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 Saccharomy....

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 ....

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

List of materials used in this article

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