SummaryThe short-time transcriptional response of yeast cells to a mild increase in external pH (7.6) has been investigated using DNA microarrays. A total of 150 genes increased their mRNA level at least twofold within 45 min. Alkalinization resulted in the repression of 232 genes. The response of four upregulated genes, ENA1 (encoding a Na + + + + -ATPase also induced by saline stress) and PHO84 , PHO89 and PHO12 (encoding genes upregulated by phosphate starvation), was characterized further. The alkaline response of ENA1 was not affected by mutation of relevant genes involved in osmotic or oxidative signalling, but was decreased in calcineurin and rim101 mutants. Mapping of the ENA1 promoter revealed two pHresponsive regions. The response of the upstream region was fully abolished by the drug FK506 or mutation of CRZ1 (a transcription factor activated by calcium/calcineurin), whereas the response of the downstream region was essentially calcium independent. PHO84 and PHO12 responses were unaffected in crz1 cells, but required the presence of Pho2 and Pho4. In contrast, part of the alkali-induced expression of PHO89 was maintained in pho4 or pho2 cells, but was fully abolished in a crz1 strain or in the presence of FK506. Heterologous promoters carrying the minimal calcineurin-dependent response elements found in ENA1 or FKS2 were able to drive alkaline pH-induced expression. These results demonstrate that the transcriptional response to alkaline pH involves different signalling mechanisms, and that calcium signalling is a relevant component of this response.

Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.Calcium-mediated signaling mechanisms are used by virtually every eukaryotic cell to regulate a wide variety or cellular processes, including gene expression. Transient increases in cytosolic calcium results in activation of diverse enzymes, such as the protein phosphatase calcineurin. Calcineurin is a heterodimer of catalytic subunit and regulatory subunits. In the yeast Saccharomyces cerevisiae, the catalytic subunit is encoded by two genes, CNA1 and CNA2 (1), whereas a single gene, CNB1, encodes the regulatory subunit (2). Cells lacking the catalytic subunits, or the regulatory subunit, are deficient in calcineurin activity.Exposure of yeast cells to a number of signals, such as ␣-factor (3, 4), glucose (5), sphingosine (6), and certain stress conditions (7-9), triggers a rise in cytoplasmic calcium. This increase in calcium can be a consequence of external calcium influx or release from internal stores, such as the vacuole, and results in activation of calcineurin. For instance, hyperosmotic shock has been reported to provoke calcium release from vacuolar stores (8) through Yvc1, a member of the transient receptor potential channel family, and to trig...

Adaptive response of the yeast Saccharomyces cerevisiae to environmental alkalinization results in remodeling of gene expression. A key target is the gene ENA1, encoding a Na ؉ -ATPase, whose induction by alkaline pH has been shown to involve calcineurin and the Rim101/Nrg1 pathway. Previous functional analysis of the ENA1 promoter revealed a calcineurin-independent pH responsive region (ARR2, 83 nucleotides). We restrict here this response to a small (42 nucleotides) ARR2 5-region, named MCIR (minimum calcineurin independent response), which contains a MIG element, able to bind Mig1,2 repressors. High pH-induced response driven from this region was largely abolished in snf1 cells and moderately reduced in a rim101 strain. Cells lacking Mig1 or Mig2 repressors had a near wild type response, but the double mutant presented a high level of expression upon alkaline stress. Deletion of NRG1 (but not of NRG2) resulted in increased expression. Induction from the MCIR region was marginal in a quadruple mutant lacking Nrg1,2 and Mig1,2 repressors. In vitro band shift experiments demonstrated binding of Nrg1 to the 5 end of the ARR2 region. Furthermore, we show that Nrg1 binds in vivo around the MCIR region under standard growth conditions, and that binding is largely abolished after high pH stress. Therefore, the calcineurin-independent response of the ENA1 gene is under the regulation of Rim101 (through Nrg1) and Snf1 (through Nrg1 and Mig2). Accordingly, induction by alkaline stress of the entire ENA1 promoter in a snf1 rim101 mutant in the presence of the calcineurin inhibitor FK506 is completely abolished. Thus, the transcriptional response to alkaline stress of the ENA1 gene integrates three different signaling pathways.

Type 2C protein phosphatases are encoded in Saccharomyces cerevisiae by several related genes (PTC1-5 and PTC7). To gain insight into the functions attributable to specific members of this gene family, we have investigated the transcriptional profiles of ptc1-5 mutants. Two main patterns were obtained as follows: the one generated by the ptc1 mutation and the one resulting from the lack of Ptc2-5. ptc4 and ptc5 profiles were quite similar, whereas that of ptc2 was less related to this group. Mutation of PTC1 resulted in increased expression of numerous genes that are also induced by cell wall damage, such as YKL161c, SED1, or CRH1, as well as in higher amounts of active Slt2 mitogen-activated protein kinase, indicating that lack of the phosphatase activates the cell wall integrity pathway. ptc1 cells were even more sensitive than slt2 mutants to a number of cell wall-damaging agents, and both mutations had additive effects. The sensitivity of ptc1 cells was not dependent on Hog1. Besides these phenotypes, we observed that calcineurin was hyperactivated in ptc1 cells, which were also highly sensitive to calcium ions, heavy metals, and alkaline pH, and exhibited a random haploid budding pattern. Remarkably, many of these traits are found in certain mutants with impaired vacuolar function. As ptc1 cells also display fragmented vacuoles, we hypothesized that lack of Ptc1 would primarily cause vacuolar malfunction, from which other phenotypes would derive. In agreement with this scenario, overexpression of VPS73, a gene of unknown function involved in vacuolar protein sorting, largely rescues not only vacuolar fragmentation but also sensitivity to cell wall damage, high calcium, alkaline pH, as well as other ptc1-specific phenotypes.

Unlike most other organisms, the essential five-step Coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the gene(s) encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remains unidentified.Sequence homology analyses suggest three candidates, namely Ykl088w, Hal3 and Vhs3, as putative PPCDC enzymes in Saccharomyces cerevisiae. Interestingly, Hal3 and Vhs3 have been characterized as negative regulatory subunits of the Ppz1 protein phosphatase. Here we show that YKL088w does not encode a third Ppz1 regulatory subunit, and that the essential roles of Ykl088w and the Hal3/Vhs3 pair are complementary, cannot be interchanged and can be attributed to PPCDC-related functions. We demonstrate that while known eukaryotic PPCDCs are homotrimers, the active yeast enzyme is a heterotrimer which consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues.Our results unveil Hal3/Vhs3 as moonlighting proteins, involved in both CoA biosynthesis and protein phosphatase regulation. 3Coenzyme A (CoA, 1) is a ubiquitous and essential cofactor that is utilized by a wide variety of enzymes in reactions where it mainly acts as a carrier and activator of acyl groups 1, 2 . The CoA biosynthetic pathway has been elucidated in various diverse species, including eubacteria (Escherichia coli), plants (Arabidopsis thaliana), and mammals (Homo sapiens) 1,3,4 . These studies have shown that the pathway is universal and consists of the same five enzymatic transformations in all cases, although some diversity exist among the specific proteins that catalyze certain steps 5 . Nonetheless, bioinformatic approaches allow identifying (with a few exceptions 6 ) candidate genes encoding the CoA biosynthetic proteins in nearly all organisms, including Saccharomyces cerevisiae.Interestingly, sequence homology searches suggest three proteins, namely Hal3, Vhs3 and Ykl088w, as candidates that may exhibit phosphopantothenoylcysteine decarboxylase (PPCDC) activity in S. cerevisiae. PPCDC is a flavoprotein that catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine (PPC; 2) to yield 4'-phosphopantetheine (PP; 3), the third step in CoA biosynthesis. While previous studies on PPCDCs have shown some diversity among these enzymes (e.g. the bacterial variants are usually bifunctional proteins that also have phosphopantothenoylcysteine synthetase (PPCS) activity), they all share a common mechanism and active site architecture [7][8][9][10][11] . Furthermore, all PPCDCs characterized so far are monogenic. While the three PPCDC candidates in S. cerevisiae are indeed very similar (Vhs3 and Ykl088w have 49% and 28% sequence identity to the Hal3 protein respectively, see Figure 1a), the identification of Hal3 and Vhs3 as potential PPCDCs is in fact surprising as both proteins have previously been shown to have functions completely unrelated to CoA biosynthesis.Specifically, Hal3 (also known as Sis2) is a conserved protein originally identified as a halotoler...