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

Departament de Bioquímica i Biologia Molecular, Ed. V, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain.
Molecular and Cellular Biology (Impact Factor: 4.78). 04/2009; 29(10):2876-88. DOI: 10.1128/MCB.01740-08
Source: PubMed


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.

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Available from: Antonio Casamayor, Oct 09, 2015
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    • "A) A simplified model of signaling through the TOR pathway, focused on the regulation of NCR genes and displaying the proposed role for Ptc1 [19]. B) Growth in the presence of the indicated concentrations of rapamycin of diverse mutants in genes involved in the TOR pathway in combination with deletion of PTC6. "
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    ABSTRACT: Ptc6 is one of the seven components (Ptc1-Ptc7) of the protein phosphatase 2C family in the yeast Saccharomyces cerevisiae. In contrast to other type 2C phosphatases, the cellular role of this isoform is poorly understood. We present here a comprehensive characterization of this gene product. Cells lacking Ptc6 are sensitive to zinc ions, and somewhat tolerant to cell-wall damaging agents and to Li(+). Ptc6 mutants are sensitive to rapamycin, albeit to lesser extent than ptc1 cells. This phenotype is not rescued by overexpression of PTC1 and mutation of ptc6 does not reproduce the characteristic genetic interactions of the ptc1 mutation with components of the TOR pathway, thus suggesting different cellular roles for both isoforms. We show here that the rapamycin-sensitive phenotype of ptc6 cells is unrelated to the reported role of Pt6 in controlling pyruvate dehydrogenase activity. Lack of Ptc6 results in substantial attenuation of the transcriptional response to rapamycin, particularly in the subset of repressed genes encoding ribosomal proteins or involved in rRNA processing. In contrast, repressed genes involved in translation are Ptc6-independent. These effects cannot be attributed to the regulation of the Sch9 kinase, but they could involve modulation of the binding of the Ifh1 co-activator to specific gene promoters.
    PLoS ONE 05/2013; 8(5):e64470. DOI:10.1371/journal.pone.0064470 · 3.23 Impact Factor
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    • "The CCV37 and CCV38 strains were obtained transforming W303-1A wild-type and MCY5278 strains with a SNF1::LEU2 disruption cassette [21] and the CCV174 and CCV175 strains were generated by transforming W303-1A and MCY5278 strains with the 2.1 kbp nrg1::nat1 cassette from the plasmid pBS-nrg1::nat1 as described previously [28]. The AGS66 strain contained an integrated STRE(7×)–lacZ reporter system at the URA3 locus [29]. "
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    ABSTRACT: 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.
    Biochemical Journal 07/2011; 438(3):523-33. DOI:10.1042/BJ20110607 · 4.40 Impact Factor
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    • "Ptc1p, Ptc2p, Ptc3p and Ptc4p are all shown to negatively regulate the mitogen-activated protein kinase (MAPK) Hog1p (Warmka et al., 2001; Young et al., 2002; Shiozaki and Russell, 1995; Shitamukai et al., 2004). It is interesting that Ptc1p has additional important functions, including cell survival of heat shock, tRNA splicing, sporulation , lithium tolerance, normal functioning of the target of rapamycin (TOR) pathway, and transportation of mitochondria, vacuoles and endoplasmic reticulum from the mother cell to the daughter cell (Shiozaki et al., 1994; Robinson et al., 1994; Jin et al., 2009; Du et al., 2006; González et al., 2006 and 2009; Ruiz et al., 2006). Both Ptc2p and Ptc3P are responsible for the dephosphorylation of Cdc28p, the major budding yeast cyclindependent kinase (CDK) (Cheng et al., 1999). "
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    ABSTRACT: Type 2C protein phosphatases (PP2C) are monomeric enzymes and their activities require the presence of magnesium or manganese ions. There are seven PP2C genes, ScPTC1, ScPTC2, ScPTC3, ScPTC4, ScPTC5, ScPTC6 and ScPTC7, in Saccharomyces cerevisiae. PTC6 is highly conserved in pathogenic and nonpathogenic yeasts. In the current study we have demonstrated that the Candida albicans CaPTC6 gene could complement the functions of ScPTC6 in the rapamycin and caffeine sensitivities of S. cerevisiae cells, indicating that they are functional homologues. We have also demonstrated that the CaPTC6-encoded protein is a typical PP2C enzyme and that CaPtc6p is localized in the mitochondrion of yeast-form and hyphal cells. However, deletion of CaPTC6 neither affects cell and hyphal growth nor renders Candida cells sensitive to rapamycin and caffeine. Therefore, possibly with a functional redundancy to other mitochondrial phosphatases, CaPtc6p is likely to be involved in the regulation of a mitochondrial physiology.
    Yeast 01/2009; 27(4):197-206. DOI:10.1002/yea.1743 · 1.63 Impact Factor
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