It has been reported that the addition of glucose, uncouplers and nystatin to yeast cells grown in a sugarfree medium causes trehalase activation; it has been postulated that this activation might be mediated by the depolarization of the plasma membrane. In this article the values of membrane potential and pH gradient across the plasma membrane of Saccharomyces cerevisiae have been determined under the same conditions as those in which trehalase is activated. Membrane potential was evaluated from the distribution of triphenylmethylphosphonium, the pH gradient from the distribution of benzoic acid across the plasma membrane. When the effect of several agents on the two components of the electrochemical proton gradient across the plasma membrane of ethanol-grown yeast cells were studied, under trehalase activation conditions, the following observations were made. (a) The addition of glucose activated trehalase and caused internal acidification of the cells, but had practically no effect on the membrane potential. (b) The addition of 200 mM KCI depolarized the cell membrane but did not affect the internal pH, nor trehalase activity. (c) Although carbonyl cyanide mchlorophenylhydrazone depolarized the cells at external pH 6.0 and 7.0, it only activated trehalase at an external pH 6.0, leading to the acidification of the internal medium at this pH. (d) Nystatin caused an increase in the triphenylmethylphosphonium accumulation at external pH 6.0 and 7.0, but only activated trehalase at external pH 6.0, causing acidification of the cell interior at this pH. (e) Activation of trehalase was also observed when the internal acidification was caused by addition of a weak acid such as acetate. It is concluded that trehalase activation is mediated by an intracellular acidification and is independent of the membrane potential.It has been established by several authors that glucose, proton conductors and nystatin cause trehalose breakdown [l], gluconeogenesis inhibition [2, 31 and glycolysis stimulation [4] in Succharomyces cerevisiae. The regulatory effects of these agents is believed to be mediated by an increase in the levels of intracellular CAMP which leads to an activation of the CAMP-dependent protein kinase. This enzyme causes the subsequent activation of trehalase and phosphofructo-2-kinase and the partial inactivation of fructose-l,6-bisphosphatase among other enzymes [l -41. It has been postulated that the increase in internal CAMP is caused by the depolarization of the plasma membrane, since glucose, uncouplers and nystatin produce rapid depolarization in Neurospora crama [5, 61. Although these agents elicited an increase of the CAMP levels in several yeast strains [2 -51, proof of a direct relationship between changes in membrane potential ( A y ) and fluctuations in CAMP concentrations in S. cerevisiue is still lacking. Proton conductors and nystatin [7] are known to cause an influx of protons which could affect both parameters of the electrochemical proton gradient. Therefore, the increase in internal CAMP leve...

In glucose-grown cells of Saccharomyces cerevisiae, collected during the stationary phase of growth, the addition of K+ to the external medium reversed glucose-induced internal acidification in 2 min. However, in ethanol-grown cells external K+ did not reverse the effect of glucose even after 20 min. The presence or absence of external K+ did not alter the modification of trehalase and fructose-1,6-bisphosphatase induced by glucose. It is concluded that transient acidification may be sufficient to cause the associated transient increase in cAMP.

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