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1.
FIG. 7.

FIG. 7. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Effect of the ptc1 and sit4 mutations in the transcriptional response to the ammonium starvation of several NCR-sensitive genes. Strains BY4741 (PTC1 SIT4) and its ptc1, sit4, and ptc1 sit4 derivatives were transformed with the indicated NCR-sensitive reporters. Cells were grown overnight on either standard synthetic (closed bars) or low ammonium medium (open bars), and β-galactosidase activity was measured. Data are means ± standard errors of the means from three independent clones.

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
2.
FIG. 5.

FIG. 5. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Lack of Ptc1 affects Npr1 levels and phosphorylation state. Wild-type BY4741 (WT) and its ptc1 (MAR143) or sit4 derivative were transformed with plasmid pEJ23, which carries an N-terminally HA-tagged version of Npr1. Cells were incubated for 30 min with 100 ng/ml rapamycin or vehicle, and extracts were prepared. Thirty micrograms of protein [90 μg in the case of ptc1 cells, denoted ptc1(3x)] was electrophoresed, transferred, and incubated with anti-HA monoclonal antibodies. Faster-migrating bands correspond to the dephosphorylated forms of Npr1.

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
3.
FIG. 6.

FIG. 6. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Epistatic analysis of ptc1 and mutations affecting the TOR pathway. (A) A simplified model of signaling through the TOR pathway (focused on the regulation of NCR genes) based on previous models (15, 29, 33). (B) Rapamycin and caffeine sensitivity of diverse mutants in genes relevant in the TOR pathway in the presence (+) or the absence of PTC1 (−). Cultures were spotted on YPD plates containing the indicated concentrations of the drugs, and growth was monitored after 2 days. WT, wild type.

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
4.
FIG. 1.

FIG. 1. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Sensitivity to rapamycin and caffeine of the diverse yeast type 2C protein phosphatase mutants. (A) Wild-type strain BY4741 and the different ptc mutants were spotted onto YPD plates containing the indicated concentrations of rapamycin or caffeine. Growth was monitored after 60 h of incubation at 28°C. (B) The BY4741 strain and its ptc1 derivative were transformed with an empty centromeric YCplac33 vector (YCp-Ø) or with the wild type or a catalytically inactive form of Ptc1 (PTC1[D58N]) cloned in either centromeric (YCp) or high-copy-number (YEp) vectors. Cells were spotted as described above and incubated for 48 h.

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
5.
FIG. 3.

FIG. 3. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Decreased response of diverse NCR-sensitive genes to rapamycin treatment. (A) The indicated constructs were introduced into wild-type BY4741 (WT) and its ptc1 derivative, and cells were treated with 200 ng/ml rapamycin (Rap) (open bars) for 60 (for GAP1, GLN1, and GDH1 promoters) or 90 min (for MEP1). Control cells (closed bars) received only the solvent. β-Galactosidase activity was measured as indicated in the text. Data are means ± standard errors of the means from six independent clones. (B) RT-PCR experiments were performed using oligonucleotides specific for the indicated genes (see Materials and Methods). Amplification fragments were run on 2% agarose gels. ACT1 is included for comparison.

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
6.
FIG. 4.

FIG. 4. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

ptc1 mutation impairs rapamycin-induced Gln3 and Msn2 entry into the nucleus. (A) TB123 (PTC1 GLN3-myc13-kanMX) and AGS39 (ptc1 GLN3-myc13-kanMX) cells were exposed to 200 ng/ml rapamycin (Rap) or to solvent for 10 min and processed for indirect immunofluorescence using anti-Myc antibodies. Samples also were stained with DAPI to reveal the position of the nuclei (magnification, ×1,000). (B) The upper panel shows wild-type (WT) strain AGS66 and its ptc1 isogenic derivative AGS67, which contain integrated STRE-LacZ reporters. They were treated for 1 h with 200 ng/ml rapamycin (open bars) or solvent (closed bars), and β-galactosidase activity was measured. Data are means ± standard errors of the means from six independent clones. The lower panel shows strains BY4741 (WT) and MAR143 (ptc1), which were transformed with plasmid pMsn2-GFP (24) and incubated with 200 ng/ml rapamycin for 15 min. The localization of Msn2 was followed by fluorescence microscopy (magnification, ×1,000).

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
7.
FIG. 9.

FIG. 9. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Phosphorylation pattern of Tip41 in Ptc1-deficient cells. The upper panel shows wild-type BY4741 (WT) and ptc1 cells transformed with plasmid pEJ120, which were incubated for 30 min with 100 ng/ml rapamycin (Rap) or vehicle. Extracts were prepared for isoelectrofocusing as described in Materials and Methods. The first dimension was run using Immobiline DryStrip (pH 4 to 7) strips, and the second dimension was performed in SDS-10% polyacrylamide gels. Gels were transferred to membranes, and Tip41 was detected using anti-HA antibodies. Only the relevant region of the immunoblot is shown, and the different Tip41 forms are labeled (A to I). pHs are indicated on the top, and the molecular mass standard is on the right. The lower panel shows the vol% parameter (i.e., relative volume of a spot) for each spot, calculated using Melanie 7.0 software. The means ± standard errors from the means from three independent experiments are represented. The intensity of spots G, F, and E in the ptc1 mutant was too low to be integrated.

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
8.
FIG. 2.

FIG. 2. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Global transcriptional response analysis to rapamycin in wild-type (WT) and ptc1 cells. (A) Venn diagram showing the number of genes whose expression was considered to be induced (top) or repressed (bottom) by rapamycin in wild-type ptc1 cells for a set of 4,677 genes, with valid data for both strains. (B) Plots of the log2 values for the changes in the level of expression induced by rapamycin in both wild-type (open circles) and ptc1 strains for the 150 most upregulated (top) and 150 most downregulated (bottom) genes in the wild-type strain. The expression values for the ptc1 strain are shown according to the TOR-dependent regulon to which each gene belongs. For the induced genes, the following categories were used: the NCR family (closed squares), as defined previously (21); the RTG group (open squares) comprises the genes described as documented targets for the Rtg1 or Rtg3 transcription factors in YEASTRACT (44), as well as those identified elsewhere (19); and the documented targets of Msn2 or Msn4 described in YEASTRACT, plus those identified elsewhere (9), are represented as open diamonds. Genes not included in these categories are designated others (closed triangles). The localization of representative genes for each family in the plot is shown. The genes downregulated by rapamycin in the wild-type strain are classified into one of three possible families: Ribi regulon (closed squares), which include the genes described previously (31), ribosomal proteins (open squares), and others (closed triangles).

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.
9.
FIG. 8.

FIG. 8. From: Normal Function of the Yeast TOR Pathway Requires the Type 2C Protein Phosphatase Ptc1 .

Lack of Ptc1 decreases Tip41 stability and Tip41-Tap42 rapamycin-induced interaction. (A) BY4741 and its ptc1 derivative were transformed with plasmids pEJ27 (GST-Tap42) and pEJ120 (Tip41-HA). Cultures were grown overnight on synthetic medium lacking leucine and uracil and then inoculated into YPD (A660, 0.2 to 0.3) and grown for 4 h. Cells were treated with rapamycin (100 ng/ml) or drug vehicle for 30 min, and cell extracts were prepared and GST-Tap42 affinity purified as described in Materials and Methods. Purified material (upper) or cell extracts (lower; 40 μg of proteins) were subjected to SDS-PAGE and immunoblotted with anti-HA antibodies to reveal the presence of Tip41. Membranes were stripped and incubated with anti-GST antibody to detect Tap42. (B) A BY4741 strain lacking the TIP41 gene (PTC1) was deleted for the PTC1 gene (strain MAR164; ptc1), and both strains were transformed with plasmid YCp111-TIP41-HA. Cultures received 200 ng/ml of rapamycin or vehicle, and incubation was resumed for 10 min. Cycloheximide then was added to the cultures (25 μg/ml) to halt translation, and samples were taken at the indicated times and processed for immunoblotting using anti-HA antibodies. Membranes were stripped and incubated with anti-actin antibodies as a loading and transfer control. The experiment was repeated three times, with similar results. (C) Wild-type BY4741 (WT) and MAR143 (ptc1) were transformed with plasmid pEJ120 (expressing Tip41-HA in high copy number; filled bars) or the equivalent empty plasmid (YEplac181; open bars). These strains then were transformed with plasmid pGAP1-lacZ. Cells were grown and treated with rapamycin as illustrated in the legend to Fig. 3. β-Galactosidase activity generated from the GAP1 promoter was measured as described in the text. Data are means ± standard errors of the means from nine independent clones. The inset shows the levels of Tip41-HA in tip41 cells in the presence (lanes 2 and 3) or the absence (lanes 4 and 5) of the PTC1 gene when the gene is expressed from low-copy-number (lanes 2 and 4) or high-copy-number plasmids (lanes 3 and 5). Lane 1 corresponds to tip41 cells carrying an empty plasmid. Actin levels are included as loading controls.

Asier González, et al. Mol Cell Biol. 2009 May;29(10):2876-2888.

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