Asier González

Transcriptional Profiling of the Protein Phosphatase 2C Family in Yeast Provides Insights into the Unique Functional Roles of Ptc1

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.

Role of protein phosphatases 2C on tolerance to lithium toxicity in the yeast Saccharomyces cerevisiae

Protein phosphatases 2C are a family of conserved enzymes involved in many aspects of the cell biology. We reported that, in the yeast Saccharomyces cerevisiae, overexpression of the Ptc3p isoform resulted in increased lithium tolerance in the hypersensitive hal3 background. We have found that the tolerance induced by PTC3 overexpression is also observed in wild‐type cells and that this is most probably the result of increased expression of the ENA1 Na+‐ATPase mediated by the Hog1 MAP kinase pathway. This effect does not require a catalytically active protein. Surprisingly, deletion of PTC3 (similarly to that of PTC2, PTC4 or PTC5) does not confer a lithium‐sensitive phenotype, but mutation of PTC1 does. Lack of PTC1 in an ena1–4 background did not result in additive lithium sensitivity and the ptc1 mutant showed a decreased expression of the ENA1 gene in cells stressed with LiCl. In agreement, under these conditions, the ptc1 mutant was less effective in extruding Li+ and accumulated higher concentrations of this cation. Deletion of PTC1 in a hal3 background did not exacerbate the halosensitive phenotype of the hal3 strain. In addition, induction from the ENA1 promoter under LiCl stress decreased similarly (50%) in hal3, ptc1 and ptc1 hal3 mutants. Finally, mutation of PTC1 virtually abolishes the increased tolerance to toxic cations provided by overexpression of Hal3p. These results indicate that Ptc1p modulates the function of Ena1p by regulating the Hal3/Ppz1,2 pathway. In conclusion, overexpression of PTC3 and lack of PTC1 affect lithium tolerance in yeast, although through different mechanisms.

Type 2C protein phosphatases in fungi.

ABSTRACT Type 2C Ser/Thr phosphatases are a remarkable class of protein phosphatases, which are conserved in eukaryotes and involved in a large variety of functional processes. Unlike in other Ser/Thr phosphatases, the catalytic polypeptide is not usually associated with regulatory subunits, and functional specificity is achieved by encoding multiple isoforms. For fungi, most information comes from the study of type 2C protein phosphatase (PP2C) enzymes in Saccharomyces cerevisiae, where seven PP2C-encoding genes (PTC1 to -7) with diverse functions can be found. More recently, data on several Candida albicans PP2C proteins became available, suggesting that some of them can be involved in virulence. In this work we review the available literature on fungal PP2Cs and explore sequence databases to provide a comprehensive overview of these enzymes in fungi.

Moonlighting proteins Hal3 and Vhs3 form a heteromeric PPCDC with Ykl088w in yeast CoA biosynthesis

Unlike most other organisms, the essential five-step coenzyme A biosynthetic pathway has not been fully resolved in yeast. Specifically, the genes encoding the phosphopantothenoylcysteine decarboxylase (PPCDC) activity still remain unidentified. Sequence homology analyses suggest three candidates-Ykl088w, Hal3 and Vhs3-as putative PPCDC enzymes in Saccharomyces cerevisiae. Notably, 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 and 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 that consists of Ykl088w and Hal3/Vhs3 monomers that separately provides two essential catalytic residues. Our results unveil Hal3 and Vhs3 as moonlighting proteins involved in both CoA biosynthesis and protein phosphatase regulation.

Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1

ABSTRACT Yeast ptc1 mutants are rapamycin and caffeine sensitive, suggesting a functional connection between Ptc1 and the TOR pathway that is not shared by most members of the type 2C phosphatase family. Genome-wide profiling revealed that the ptc1 mutation largely attenuates the transcriptional response to rapamycin. The lack of Ptc1 significantly prevents the nuclear translocation of Gln3 and Msn2 transcription factors to the nucleus, as well as the dephosphorylation of the Npr1 kinase, in response to rapamycin. This could explain the observed decrease in both the basal and rapamycin-induced expression of several genes subjected to nitrogen catabolite repression (GAT1, MEP1, and GLN1) and stress response element (STRE)-driven promoters. Interestingly, this decrease is abolished in the absence of the Sit4 phosphatase. Epitasis analysis indicates that the mutation of SIT4 or TIP41, encoding a Tap42-interacting protein, abolishes the sensitivity of the ptc1 strain to rapamycin and caffeine. All of these results suggest that Ptc1 is required for normal TOR signaling, possibly by regulating a step upstream of Sit4 function. According to this hypothesis, we observe that the mutation of PTC1 drastically diminishes the rapamycin-induced interaction between Tap42 and Tip41, and this can be explained by lower-than-normal levels of Tip41 in ptc1 cells. Ptc1 is not necessary for the normal expression of the TIP41 gene; instead, its absence dramatically affects the stability of Tip41. The lack of Ptc1 partially abolishes the rapamycin-induced dephosphorylation of Tip41, which may further decrease Tap42 binding. Reduced Tip41 levels contribute to the ptc1 phenotypes, although additional Ptc1 targets must exist. All of these results provide the first evidence showing that a type 2C protein phosphatase is required for the normal functioning of the TOR pathway.

The role of the protein kinase A pathway in the response to alkaline pH stress in yeast.

Exposure of Saccharomyces cerevisiae to alkaline pH provokes a stress condition that generates a compensatory reaction. In the present study we examined a possible role for the PKA (protein kinase A) pathway in this response. Phenotypic analysis revealed that mutations that activate the PKA pathway (ira1 ira2, bcy1) tend to cause sensitivity to alkaline pH, whereas its deactivation enhances tolerance to this stress. We observed that alkalinization causes a transient decrease in cAMP, the main regulator of the pathway. Alkaline pH causes rapid nuclear localization of the PKA-regulated Msn2 transcription factor which, together with Msn4, mediates a general stress response by binding with STRE (stress response element) sequences in many promoters. Consequently, a synthetic STRE–LacZ reporter shows a rapid induction in response to alkaline stress. A msn2 msn4 mutant is sensitive to alkaline pH, and transcriptomic analysis reveals that after 10 min of alkaline stress, the expression of many induced genes (47%) depends, at least in part, on the presence of Msn2 and Msn4. Taken together, these results demonstrate that inhibition of the PKA pathway by alkaline pH represents a substantial part of the adaptive response to this kind of stress and that this response involves Msn2/Msn4-mediated genome expression remodelling. However, the relevance of attenuation of PKA in high pH tolerance is probably not restricted to regulation of Msn2 function.

Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans

The opportunistic pathogen Candida albicans has a single protein phosphatase Z (PPZ) candidate gene termed CaPPZ1, which shows significant allele variability. We demonstrate here that bacterially expressed CaPpz1 protein exhibits phosphatase activity which can be inhibited by recombinant Hal3, a known inhibitor of Saccharomyces cerevisiae Ppz1. Site-directed mutagenesis experiments based on natural polymorphisms allowed the identification of three amino acid residues that affect enzyme activity or stability. The expression of CaPPZ1 in ppz1 S. cerevisiae and pzh1 Schizosaccharomyces pombe cells partially rescued the salt and caffeine phenotypes of the deletion mutants. CaPpz1 also complemented the slt2 S. cerevisiae mutant, which is crippled in the mitogen-activated protein (MAP) kinase that mediates the cell wall integrity signalling pathway. Collectively, our results suggest that the orthologous PPZ enzymes have similar but not identical functions in different fungi. The deletion of the CaPPZ1 gene in C. albicans resulted in a mutant that was sensitive to salts such as LiCl and KCl, to caffeine, and to agents that affect cell wall biogenesis such as Calcofluor White and Congo red, but was tolerant to spermine and hygromycin B. Reintegration of the CaPPZ1 gene into the deletion mutant alleviated all of the mutant phenotypes tested. Thus CaPpz1 is involved in cation homeostasis, cell wall integrity and the regulation of the membrane potential of C. albicans. In addition, the germ tube growth rate, and virulence in the BALB/c mouse model, were reduced in the null mutant, suggesting a novel function for CaPpz1 in the yeast to hypha transition that may have medical relevance.

Functional mapping of the disparate activities of the yeast moonlighting protein Hal3.

The Saccharomyces cerevisiae Hal3 protein is a moonlighting protein, able to function both as an inhibitory subunit of the Ppz1 protein phosphatase and as a constituent protomer of an unprecedented heterotrimeric PPCDC (phosphopantothenoylcysteine decarboxylase), the third enzyme of the CoA biosynthetic pathway. In the present study we initiated the dissection of the structural elements required for both disparate cellular tasks by using a combination of biochemical and genetic approaches. We show that the conserved Hal3 core [PD (PPCDC domain)] is necessary for both functions, as determined by in vitro and in vivo assays. The Hal3 NtD (N-terminal domain) is not functional by itself, although in vitro experiments indicate that when this domain is combined with the core it has a relevant function in Hal3s heteromeric PPCDC activity. Both the NtD and the acidic CtD (C-terminal domain) also appear to be important for Hal3s Ppz1 regulatory function, although our results indicate that the CtD fulfils the key role in this regard. Finally, we show that the introduction of two key asparagine and cysteine residues, essential for monofunctional PPCDC activity but absent in Hal3, is not sufficient to convert it into such a homomeric PPCDC, and that additional modifications of Hal3s PD aimed at increasing its resemblance to known PPCDCs also fails to introduce this activity. This suggests that Hal3 has undergone significant evolutionary drift from ancestral PPCDC proteins. Taken together, our work highlights specific structural determinants that could be exploited for full understanding of Hal3s cellular functions.

Ref2, a regulatory subunit of the yeast protein phosphatase 1, is a novel component of cation homoeostasis.

Maintenance of cation homoeostasis is a key process for any living organism. Specific mutations in Glc7, the essential catalytic subunit of yeast protein phosphatase 1, result in salt and alkaline pH sensitivity, suggesting a role for this protein in cation homoeostasis. We screened a collection of Glc7 regulatory subunit mutants for altered tolerance to diverse cations (sodium, lithium and calcium) and alkaline pH. Among 18 candidates, only deletion of REF2 (RNA end formation 2) yielded increased sensitivity to these conditions, as well as to diverse organic toxic cations. The Ref2F374A mutation, which renders it unable to bind Glc7, did not rescue the salt-related phenotypes of the ref2 strain, suggesting that Ref2 function in cation homoeostasis is mediated by Glc7. The ref2 deletion mutant displays a marked decrease in lithium efflux, which can be explained by the inability of these cells to fully induce the Na+-ATPase ENA1 gene. The effect of lack of Ref2 is additive to that of blockage of the calcineurin pathway and might disrupt multiple mechanisms controlling ENA1 expression. ref2 cells display a striking defect in vacuolar morphogenesis, which probably accounts for the increased calcium levels observed under standard growth conditions and the strong calcium sensitivity of this mutant. Remarkably, the evidence collected indicates that the role of Ref2 in cation homoeostasis may be unrelated to its previously identified function in the formation of mRNA via the APT (for associated with Pta1) complex.

Heterologous Expression Implicates a GATA Factor in Regulation of Nitrogen Metabolic Genes and Ion Homeostasis in the Halotolerant Yeast Debaryomyces hansenii

ABSTRACT The yeast Debaryomyces hansenii has a remarkable capacity to proliferate in salty and alkaline environments such as seawater. A screen for D. hansenii genes able to confer increased tolerance to high pH when overexpressed in Saccharomyces cerevisiae yielded a single gene, named here DhGZF3, encoding a putative negative GATA transcription factor related to S. cerevisiae Dal80 and Gzf3. Overexpression of this gene in wild-type S. cerevisiae increased caffeine and rapamycin tolerance, blocked growth in low glucose concentrations and nonfermentable carbon sources, and resulted in lithium- and sodium-sensitive cells. Sensitivity to salt could be attributed to a reduced cation efflux, most likely because of a decrease in expression of the ENA1 Na+-ATPase gene. Overexpression of DhGZF3 did not affect cell growth in a gat1 mutant but was lethal in the absence of Gln3. These are positive factors that oppose both Gzf3 and Dal80. Genome-wide transcriptional profiling of wild-type cells overexpressing DhGZF3 shows decreased expression of a number of genes that are usually induced in poor nitrogen sources. In addition, the entire pathway leading to Lys biosynthesis was repressed, probably as a result of a decrease in the expression of the specific Lys14 transcription factor. In conclusion, our results demonstrate that DhGzf3 can play a role as a negative GATA transcription factor when expressed in S. cerevisiae and that it most probably represents the only member of this family in D. hansenii. These findings also point to the GATA transcription factors as relevant elements for alkaline-pH tolerance.