Membrane potentials in respiring and respiration-deficient yeasts monitored by a fluorescent dye

Abstract

Changes in fluorescence of 3,3′-dipropylthiodicarbocyanine iodide which had been equilibrated with suspensions of the wild-type yeast Saccharomyces cerevisiae and of respiration-deficient mutants were followed. The changes have been attributed to changes of yeast membrane potentials, since the fluorescence with wild-type yeast could be affected in a predictable manner by uncouplers and the pore-forming agent nystatin. As in other systems, a rise of steady-state fluorescence was ascribed to depolarization and a drop of the fluorescence to hyperpolarization. (1) A considerable rise in steady-state fluorescence was brought about by addition of antimycin A or some other mitochondrial inhibitors to respiring cells. A major part of the composite membrane potential monitored in intact yeast cells appeared to be represented by the membrane potential of mitochondria. (2) Addition of D-glucose and of other substrates of hexokinase, including non-metabolizable 2-deoxy-D-glucose, induced a two-phase response of fluorescence, indicating transient depolarization followed by repolarization. Such a response was not elicited by other sugars which had been reported to be transported into the cells by a glucose carrier or by D-galactose in galactose-adapted cells. The depolarization was explained by electrogenic ATP exit from mitochondria to replenish the ATP consumed in the hexokinase reaction and the repolarization by subsequent activation of respiration. (3) In non-respiring cells only a drop in fluorescence was induced by glucose and this was ascribed to an ATP-dependent polarization of the plasma membrane. (4) Steady-state fluorescence in suspensions of respiration-deficient mutants, lacking cytochrome a, cytochrome b, or both, was high and remained unaffected by uncouplers and nystatin. This indicates that membranes of the mutants may have been entirely depolarized. A partial polarization, apparently restricted to the plasma membrane, could be achieved by glucose addition.