Measurement of Vacuolar and Cytosolic pH <em>In Vivo</em> in Yeast Cell Suspensions

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.

Homotypic Vacuole Fusion in Yeast Requires Organelle Acidification and Not the V-ATPase Membrane Domain

“…Eighty-four percent of the parental “wild type” cells had 1-3 vacuoles and the vacuolar pH measured using the ratiometric, pH sensitive dye BCECF was 5.67 (Figure 4A, B, C). This was consistent with our previous vacuole morphology results (Figure 1) and with measurements of vacuolar pH by others (pH 5.5 to 5.9) (Diakov and Kane, 2010; Diakov et al, 2013; Martinez-Munoz and Kane, 2008; Plant et al, 1999; Tarsio et al, 2011). As seen previously, vph1Δ cells displayed fragmented vacuoles and, as expected, 98% of double mutant vph1 ' ipp1 ' cells expressing IPP1 also displayed fragmented vacuoles (Figure 4A, B).…”

“…Vacuolar pH was measured using the ratiometric pH indicator dye BCECF (2’,7’-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) (Life Technologies, Grand Island, NY) as described (Diakov et al, 2013). Data were collected using a FluoroLog-3 spectrofluorometer and FluorEssence TM software (HORIBA, Edison, NJ).…”

Loss of Vacuolar H+-ATPase Activity in Organelles Signals Ubiquitination and Endocytosis of the Yeast Plasma Membrane Proton pump Pma1p

“…Measurement of Vacuolar pH-Vacuolar pH was measured using the BCECF method as described (30). Cells were grown in YEPD, pH 5, washed, and resuspended in medium without glucose before initiating the fluorescence measurements in a Horiba FluoroMax-4 spectrometer.…”

“…Fluorescence was measured before and 5 min after the addition of glucose to a final concentration of 2%. Calibration of fluorescence ratios to pH was performed as described (30).…”

ALL2 , a Homologue of ALL1 , Has a Distinct Role in Regulating pH Homeostasis in the Pathogen Cryptococcus neoformans

“…BCECF accumulates in yeast vacuoles, and excitation spectra are sensitive to pH (22). pH i measurement was performed by collecting the ratio of fluorescence at two excitation wavelengths (450 and 490 nm) and measuring at a single emission wavelength (535 nm) using protocols described elsewhere (23).…”