Cytosolic pH Regulates Cell Growth through Distinct GTPases, Arf1 and Gtr1, to Promote Ras/PKA and TORC1 Activity  Reinhard Dechant, Shady Saad, Alfredo J. Ibáñez, Matthias Peter  Molecular Cell  Volume 55, Issue 3, Pages 409-421 (August 2014) DOI: 10.1016/j.molcel.2014.06.002 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Cytosolic pH Tightly Correlates with Growth Rate with Different C-Sources (A and B) Correlation of growth rate and cytosolic pH with glucose concentration. Cells expressing pHluorin were grown in YNB media with 0.1% Gln and glucose as indicated, and growth rate (A) and cytosolic pH (B) were determined. Data were fitted to Michaelis Menten kinetics. Optimal fit (red line) and 95% confidence intervals (gray line) are indicated. (C) Correlation of growth rate and cytosolic pH in media containing alternative carbon sources or amino acids. Cells as in (A) were grown in YNB media with 1% C-source and 0.1% Gln, or with 1% glucose and 0.1% amino acids. Cytosolic pH is plotted as a function of growth rate. Dashed lines represent a linear fit of the data to better indicate proposed correlations. (D) Regulation of cell growth by 2-DOG. Cells as in (A) were grown with 0.2% glucose and growth was monitored by following OD600 over time with or without addition of 2-DOG (2%) at the indicated time. (E) Schematic representation of metabolic signals that might promote cell growth in response to C and N-sources. (F) Modulation of growth rate by extracellular pH. Cells as in (A) were grown in media containing 1% of C-source and 0.1% Gln adjusted to pH of 4.6 and 7.0, respectively, and growth rates were determined. Error bars represent SEM. See also Figure S1. Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 High Cytosolic pH Promotes Growth and Cell Cycle Progression (A) Measurement of net-proton export from cells. Cells were grown in YNB-based medium with 0.1% Gln and 1% C-source. See Experimental Procedures for details. (B) Cells harboring a doxycycline (dox) repressible allele of PMA1 (tetO7-PMA1) were grown in SD media, treated for 8 hr with (+) or without (−) dox and net-proton export was determined. (C) Time-lapse analysis of tetO7-PMA1 cells. Time after addition of dox is indicated for each frame and a mother cell (m) and daughter cells (arrowheads) are marked. (D) Measurements of cytosolic pH of mother cells and daughter cells. tetO7-PMA1 cells expressing pHluorin were followed for several divisions in the presence of dox and pH was determined in mothers and daughters at the time of the appearance of the following bud to assure completion of cytokinesis. (E) tetO7-PMA1 cells were grown in medium adjusted to the indicated pH and containing dox. Growth was followed by measurement of OD600 over time; dox was used at 10 μg/ml. Error bars represent SEM. See also Figure S2. Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 High Cytosolic pH Promotes Growth through Activation of Ras (A and B) Regulation of Ras activity by glucose metabolism. (A) Cells expressing RBD-GFP were grown in SD media and analyzed for localization of RBD-GFP before treatment, 15 min after glucose starvation, and 15 min after glucose readdition. (B) Cells as in (A) were grown in media containing 0.2% glucose and analyzed for localization of RBD-GFP before and 15 min after addition of 2-DOG (2%). (C) Regulation of Ras activity by V-ATPase. Wild-type (WT) and vma2Δ cells expressing RBD-GFP were grown in SD media and analyzed for RBD-GFP localization. (D) Time-lapse analysis of tetO7-PMA1 cells expressing RBD-GFP after addition of doxycycline (dox). A mother cell (m) generating multiple daughter cells is shown. At the indicated time, medium supply is switched to medium adjusted to pH 7.0. Two daughter cells (1 and 2) that were born early during the time course and resume growth after switching the media are indicated. (E and F) Influence of Ras activity on cell growth upon inactivation of Pma1. tetO7-PMA1 cells expressing the dominant active Ras2-V19 allele or a control plasmid were grown with or without dox. (E) Growth was determined by measuring OD600 over time and (F) the mean cell size was determined 10 hr after addition of dox, which was used at 10 μg/ml. Error bars represent SEM. See also Figure S3. Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 V-ATPase Activates Ras through Its Interaction with the GTPase Arf1 (A and B) Arf1 localization is regulated by glucose metabolism. (A) Cells expressing Arf1-GFP were grown in SD media and analyzed for localization of Arf1-GFP before treatment, 15 min after glucose starvation, and 15 min after glucose readdition. (B) Cells as in (A) were grown in media containing 0.2% glucose and analyzed for localization of Arf1-GFP before and 15 min after addition of 2-DOG (2%). (C) Regulation of Arf1 by V-ATPase and glucose availability. Wild-type (WT) and vma2Δ cells expressing Arf1-GFP were grown in SD medium, loaded in a microfluidic chip and followed during glucose starvation and readdition (red line). (D) Regulation of V-ATPase assembly by glucose. WT and arf1Δ cells expressing Vma5-GFP were grown in SD media, loaded into a microfluidic chip, and followed during glucose starvation and readdition (red line). (E and F) Arf1 and V-ATPase are required for Ras activity. Cells expressing RBD-GFP were grown in SD media and followed during glucose starvation and readdition (red line) in a microfluidic chip. (G) Regulation of Ras activity by Arf1. Cells expressing RBD-GFP and dominant active alleles of Arf1 (Arf1-GTP) or a control plasmid were grown in SD media and scored for localization of RBD-GFP. (H) Arf1 interacts with V-ATPase component Stv1. Cells expressing Arf1-GST were grown in SC media with 2% galactose. TAP-tagged proteins were purified using IgG beads and the amount of coimmunoprecipitated Arf1-GST was analyzed with western blotting. (I) Schematic representation of the regulation of Ras activity by cytosolic pH and V-ATPase. Error bars represent SEM. See also Figure S4. Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Glucose Regulates TORC1 Activity toward Sch9 through Cytosolic pH (A and B) Activation of Sch9 is regulated by glucose metabolism and Gtr1. (A) Cells expressing Sch9-HA were grown in SD media and analyzed for Sch9 phosphorylation upon glucose starvation and glucose readdition at the indicated time points. Relative phosphorylation levels of Sch9 are indicated. Shown is the mean of three independent experiments, SEM generally < 10%. (B) Wild-type cells as in (A) were grown in SD media containing 0.2% glucose and analyzed for Sch9-phosphorylation before and 15 min after addition of 2-DOG (2%). (C) Activation of Sch9 is regulated by cytosolic pH through Gtr1. tetO7-PMA1; gtr1Δ cells expressing wild-type Gtr1 or Gtr1-Q65L (GTR1-GTP) and Sch9-HA were grown in SD media with or without doxycycline (dox) for 10 hr and analyzed for Sch9 phosphorylation. (D) Cells as in (C) were grown with or without dox and growth was recorded by monitoring OD600 over time. Error bars represent SEM. (E and F) Hyperactivation of Gtr1 alleviates growth defects on galactose media. (E) gtr1Δ cells expressing wild-type Gtr1 or Gtr1-Q65L (GTR1-GTP) were grown in synthetic media containing glucose or galactose and the growth rate was determined by measurement of OD600 over time. (F) Wild-type and npr2Δ cells were grown as in (E) and growth rates were determined. Asterisks indicate a p < 0.02 based on a Student’s t test. “n.s.” not significantly different (p > 0.1); dox was used at 10 μg/ml. See also Figures S5 and S6. Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Glucose Regulation of TORC1 Is Mediated by V-ATPase and the GTPase Gtr1 (A) V-ATPase is required for TORC1 activation. Wild-type (WT) and vma2Δ cells were grown in SD media adjusted to the indicated pH, and Sch9 phosphorylation was analyzed. Relative phosphorylation levels of Sch9 are indicated as in Figure 5A. (B) Gtr1 interacts with V-ATPase in vivo. Cells expressing GST-Gtr1 were grown in SC media containing 2% galactose. TAP-tagged proteins were purified using IgG beads and the amount of coimmunoprecipitated Gtr1-GST was analyzed with western blotting. (C) Gtr1 is not required for V-ATPase assembly. WT and gtr1Δ cells expressing Vma5-GFP were grown in SD media, loaded into a microfluidic chip, and followed during glucose starvation and readdition. Error bars represent SEM. (D) Activation of Gtr1 rescues growth defects of cells lacking V-ATPase activity. Cells expressing Gtr1 or Gtr1-Q65L (GTR1-GTP) were grown in SD media and growth rates were determined. Asterisks indicate p < 0.02 based on a Student’s t test. (E) Schematic representation of the regulation of TORC1 activity by cytosolic pH and V-ATPase. See also Figures S5 and S7. Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 TORC1 Retains Activity toward Some Targets upon Glucose Starvation (A) Regulation of Rtg1 and Gat1 localization by TORC1 and glucose. Cells expressing Rtg1-GFP or Gat1-GFP were grown in SD medium, treated with Rapamycin, starved for glucose, or starved for glucose in the presence of Rapamycin. Localization of GFP fluorescence was analyzed 15 min after treatment. (B–D) Regulation of bulk autophagy by TORC1 and glucose. (B and C) Cells expressing GFP-Atg8 were grown, loaded into a microfluidic chip, and analyzed for GFP-Atg8 localization while treated as in (A). Representative images taken 1 hr after treatment (B) and quantification of GFP-Atg8 accumulation (C) is shown. Error bars represent SEM (C). atg1Δ cells expressing GFP-Atg8 serve as specificity control for the readout. (D) Cells expressing Pgk1-GFP were grown and treated as in (A). One hour after treatment, autophagy was assayed by appearance of a free GFP as a result of vacuolar degradation of Pgk1-GFP. (E) Model depicting the hypothesized glucose sensitive and glucose-insensitive TORC1 complexes with differential regulation and specificity. See Discussion for details. See also Figure S7. Molecular Cell 2014 55, 409-421DOI: (10.1016/j.molcel.2014.06.002) Copyright © 2014 Elsevier Inc. Terms and Conditions