Differential Sensitivity of the Cellular Compartments of Saccharomyces cerevisiae to Protonophoric Uncoupler under Fermentative and Respiratory Energy Supply

Abstract

The effect of a protonophoric uncoupler (CCCP) on the different cellular compartments was investigated in yeast grown aerobically on lactate. These cells were incubated in a resting cell medium under three conditions; in aerobiosis with lactate or glucose or in anaerobiosis with glucose as energetic substrate. For each condition, in vivo 31P NMR was used to measure pH gradients across vacuolar and plasma membrane and phosphorylated compound levels. Respiratory rate (aerobic conditions) and TPP+ uptake were measured independently. Concerning the polyphosphate metabolism, spontaneous NMR-detected polyphosphate breakdown occurred, in anaerobiosis and in the absence of CCCP. In contrast, in aerobiosis, polyphosphate hydrolysis was induced by addition of either CCCP or a vacuolar membrane ATPase-specific inhibitor, bafilomycin A1. Moreover, polyphosphates were totally absent in a null vacuolar ATPase activity mutant. The vacuolar polyphosphate content depended on two factors: vacuolar pH value, strictly linked to the vacuolar H+-ATPase activity, and inorganic phosphate concentration. CCCP was more efficient in dissipating the proton electrochemical gradient across vacuolar and mitochondrial membranes than across the plasma membrane. This discrepancy can be essentially explained by a difference of stimulability of each proton pump involved. As long as the energetic state (measured by NDP + NTP content) remains high, the plasma membrane proton ATPase is able to compensate the proton leak. Moreover, this ATPase contributes only partially to the generation of ΔpH. The maintenance of the ΔpH across the plasma membrane, that of the energetic state, and the cellular TPP+ uptake depend on the nature of the ATP-producing process. As expected, these parameters are more sensitive to CCCP when the ATP-producing process is only oxidative phosphorylation (lactate condition) than when in the aerobic glucose condition, thus suggesting the role of the glycolytic pathway in the resistance to this compound. However, the resistance of cells to CCCP is not effective when the ATP-producing process is only fermentation (anaerobic glucose condition), although under these conditions glycolysis is optimal. The role of mitochondrial uncoupled respiration in maintaining the cellular ATP level in the presence of glucose is discussed. © 1991, American Chemical Society. All rights reserved.