Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: Inhibition of TORC1 reduces mating GPCR levels at the PM and represses the mating pathway. A - D , representative images of cells expressing ( A ) Ste2-mEnvy, ( B ) Ste3-mEnvy, ( C ) Gpr1-mEnvy, and ( D ) Pma1-mRuby2 treated with or without 0.2 μM rapamycin for 2 h. Scale bars represent 4 μm. E , quantification of Ste2-mEnvy, Ste3-mEnvy, Gpr1-mEnvy, and Pma1-mRuby2 mean fluorescent intensities at the plasma membrane (-Rap: Ste2 n = 212 cells, Ste3 n = 285 cells, Gpr1 n = 197 cells, Pma1 n = 270 cells. +Rap: Ste2 n = 277 cells, Ste3 n = , Gpr1 n = 214 cells, Pma1 n = ). Measurements were normalized to the mean of the untreated groups to produce percentages. Error bars represent ±SEM. ∗ = p < 0.05, ∗∗ = p < 0.01, and ∗∗∗ = p < 0.001. F , β-galactosidase (pFUS1-LacZ) mating transcription assays of cell cultures pretreated with or without 0.2 μM rapamycin for 2 h (n > 10 colonies). Error bars represent ±SEM. EC 50 and E max are reported with ±95% confidence intervals. G , quantitative mating assays in cells pretreated with or without 0.2 μM rapamycin for 2 h (n = 5 assays). Error bars represent ±SEM. ∗ = p < 0.05 by two-sample one-tailed unequal variance t test. GPCR, G-protein–coupled receptor; mEnvy, monomeric Envy; PM, plasma membrane; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Inhibition, Expressing, Clinical Proteomics, Membrane, One-tailed Test

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: TORC1 inhibition leads to CME of Ste2 through α-arrestins and Ypk1. A , to identify candidates involved in TORC1-mediated endocytosis, we deleted multiple genes that are involved in TORC signaling, receptor endocytosis, and CME. Casein kinases ( , , ), α-arrestins ( , , , ), and epsin-like proteins ( , ) are all involved in priming the pheromone receptors for CME during mating. CME utilizes motors and cytoskeletal organizers ( , ) to form then endocytic pit that matures into an endosome. During starvation, TORC1 activity is reduced, downregulating PP2A activity . TORC2 activates Ypk1 and Ypk2 ( , , ), which repress major cell regulators such as calcineurin . B , quantification of mean Ste2-mEnvy on the plasma membrane in the indicated strain, with or without 0.2 μM rapamycin for 2 h (n > 170 cells). Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA was used to analyze variance between treated and untreated mutant and WT cells. Tukey’s HSD test was used as a multiple comparison test to compare means between groups. Based on ANOVA results, genetic mutants treated with rapamycin were grouped into one of three categories: no rescue ( red ), partial rescue ( yellow ), and complete rescue ( green ). Mutants in the no rescue category were only significantly different ( p < 0.05) from untreated WT cells. Mutants in the partial rescue category were significantly different from both treated and untreated WT cells. Mutants in the complete rescue category were only significantly different from rapamycin-treated WT cells. Error bars represent ±SEM. Bars with dashed outlines show data reported in E . C – G , representative images of ( C ) WT, ( D ) end 3Δ, ( E ) rog Δ, ( F ) rod 1Δ, and ( G ) ypk 1Δ cells expressing Ste2-mEnvy treated with or without 0.2 μM rapamycin for 2 h. Scale bars represent 4 μm. CME, clathrin-mediated endocytosis; HSD, honestly significant difference; mEnvy, monomeric Envy; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Inhibition, Activity Assay, Clinical Proteomics, Membrane, Mutagenesis, Comparison, Expressing

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: The C terminus of Ste2 is required for TORC1-mediated endocytosis. A , representative images of cells expressing Ste2-mEnvy and Ste2 T326 -EGFP treated with (n = 184 cells) and without (n = 132 cells) 0.2 μM rapamycin for 2 h. Scale bar represents 4 μm. B , quantification of Ste2-mEnvy and Ste2 T326 -EGFP mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. Error bars represent ±SEM. ∗ = p < 0.001. Bars with dashed outlines show data reported in E . HSD, honestly significant difference; mEnvy, monomeric Envy; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: The Ypk1 activators Pkh1 and Pkh2 contribute to TORC1-mediated Ste2 endocytosis. A , representative images of pkh1Δ cells expressing Ste2-mEnvy treated with (n = 186 cells) or without (n = 195 cells) 0.2 μM rapamycin for 2 h. B , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. C , representative images of pkh2Δ cells expressing Ste2-mEnvy treated with (n = 135 cells) or without (n = 162 cells) 0.2 μM rapamycin for 2 h. D , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. All error bars represent ±SEM. ∗ = p < 0.01, ∗∗ = p < 0.001. All bars with dashed outlines show data reported in E . Scale bars represent 4 μm. mEnvy, monomeric Envy; HSD, honestly significant difference.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: TORC2 activity directs TORC1-mediated Ste2 endocytosis. A , representative images of cells expressing Ste2-mEnvy and avo3 T1273 treated with (n = 233 cells) and without (n = 139 cells) 0.2 μM rapamycin for 2 h. B , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. C , representative images of cells expressing Ste2-mEnvy and avo3 T1273 grown in SCD media (+N) (n = 141 cells) or low nitrogen SCD media (-N) (n = 109 cells) for 6 h. D , quantification of Ste2-mEnvy mean fluorescent intensities at the plasma membrane. Measurements were normalized to the mean of the untreated groups to produce percentages. One-way ANOVA paired with Tukey’s HSD test was used to compare means between groups. All error bars represent ±SEM. ∗ = p < 0.05, ∗∗ = p < 0.001. All bars with dashed outlines show data reported in E . Scale bars represent 4 μm. HSD, honestly significant difference; mEnvy, monomeric Envy; SCD, Synthetic Complete plus Dextrose; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Activity Assay, Expressing, Clinical Proteomics, Membrane

Journal: The Journal of Biological Chemistry

Article Title: TOR signaling regulates GPCR levels on the plasma membrane and suppresses the Saccharomyces cerevisiae mating pathway

doi: 10.1016/j.jbc.2025.110700

Figure Lengend Snippet: Proposed mechanism of TORC1-directed endocytosis. In nutrient-deprived conditions or during rapamycin-mediated inhibition, TORC1 is repressed, leading, through an unknown pathway, to the TORC2-directed activation of Ypk1. Active Ypk1 facilitates α-arrestin–directed CME of Ste2, which ultimately traffics to the vacuole. During the pheromone response, TORC2 is also required for endocytosis, after which Atg8 promotes trafficking of the receptor to the lumen of the vacuole. CME, clathrin-mediated endocytosis; mEnvy, monomeric Envy; TORC, target of rapamycin complex.

Article Snippet: To repress TORC1 activity with rapamycin (0215934691, MP Biomedicals), cells were treated with 0.2 μM rapamycin suspended in ethanol for 2 h prior to imaging.

Techniques: Inhibition, Activation Assay