Article

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: 5.04). 04/2009; 29(10):2876-88. DOI: 10.1128/MCB.01740-08
Source: PubMed

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.

0 Followers
 · 
106 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The SNF1/AMP-activated protein kinases (AMPKs) function in energy regulation in eukaryotic cells. SNF1/AMPKs are αβγ heterotrimers that are activated by phosphorylation of the activation-loop Thr on the catalytic subunit. Protein kinases that activate SNF1/AMPK have been identified, but the protein phosphatases responsible for dephosphorylation of the activation loop are less well defined. For Saccharomyces cerevisiae SNF1/AMPK, Reg1-Glc7 protein phosphatase 1 (PP1) and Sit4 type 2A-related phosphatase function together to dephosphorylate Thr210 on the Snf1 catalytic subunit during growth on high concentrations of glucose; reg1 and sit4 single mutations do not impair dephosphorylation when inappropriate glycogen synthesis, also caused by these mutations, is blocked. We here present evidence that Ptc1 protein phosphatase 2C (PP2C) also has a role in dephosphorylation of Snf1 Thr210 in vivo. The sit4 ptc1 mutant exhibited partial defects in regulation of the phosphorylation state of Snf1. The reg1 ptc1 mutant was viable only when expressing mutant Snf1 proteins with reduced kinase activity, and Thr210 phosphorylation of the mutant SNF1 heterotrimers was substantially elevated during growth on high glucose. This evidence, together with findings on the reg1 sit4 mutant, indicates that while Reg1-Glc7 plays the major role, all three phosphatases contribute to maintenance of the Snf1 activation loop in the dephosphorylated state during growth on high glucose. Ptc1 has overlapping functions with Reg1-Glc7 and Sit4 in glucose regulation of SNF1/AMPK and cell viability.
    Journal of Biological Chemistry 09/2013; DOI:10.1074/jbc.M113.503763 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A key mechanism of signal transduction in eukaryotes is reversible protein phosphorylation mediated through protein kinases and protein phosphatases (PPases). Modulation of signal transduction by this means regulates many biological processes. Saccharomyces cerevisiae has 40 PPases, including seven protein phosphatase 2C (PP2C PPase) genes (PTC1-PTC7). However, the precise functions remain poorly understood. To elucidate their cellular functions and to identify those that are redundant, we constructed 127 strains with deletions of all possible combinations of the seven PP2C PPase genes. All 127 disruptants were viable under nutrient rich conditions, demonstrating that none of the combinations induced synthetic lethality under these conditions. However, several combinations exhibited novel phenotypes, e.g., the Δptc5Δptc7 double disruptant and the Δptc2Δptc3Δptc5Δptc7 quadruple disruptant exhibited low (13 °C) and high (37 °C) temperature sensitive growth, respectively. Interestingly, the septuple disruptant ∆ptc1∆ptc2∆ptc3∆ptc4∆ptc5∆ptc6∆ptc7 showed an essentially normal growth phenotype at 37 °C. The Δptc2Δptc3Δptc5Δptc7 quadruple disruptant was sensitive to LiCl (0.4 M). Two double disruptants Δptc1Δptc2 and Δptc1Δptc4 displayed slow growth and Δptc1Δptc2Δptc4 could not grow on medium containing 1.5 M NaCl. The Δptc1Δptc6 double disruptant showed increased sensitivity to caffeine, congo red and calcoflour white compared to each single deletion. Our observations indicate that S. cerevisiae PP2C PPases have a shared and important role in responses to environmental stresses. These disruptants also provide a means for exploring the molecular mechanisms of redundant PTC gene functions under defined conditions. This article is protected by copyright. All rights reserved.
    Yeast 10/2014; 31(10). DOI:10.1002/yea.3032 · 1.74 Impact Factor

Full-text (2 Sources)

Download
42 Downloads
Available from
Jun 2, 2014