MEASUREMENT OF CYTOSOLIC, MITOCHONDRIAL, AND GOLGI PH IN SINGLE LIVING CELLS WITH GREEN FLUORESCENT PROTEINS

Many cellular events depend on a tightly compartmentalized distributio n of H+ ions across membrane-bound organelles. However, measurements o f organelle pH in living cells have been scarce, Several mutants of th e Aequorea victoria green fluorescent protein (GFP) displayed a pH-dep endent absorbance and fluorescent emission, with apparent pKa values r anging from 6.15 (mutations F64L/S65T/H231L) and 6.4 K26R/F64L/S65T/Y6 6W/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 mitoc hondrial matrix by using the targeting signal from subunit IV of cytoc hrome c oxidase, Cells in culture transfected with these cDNAs display ed the expected subcellular localization by light and electron microsc opy and reported local pH that was calibrated in situ with ionophores. We monitored cytosolic and nuclear pH of HeLa cells, and mitochondria l matrix pH in HeLa cells and in rat neonatal cardiomyocytes. The pH o f the medial/trans-Golgi was measured at steady-state (calibrated to b e 6.58 in HeLa cells) and after various manipulations. These demonstra ted 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 previou sly inaccessible.