V-ATPase and cytosolic pH are regulated by glucose. (A) Schematic representation of V-ATPase structure and its regulation by glucose. Subunits (a: Vph1p; B: Vma2p, C: Vma5p) used to follow V-ATPase assembly are indicated. (B) Regulation of V-ATPase requires glucose metabolism. Cells were starved for 15 min and imaged 5 min after addition of the indicated carbon source. 2-DOG, 2-deoxy-glucose. Localization of Vma5p (Vma5p-RFP, red) and Vph1p (Vph1p-GFP, green) and merged images are shown. (C) Time-lapse analysis of V-ATPase assembly during glucose starvation/readdition in a microfluidic chamber. V-ATPase assembly is quantified as the coefficient of variation of the intensity of the total cellular Vma5p-RFP signal and plotted as the mean±s.e.m. together with glucose concentration as a function of time (see Materials and methods and Supplementary data for details). (D–F) Cytosolic pH is regulated by glucose, but independent of V-ATPase activity. (D) Cytosolic pH was determined in cells expressing pHluorin grown in SD medium, 10 min after glucose starvation and 10 min after readdition of glucose to starved cells. Data are represented as mean±σ of at least three independent experiments. (E, F) Time-lapse analysis of cytosolic pH during glucose starvation/readdition. The mean±s.e.m. of cytosolic pH and glucose concentration in wild type (E) and vma5Δ (F) expressing pHluorin are plotted as a function of time.

Regulation of cytosolic pH by glucose metabolism. (A) Important metabolic pathways for glucose and galactose. Genes encoding for enzymes important in this study are indicated. (B) Influence of pyruvate kinase (CDC19) on cytosolic pH. Logarithmically grown cells (25°C) were processed for SNARF-4 staining at 37°C. pH was determined 10 min after glucose addition at 37°C. The average±σ of four independent experiments is shown. Asterisks (^*) indicate P-values obtained from a t-test of P<0.05. (C) V-ATPase assembly and cytosolic pH is reduced in pgm2Δ cells on galactose (gal), but not glucose (glc). Cells were grown in YPD or YPGal medium and processed for SNARF-4 staining. Cells were resuspended in SD or SGal and pH was determined after 10 min. Data from four independent experiments are represented as in (B) and asterisks (^*) indicate P-values obtained from a t-test of P<0.05. (D, E) Excess 2-DOG leads to reduction of cytosolic pH in glucose-grown cells. Time-lapse analysis of cells expressing Vma5p-RFP and pHluorin grown in 0.2% glucose that were subjected to an excess of 2-DOG (2%). (D) cytosolic pH and (E) V-ATPase assembly were scored as in Figure 1C and E.

V-ATPase is required for activation of the Ras/PKA pathway. (A) V-ATPase is required for Msn2p regulation by glucose. Cells expressing Msn2p-GFP were scored for Msn2p localization on glucose withdrawal in a microfluidic chamber. Nuclear accumulation was measured as the coefficient of variation of a region containing the 500 brightest pixels of each cell and plotted as the mean±s.e.m. together with glucose concentration as a function of time. Asterisk denotes P<0.001 derived from a t-test for the indicated time points. (B) V-ATPase is required for efficient cAMP accumulation. cAMP levels in derepressed cells of the indicated genotypes was assayed over time after glucose addition. A representative result of three independent experiments is shown. (C) Ras2p-GTP loading is regulated by V-ATPase. Cells were grown under derepressed conditions, starved and stimulated with glucose at time 0 and the amount of Ras2p-GTP was determined by affinity purification using GST-RBD. The relative amount of Ras2p in the pull down and the input was determined by densitometry. (D) V-ATPase and Gpa2p cooperate in the activation of PKA. Cells of the indicated genotype expressing Msn2p-GFP were starved in a microfluidic chamber for 20 min and Msn2p-GFP localization was followed as in panel A over time after addition of glucose. (E) Disassembly of V-ATPase is required for timely nuclear accumulation of Msn2p-NLS-GFP. Cells expressing Msn2p-NLS-GFP were scored as in panel A for Msn2p-NLS localization on glucose withdrawal in a microfluidic chamber.

Cytosolic pH may act as a second messenger for glucose. Glucose addition to starved cells activates glucose metabolism (glycolysis) and triggers an increase of cytosolic pH, possibly through increasing ATP levels. Increasing pH promotes V-ATPase assembly. Activation of V-ATPase is required for full activation of PKA on glucose stimulation, thereby transducing, at least in part, the pH signal to PKA. In addition, other mechanisms might exist that link cytosolic pH and PKA activation.

V-ATPase is regulated by pH in vivo. (A, B) V-ATPase disassembles on intracellular acidification. Logarithmically grown cells were resuspended in fresh SD adjusted to pH 4.6 or 7.4. V-ATPase assembly was scored 2 min after addition of 2,4-DNP (2 mM) in cells expressing Vma5p-GFP and representative images are shown. Cells expressing Vph1p-GFP serve as control. (B) Cells expressing Vma5p-GFP were resuspended in fresh SD medium (pH 4.6) and scored for V-ATPase assembly as in Figure 1C 2 min after addition of 2,4-DNP (2 mM) or 15 min after addition of Antimycin A (1 μg/ml). Data are represented as mean±s.e.m. (C, D) V-ATPase disassembly is defective on starvation at high pH. Time-lapse analysis of V-ATPase assembly (C) and cytosolic pH (D) of cells expressing Vma5p-RFP and pHluorin on starvation with medium adjusted to pH 4.6 or 7.4. Data are represented as in Figure 1C and E. (E, F) Influence of the V-ATPase subunit ‘a' on V-ATPase regulation by glucose. (E) Representative images of cells after 15 min starvation. (F) Time-lapse analysis of V-ATPase assembly of cells expressing wild type or mutated Vph1p. Data are represented as described in Figure 1C.

Regulation of glucose signalling by glucose metabolism. (A) Reduction of cytosolic pH triggers nuclear accumulation of Msn2p-GFP. Cells expressing Msn2p-GFP were incubated in 0.2% glucose with or without 2-DOG for 5 min. Localization of Msn2p-GFP is shown. (B) Cells expressing Msn2p-NLS-GFP were treated as in panel A, and scored for Msn2p-NLS-GFP localization. (C) PGM2 is required for cytoplasmic localization of Msn2p-NLS-GFP on galactose, but not glucose. Cells were grown in medium containing 2% glucose or 2% galactose and scored for Msn2p-NLS-GFP localization. (D) Cells of the indicated genotypes were grown in SD (glc) or SGal (gal) medium and stimulated with 20 mM cAMP for 30 min. Msn2p phosphorylation was determined by western blot using a phospho-specific antibody against Msn2p-S582. Actin serves as a loading control.

V-ATPase is required for insulin secretion and PKA activation in the pancreatic β-cell line Min6. (A) V-ATPase activity is regulated by glucose in Min6 cells. Min6 cells were incubated with low glucose (2.5 mM) medium for 4 h before incubation with KRBH buffer (2.5 mM glucose) for 30 min in the absence or presence of Concanamycin A. Cells were incubated with KRBH containing DAMP, glucose and drug as indicated for 30 min before cells were fixed and DAMP was visualized by immunofluorescence. For control, Min6 cells were grown in high glucose (25 mM) media and incubated with DAMP for 30 min before fixation. (B) V-ATPase inhibition decreases glucose-stimulated insulin secretion. Supernatants of cells in (A) were collected immediately before fixation and assayed for insulin content. A representative result of four independent experiments is shown. Error bars indicating s.d. (σ) and P-value obtained from a t-test are shown. (C, D) V-ATPase contributes to the activation of PKA in Min6 cells. (C) Cells were grown as in (A) and samples were taken for preparation of total lysates 10 min after glucose stimulation and blotted for phospho-S122 CREB1 and phospho-ERK1/2. Equal amounts of extract were blotted for CREB1 and ERK1/2 for control. (D) Cells were incubated with serum-free medium in the presence of the phosphodieasterase inhibitor IBMX and treated with Concanamycin A or drug vehicle for 30 min before determination of cAMP. A representative result of three independent experiments is presented as in (B).