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

Department of Biochemistry and Applied Molecular Biology, University of Manchester Institute of Science and Technology, PO Box 88, Manchester M60 1QD, UK

^* Author for correspondence: A. A. Eddy. Tel: +44 161 200 4202. Fax: +44 161 236 0409.

ABSTRACT

The magnitude of the proton gradient (µ_H⁺) 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 (). In the present work, the size of the cytosine gradient (µ_H⁺) that the yeast generated was used as a measure of the driving gradient (µ_H⁺). The selected yeast lacked cytosine deaminase and overexpressed cytosine permease, a 1 H⁺/cytosine system. µ_cyt, assayed in washed cell suspensions fermenting glucose and containing 0.5 or 50 mM KCI, was about 260 mV at pH4 or 5, falling to about 194 mV at pH 7. As a first estimate, -µ_H⁺ was thus at least as large at the respective pH value. A 20 mM solution of the lipophilic cation tetraphenylphosphonium lowered µ_cyt to a value roughly equal to the magnitude of the pH gradient (pH). A mathematical model was used to correct the first estimates of µ_H^- for the effect of cytosine leakage outside the symport. In such a system, µ_cyt cannot exceed the equivalent ratio V_maxK_mL where V_max and K_m are kinetic parameters of the symport and L is the rate coefficient for leakage. The feasibility of assaying µ_H⁺ 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, -µ_H⁺ during fermentation was estimated to be up to 25 mV larger than µ_cyt and at least 70 mV larger than previous estimates in the literature involving lipophilic cations. From a knowledge of pH, was in turn deduced and compared with the maximum methylamine gradient (µ_M) the yeast formed. The results supported the claim in the literature that, at acid pH, µ_M is a measure of PS.

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