Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Summary

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Abstract

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Introduction

pH homeostasis is a dynamic and highly regulated process in all organisms ^1,2. Biochemical processes are tightly regulated by pH, and intracellular environments are tuned to narrow pH ranges to allow optimal activity of the resident enzymes. However, intracellular pH homeostasis can be challenged by rapid changes in environmental pH, metabolic shifts, and certain signaling pathways. In addition, intracellular pH can itself serve as an important signal. Finally, many organelles maintain lumenal pH values that are distinct from the surrounding cytosol and essential for organelle-specific functions.

The yeast Saccharomyces cerevisiae shares a number of pH homeostasis mechanisms with higher eukaryotes ². In the acidic organelles of the endocytic/lysosomal pathway, pH is primarily controlled by the highly conserved vacuolar proton-translocating ATPase (V-ATPase), acting in tandem with many exchangers dependent on the pH gradient. All eukaryotic cells also have proton export mechanisms. In fungi and plants, a second, distinct proton pump at the plasma membrane, Pma1, exports metabolic protons and is believed to be the major determinant of cytosolic pH and plasma membrane potential. The genetic flexibility of S. cerevisiae and its commercial importance, have made it a very interesting and important model for studying pH homeostasis ².

In addition to being the primary drivers of organelle acidification, V-ATPases are highly regulated enzymes and our lab is interested in understanding mechanisms of V-ATPase regulation. Towards this goal, we have been using in vivo pH measurements of vacuolar and cytosolic pH: 1) to monitor responses to changing extracellular conditions, such as glucose deprivation and readdition, 2) to examine the effects of mutations that compromise V-ATPase activity, and 3) to explore the coordination of organelle and plasma membrane proton pumps ^3-5. These experiments only became possible through the development of robust ratiometric pH indicators amenable to use in yeast cells. Plant et al. first showed that BCECF (2'7'-Bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein), which has been used widely to measure cytosolic pH in mammalian cells, accumulates in the yeast vacuole instead of the cytosol ⁶. This difference in BCECF localization has been attributed to the many hydrolytic enzymes in the vacuole, which are likely responsible for cleavage of the acetoxy methyl ester from BCECF-AM (acetoxymethyl ester of BCECF) and vacuolar retention ⁶. Ali et al. ⁷ further developed vacuolar pH measurement using BCECF and adapted these measurements to a fluorescence plate reader format. Brett et al. introduced yeast pHluorin as a means of measuring cytosolic pH in yeast by expressing a plasmid-borne ratiometric pH-sensitive GFP ⁸ under control of a yeast-specific promoter ⁹.

The excitation spectra of both BCECF and yeast pHluorin are sensitive to pH, so they are used as ratiometric pH indicators in which the ratio of fluorescence at two excitation wavelengths, measured at a single emission wavelength, provides a measure of pH ^8,10. These yeast vacuolar and cytosolic pH sensors have been used for both single-cell and population-based measurements. Single-cell measurements ^6,11 are performed by fluorescence microscopy and image analysis. Vacuolar or cytosolic fluorescence at the two wavelengths is measured for each cell. The population-based measurements are performed in either a microplate reader with appropriate fluorescence capabilities or in a fluorimeter. We have generally done our measurements in a fluorimeter, because it provides easy access for addition of components such as glucose during continuous kinetic measurements. Our current lab protocols for measurement of vacuolar and cytosolic pH are listed below; both are also easily adapted to microplate assays.

Protocol

1. Measurement of Vacuolar pH In Vivo Using BCECF-AM

1. Grow a 50 ml liquid culture of the yeast strain to be measured in the desired medium overnight. The goal is to have cells in mid-log phase (OD600 (optical density at 600 nm) measurement of approximately 0.8 for the suspension).
2. Pellet the yeast cells by centrifugation. Resuspend the pellet in 0.6 ml of the growth medium and transfer to a microcentrifuge tube that has been weighed previously. Pellet the cells again in a microcentrifuge at 2,000 x g for 60 sec. Remove the supernatant as completely as possible and then weigh the cell pellet. Resuspend the pellet to a final density of 0.5 g/ml (w/v); a culture of this volume will give a cell pellet of approximately 200 mg, and 200 μl of buffer would be added to give a final volume of 400 μl.
3. Add BCECF-AM to the cell suspension at a final concentration of 50 mM from a 12 mM stock prepared in DMSO. Mix well and then incubate the cells at 30 °C for 30 min. on a rocking platform or roller drum.
4. While the cells are incubating, prepare calibration buffers. The calibration buffer contains 50 mM MES (2-(N-morpholino)ethanesulfonic acid), 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 50 mM KCl, 50 mM NaCl, 0.2 M ammonium acetate, 10 mM sodium azide, and 10 mM 2-deoxyglucose, and is adjusted to the pH values appropriate for your calibration range with NaOH or HCl. (Caution: Sodium azide is highly toxic and should be handled with care.) For measurement of pH in wild-type cells, we would prepare several calibration mixtures at pH 5.5 to 6.5, but for mutants expected to have a more alkaline vacuolar pH (vma mutants), we would prepare additional, higher pH buffers.
5. Aliquot 2 ml of calibration buffer for each pH into 15 ml conical tubes, and add monensin (15 mM stock) and nigericin (2 mM stock) to give final concentrations of 110 μM monensin and 15 μM nigericin, respectively. Mix well on a vortex mixer. (Caution: Both monensin and nigericin are toxic and should be handled with care.)
6. When the BCECF-AM incubation time is completed, pellet the cell suspension by centrifugation for 30 sec at 2,000 x g in a microcentrifuge. Resuspend the cells is 1 ml of growth medium lacking glucose and centrifuge as above. Repeat this wash step once, and then resuspend the pellet in 200 μl of growth medium without glucose. Place on ice.
7. Add 20 μl of the cell suspension to each pH calibration tube prepared above. Incubate at 30 °C for 30-60 min. on a roller drum.
8. Set the fluorimeter to alternately measure at excitation wavelengths 450 nm and 490 nM, both with an emission wavelength of 535 nm. Set the sample chamber temperature to 30 °C. Perform all measurements with continuous stirring of the mixture in the cuvette.
9. Add 1.96 ml of 1 mM MES (adjusted to pH 5 or 7 depending on the pH of the cells' growth medium and the experimental design). Add 20 μl of cell suspension into the cuvette. Initiate fluorescence measurements. We collect both continuous kinetic and time-point data in different experiments. For continuous kinetic data, we take measurements every 6 sec for 5 min, then add glucose to a final glucose concentration of 50 mM, and continue measurement for 5-10 min. For single time-point measurements, we generally take measurements at 1 and 5 min. after addition of cells to the cuvette, add glucose as in the kinetic measurements, and then take another measurement 5 min. after glucose addition. These additions can be varied, and additional components (inhibitors, etc) can also be added.
10. After the experimental measurements are complete, remove the calibration tubes from the 30 °C incubation and transfer the entire 2 ml volume for each to the fluorimeter cuvette. Measure fluorescence at the same settings (see 1.8) every 5 sec over a total of 30 sec for each sample.
11. Export fluorescence data to Microsoft Excel. (For our fluorimeter, this requires export of data as text in tab-delimited form and import into Excel.) Obtain a calibration curve by calculating the ratio of fluorescence at 490 nm to 450 nm for each calibration mixture. The fluorescence ratio is then plotted vs. pH to get a calibration curve.
12. Convert the experimental data to fluorescence ratio and calculate pH using the standard curve. Vacuolar pH can then plotted against time (kinetics of pH changes) or compared under various conditions (Figure 1) or in different mutant strains.

2. Measurement of Cytosolic pH In Vivo Using Yeast pHluorin

1. Transform the desired yeast strain with the yeast pHluorin plasmid by standard protocols, and select for transformants on supplemented minimal medium lacking uracil (SC-uracil).
2. Grow a 50 ml liquid culture of transformed cells in SC-uracil to mid-log phase (OD₆₀₀ = 0.8 or lower).
3. Prepare calibration standards as described for vacuolar pH, but buffer to a pH range appropriate for cytosolic pH measurements, generally pH 6-8. Aliquot 2 ml of calibration buffer adjusted to the desired pH into several 15 ml conical tubes. Add monensin and nigericin as described above (1.4) and mix well.
4. Harvest the cells by centrifugation as described above, and resuspend the pellet in 600 μl of the growth medium. Transfer to a weighed microcentrifuge tube, and pellet cells by centrifugation at 5,000 rpm for 30 sec. Resuspend the pellet in 1 ml of growth medium without glucose (SC-uracil, -glucose), pellet cells again and repeat. After the final centrifugation, remove the supernatant as thoroughly as possible and weigh the cell pellet. Resuspend cells to a final density of 0.5 g/ml in SC-uracil with no glucose.
5. Add 20 μl of cell suspension to each tube of calibration buffer and mix well on a vortex mixer. Incubate at 30 °C on a spinning drum rotor for 60 min.
6. Set up the fluorimeter for excitation at wavelengths 405 and 485 nm and an emission wavelength of 508 nm. Proceed with single time point or kinetic measurements and addition of glucose as described above for BCECF measurement.
7. Measure fluorescence of calibration samples, and construct a calibration curve as for BCECF (see 1.10-1.11). A plot of fluorescence ratio vs. pH is linear over the range of most cytosolic pH measurements (pH 6.0-8.0) ⁹, so experimental fluorescence ratio measurements are easily converted to pH.

Results

Figure 1 presents vacuolar pH data obtained on wild-type yeast cells grown in rich medium (yeast extract-peptone-dextran; YEPD) buffered to pH 5 with 50 mM MES. We often grow the cells in buffered medium because the pH of the medium can change quite dramatically during overnight growth, particularly for minimal medium, and we have found that the pH of the growth medium can affect vacuolar pH responses ³. However, it is also acceptable for many experiments to grow the cells in unbuffered...

Discussion

We have utilized these protocols to address a number of aspects of pH homeostasis. For example, we have compared cytosolic and pH responses of wild-type and V-ATPase-deficient mutant cells ^4,5. We have also examined the effects of altered growth conditions, particularly extracellular pH, on vacuolar pH response to glucose ³. Importantly, the responses we observe are both consistent with other methods of quantitative pH measurement and with biochemical data describing altered activities of the ...

Materials

Name	Company	Catalog Number	Comments
Spectrofluorometer	Horiba Jobin Yvon	Model Fluoromax-4	Temperature control and stirring capability are desirable.
BCECF-AM	Invitrogen/Molecular Probes	B1150	Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 °C
monensin	Sigma	M5273	Toxic.
nigericin	Sigma	N7143	Toxic.
MES	Sigma	M8250