Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast

Abstract

Vacuolar proton-translocating ATPases (V-ATPases) play a central role in organelle acidification in all eukaryotic cells. To address the role of the yeast V-ATPase in vacuolar and cytosolic pH homeostasis, ratiometric pH-sensitive fluorophores specific for the vacuole or cytosol were introduced into wild-type cells and vma mutants, which lack V-ATPase subunits. Transiently glucose-deprived wild-type cells respond to glucose addition with vacuolar acidification and cytosolic alkalinization, and subsequent addition of K(+) ion increases the pH of both the vacuole and cytosol. In contrast, glucose addition results in an increase in vacuolar pH in both vma mutants and wild-type cells treated with the V-ATPase inhibitor concanamycin A. Cytosolic pH homeostasis is also significantly perturbed in the vma mutants. Even at extracellular pH 5, conditions optimal for their growth, cytosolic pH was much lower, and response to glucose was smaller in the mutants. In plasma membrane fractions from the vma mutants, activity of the plasma membrane proton pump, Pma1p, was 65-75% lower than in fractions from wild-type cells. Immunofluorescence microscopy confirmed decreased levels of plasma membrane Pma1p and increased Pma1p at the vacuole and other compartments in the mutants. Pma1p was not mislocalized in concanamycin-treated cells, but a significant reduction in cytosolic pH under all conditions was still observed. We propose that short-term, V-ATPase activity is essential for both vacuolar acidification in response to glucose metabolism and for efficient cytosolic pH homeostasis, and long-term, V-ATPases are important for stable localization of Pma1p at the plasma membrane.

FIGURE 1.

vma mutants regulate vacuolar and cytosolic pH differently than wild-type cells. Vacuolar and cytosolic pH were measured in wild-type, vma2Δ, and vma3Δ mutants. Cultures of isogenic wild-type (WT) or vma null mutants were grown in YEPD, pH 5, at 30 °C to early log phase (A₆₀₀ 0.6–0.8). For A and B, vacuolar and cytosolic pH were measured directly; for C and D, cells were harvested at A₆₀₀ 0.6 then shifted to pH 7.5 medium and grown for an additional 3–4 h. To measure vacuolar pH (A and C), cultures were loaded with BCECF-AM as described (30) (vacuolar labeling was confirmed by fluorescence microscopy). Fluorescence intensity values were collected at excitation wavelengths 450 and 490 nm and emission wavelength 535 nm, and the ratio of fluorescence signals from the two excitation wavelengths was calculated and converted to pH via a calibration curve (see “Experimental Procedures”). In the case of cytosolic pH (B and D), cells transformed with pHluorin (31) were used, and fluorescence intensity was measured at excitation wavelengths 405 and 485 nm and emission wavelength of 508 nm, then converted to cytosolic pH after calibration (see “Experimental Procedures”). Assays for both vacuolar and cytosolic pH were done by adding 25 μl of yeast suspended at 1:2 (w/v) to 2 ml of 1 mm MES buffer adjusted to at pH 5 or in MOPS buffer at pH 7.5 (depending on final growth condition) with TEA. Fluorescence responses were recorded after 5 min of incubation without any further addition (black bars), 5 min after the addition of glucose to 50 mm final (dark gray bar), and 5 min after the addition of KCl to 50 mm final (light gray bar). Samples were stirred and maintained at 30 °C during the measurements. The asterisk indicates a significant difference (p < 0.05) for the mutant sample relative to comparable wild-type sample.

FIGURE 2.

Proton pumping from vma mutant cells is lower than from wildtype cells. Proton pumping was measured by recording the extracellular pH with a selective electrode connected to a potentiometer (Beckman Selection 2000). Cells were grown to log phase in YEPD, pH 5 medium, collected by centrifugation, washed with YEP pH 5, and resuspended to a final density of 1:2 (w/v) in the same medium. 25 μl of the cell suspension was added to 15 ml of buffer (1 mm MES-TEA, pH 5) and incubated 16 min at 30 °C with stirring. pH was recorded every 30 s by hand. For experimental samples (closed symbols) glucose (final concentration 40 mm) and KCl (final concentration 40 mm) were added at 3 and 8 min of incubation, respectively. Control samples (open symbols) received no additions. Strains are shown as follows: wild-type (squares), vma2Δ (triangles), vma3Δ (circles). A representative of several experiments with very similar results is shown.

FIGURE 3.

Comparison of Pma1 protein levels in partially purified plasma membrane fractions. Immunoblots were prepared from wild-type (wt), vma2Δ, and vma3Δ cells as described under “Experimental Procedures” and probed for Pma1p, Pep12p, and alkaline phosphatase (ALP). As described under “Experimental Procedures,” equal amounts of protein from each strain were loaded to allow comparison and quantitation by NIH Image J.

FIGURE 4.

Pma1p is mislocalized to the vacuole in vma mutants lacking vacuolar protease activity. Pma1p was localized by immunofluorescence microscopy in both wild-type and vma mutants that contained a pep4-3 mutation, which inhibits most vacuolar protease activity. Nomarski images (left panels) and indirect immunofluorescence using anti-Pma1p antibody followed by Alexa Fluor 488-conjugated secondary antibody (right panels) are shown for each field. All fluorescence images were recorded with identical exposure times.

FIGURE 5.

Pma1p mislocalization occurs in vma strains containing vacuolar proteases but not in wild-type cells treated with concanamycin A. Pma1p was localized as described in Fig. 4 in wild-type (wt), vma2Δ, and vma3Δ mutant cells that are Pep4+. The wild-type cells were also treated for 30 min with 1 μm concanamycin A (wt + conA) and then visualized. All fluorescence images were recorded with identical exposure times.

FIGURE 6.

Acute inhibition of V-ATPase activity with concanamycin A affects pH homeostasis. Vacuolar (A) and cytosolic (B) pH responses were measured as described in Fig. 1 for wild-type cells grown at pH 5, labeled with BCECF in YEPD as in Fig. 1, and then incubated with 1 μm concanamycin A or an equivalent concentration of DMSO (control) for 30 min at 30 °C in YEP, pH 5. Assays for both vacuolar and cytosolic pH were done by adding 25 μl of yeast suspended at 1:2 (w/v) to 2 ml of 1 mm MES buffer adjusted to pH 5 with TEA. Fluorescence responses were recorded after 5 min of incubation without any further addition (black bars), 5 min after the addition of glucose to 50 mm final (dark gray bar), and 5 min after the addition of KCl to 50 mm final (light gray bar). Samples were stirred and maintained at 30 °C during the measurements. Concanamycin was present throughout the measurements for the concanamycin-treated cells. The asterisk indicates a significant difference (p < 0.5) for the concanamycin-treated cells relative to the untreated control under the same conditions. C, proton export from concanamycin-treated or control cells was measured as described in Fig. 2, except that wild-type cells were preincubated with 1 μm concanamycin A (circles) or DMSO (triangles) before initiating the export assay. For experimental samples (closed symbols) glucose (final concentration 40 mm) and KCl (final concentration 40 mm) were added at 3 and 8 min of incubation, respectively. Control samples (open symbols) received no additions. A representative of several experiments with very similar results is shown.

FIGURE 7.

Model for collaboration between V-ATPase and Pma1p in cytosolic pH homeostasis. Cellular positions of the V-ATPase and Pma1p and potential mechanisms driving pH responses to glucose and KCl addition are highlighted.