Cytosine Accumulation as a Measure of the Proton Electrochemical Gradient Acting on the Overexpressed Cytosine Permease of Saccharomyces Cerevisiae

Abstract

The magnitude of the proton gradient (Δμ̄H+ ) driving solute accumulation in Saccharomyces Cerevisiae has long been in doubt, principally because of the lack of an agreed method for assaying its electrical component, the membrane potential (Δψ). In the present work, the size of the cytosine gradient (Δμ̄cyt) that the yeast generated was used as a measure of the driving gradient (Δμ̄H+ ). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H+Zcytosine system, Δμ̄cyt, assayed in washed cell suspensions fermenting glucose and containing 0·5 or 50 mM KCI, was about 260 mV at pH 4 or 5, falling to about 194 mV at pH 7. As a first estimate, –Δμ̄H+ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered Δμ̄cyt to a value roughly equal to the magnitude of the pH gradient (ΔpH). A mathematical model was used to correct the first estimates of Δμ̄H+ for the effect of cytosine leakage outside the symport. In such a system, Δμ̄cyt cannot exceed the equivalent ratio V max/K m L, where V max and K m are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying Δμ̄H+ depends on it not being much larger than that ratio. The model was tested successfully against observations made with yeast preparations depleted of ATP. After correction, –Δμ̄H+ during fermentation was estimated to be up to 25 mV larger than Δμ̄cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of ΔpH, Δψ was in turn deduced and compared with the maximum methylamine gradient (Δμ̄M) the yeast formed. The results supported the claim in the literature that, at acid pH, Δμ̄M is a measure of Δψ.