Transport of an anionic substrate by the H⁺/monosaccharide symport inRhodotorula gracilis: Only the protonated form of the carrier is catalytically active

Summary

The yeastRhodotorula gracilis accumulated glucuronate by an H⁺/symport. The transport was electroneutral, driven by the chemical gradient of protons Δ pH. The observed stoichiometry amounted to 1 proton per molecule glucuronate. At pH 4, the half-saturation constantK _T was at its lowest value (K _T =8mm), whereas the maximal velocityV _T reached a maximum (V _T =15 nmol/min×mg dry wt). Monosaccharides competitively inhibited the uptake of glucuronate and vice versa. Hence, the two substrates share the same transport system. The steady-state accumulation of glucuronate reflected the course of the pH gradient. It is concluded that glucuronate is transported as an anionic substrate by the protonated carrier, the driving force being the chemical gradient of the H⁺ (ΔpH). The ternary carrier/H⁺/glc-COOO-complex is electroneutral and independent of the membrane potential. Simultaneous uptake of organic acids (acetic or propionic acid) which is also energized by the pH gradient led to a noncompetitive inhibition of glucuronate transport. Thus, manipulation of the driving force, ΔpH, reducedV _T without affectingK _T . Kinetic and energetic arguments are presented which stronly suggest that only the protonated carrier is catalytically active inR. gracilis.