Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins

Abstract

Many cellular events depend on a tightly compartmentalized distribution of H+ ions across membrane-bound organelles. However, measurements of organelle pH in living cells have been scarce. Several mutants of the Aequorea victoria green fluorescent protein (GFP) displayed a pH-dependent absorbance and fluorescent emission, with apparent pKa values ranging from 6.15 (mutations F64L/S65T/H231L) and 6.4 (K26R/F64L/S65T/Y66W/N146I/M153T/ V163A/N164H/H231L) to a remarkable 7.1 (S65G/S72A/T203Y/H231L). We have targeted these GFPs to the cytosol plus nucleus, the medial/trans-Golgi by fusion with galactosyltransferase, and the mitochondrial matrix by using the targeting signal from subunit IV of cytochrome c oxidase. Cells in culture transfected with these cDNAs displayed the expected subcellular localization by light and electron microscopy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondrial matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH of the medial/trans-Golgi was measured at steady-state (calibrated to be 6.58 in HeLa cells) and after various manipulations. These demonstrated that the Golgi membrane in intact cells is relatively permeable to H+, and that Cl- serves as a counter-ion for H+ transport and likely helps to maintain electroneutrality. The amenability to engineer GFPs to specific subcellular locations or tissue targets using gene fusion and transfer techniques should allow us to examine pH at sites previously inaccessible.

Figure 1

(a) pH-dependent absorbance of EYFP. (b) pH dependency of fluorescence of various GFP mutants in vitro and in cells. The fluorescence intensity of purified recombinant GFP mutant protein (solid symbols) as a function of pH was measured in a microplate fluorometer as indicated in Materials and Methods. The fluorescence of the Golgi region of HeLa cells expressing either GT-EYFP or GT-EGFP (open symbols) was determined during pH titration with the ionophores monensin/nigericin in high KCl solutions.

Figure 2

Constructs for compartment-specific expression in mammalian cells. Kz, Kozak consensus sequence; coxIV, cytochrome c oxidase subunit IV targeting signal.

Figure 3

(a) Fluorescence image of living HeLa cells transfected with EYFP shows cytosolic plus nuclear localization. (Bar = 10 μm in all fluorescence images.) (b) pH measurements in cytosol and nucleus of HeLa cells (mean ± SE of n cells). Perfusion of permeant base and acid changes cytosolic pH (n = 7). (c) Confocal microscopy image of living HeLa cells transfected with EYFP-mito shows mitochondrial staining. (d) Effect of glucose and lactate/pyruvate perfusion on mitochondrial pH (n = 3). (e) The uncoupler CCCP collapses mitochondrial pH (n = 3).

Figure 4

Fluorescence and electron microscopy of Golgi-targeted EYFP. (a and b) Double-staining fluorescence image of fixed HeLa cells transfected with GT-EYFP shows the overlap of the endogenous fluorescence of EYFP (a) and the fluorescence of the Golgi marker α-manII [detected with Texas red goat anti-rabbit F(ab′)₂ conjugate]. (c) Immunogold localization of GT-EYFP (polyclonal antibodies to GFP) to the Golgi apparatus in ultra-thin cryosections of transfected HeLa cells. Note that GT-EYFP is distributed broadly across the Golgi stack, but is more concentrated toward the trans side. (d) Double-immunogold localization showing overlap between GT-EYFP (10 nm gold) and the trans-Golgi network protein TGN38 (arrows, 5 nm gold).

Figure 5

pH measurements in the medial/trans-Golgi of HeLa cells expressing GT-EYFP (mean ± SE of n cells). (a) Inhibition of V-type H⁺ ATPase by bafilomycin A1 (0.2 μM) raises pH_G (n = 3). Calibration of fluorescence as pH was performed with nigericin/monensin in high K⁺ buffers. (b) The protonophore CCCP increases the endogenous H⁺ leakage of Golgi membranes raising pH_G (n = 7). One control cell with diffuse cellular staining was selected in the same field. (c) Cl⁻ is a counter-ion for H⁺ transport in the Golgi. Effect of substitution of gluconate medium (Cl⁻-free) for Hanks’ buffered saline (n = 4). (d) Cytosolic pH decreases slightly by Cl⁻ removal in control HeLa cells loaded with the pH indicator carboxy-SNARF (n = 13). (e) Increasing K⁺ conductance with the ionophore valinomycin does not change steady-state Golgi pH (n = 10). (f) Effect of Ca²⁺ mobilization by agonists and ionomycin on pH_G (n = 3).

Figure 6

Ratiometric measurement of pH_G by cotransfecting HeLa cells with GT-ECFP and GT-EYFP. (a) Single wavelength fluorescence intensities of GT-EYFP and GT-ECFP in the Golgi region of a HeLa cell. (b) Ratio of GT-EYFP/GT-ECFP fluorescence emission of the same cell.