI, CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism. CARGRI is identical to UME6 and encodes a regulator of early meiotic genes. In this work we identify CARGRII as SIN3 and CARGRIII as RPD3. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress CAR1 expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of the UME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the ⌺1278b background.The first mutations affecting the expression of the arginine catabolic genes CAR1 and CAR2, encoding arginase and ornithine transaminase, respectively, were located in three unlinked genes, ARGRI (ARG80), ARGRII (ARG81), and ARGRIII (ARG82). The products of these genes were required for the induction of arginase and ornithine transaminase synthesis, and their loss impaired cell ability to use arginine and ornithine as nitrogen sources. The same proteins repressed the synthesis of five arginine anabolic enzymes when exogenous arginine was present in the growth medium (1, 42). Later, it was shown that the pleiotropic factor Mcm1 also participated, with the ArgR proteins, in the arginine-specific regulation (10, 28). The growth defect of an argR mutant allowed the selection of suppressor mutations falling into three complementation groups containing the CARGRI (CAR80), CARGRII (CAR81) and CARGRIII (CAR82) genes. Mutations in any of these genes led to overproduction of arginase and ornithine transaminase, even in an argR background (7, 9). The CARGRI gene was identical to UME6, a gene whose product is involved in controlling the expression of early meiotic genes (34, 41). Kadosh and Struhl (20) showed that repression by Ume6 at URS1, a sequence present in a wide variety of yeast promoters (24), involved recruitment of a Sin3-Rpd3 complex and targeted histone deacetylation.Although CAR1 and CAR2 genes are coordinately induced by arginine and the ArgR-Mcm1 complex (29) and repressed by the three CargR proteins, only CAR2 is induced by Dal82 and allophanate, the last intermediate of the allantoin-degrading pathway (17, 35), whereas CAR1 is activated by Gln3 and Nil1 through multiple GATAA sequences in the absence of optimal nitrogen sources (ammonia, glutamine) (11,40). In addition CAR1 expression is controlled not only by the quality of the nitrogen source...