CYTOSINE ACCUMULATION AS A MEASURE OF THE PROTON ELECTROCHEMICAL GRADIENT ACTING ON THE OVEREXPRESSED CYTOSINE PERMEASE OF SACCHAROMYCES-CEREVISIAE

The magnitude of the proton gradient (Delta<(mu)over bar>(H)+) driving solute accumulation in Saccharomyces cerevisiae has long been in doub t, principally because of the lack of an agreed method for assaying it s electrical component, the membrane Institute of Science and potentia l (Delta psi). In the present work, the size of the cytosine gradient (Delta<(mu)over bar>(cyt)) that the yeast generated was used as a meas ure of the driving gradient (Delta<(mu)over bar>(H)+()). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1H(+)/cytosine system. Delta<(mu)over bar>(cyt), assayed in washed ce ll suspensions fermenting glucose and containing 0.5 or 50 mM KCl, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a firs t estimate, -Delta<(mu)over bar>(H)+ was thus at least as large at the respective ph value. A 20 mM solution of the lipophilic cation tetrap henylphosphonium lowered Delta<(mu)over bar>(cyt) to a value roughly e qual to the magnitude of the pH gradient (Delta pH). A mathematical mo del was used to correct the first estimates of Delta<(mu)over bar>(H) for the effect of cytosine leakage outside the symport. In such a sys tem, Delta<(mu)over bar>(cyt) cannot exceed the equivalent ratio V-max /K(m)L(I) where V-max and K-m are kinetic parameters of the symport an d L is the rate coefficient for leakage. The feasibility of assaying D elta<(mu)over bar> (H)+ depends on it not being much larger than that ratio. The model was tested successfully against observations made wit h yeast preparations depleted of ATP. After correction, -Delta<(mu)ove r bar>(H)+ during fermentation was estimated to be up to 25 mV larger than Delta<(mu)over bar>(cyt) and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowl edge of Delta pH, Delta psi was in turn deduced and compared with the maximum methylamine gradient (Delta<(mu)over bar>(M)) the yeast formed . The results supported the claim in the literature that, at acid pH, Delta<(mu)over bar>(M), is a measure of Delta psi.