Active transport of charged substrates by a proton/sugar co-transport system. Amino-sugar uptake in the yeast Rhodotorula gracilis

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Abstract
1. In the yeast Rhodotorula gracilis several amino sugars were actively transported. Glucosamine, which is largely protonated at physiological pH (pK 7.75) was used as a model substrate. At pH 6.75 its half-saturation constant was 1 mM and the maximal velocity was 50 nmol/min per mg dry wt. 2. Amino sugars were taken up via the monosaccharide carrier. The transport of glucosamine was strongly restricted by monosaccharides. D-Xylose inhibited competitively the uptake of glucosamine. The inhibition constant was 1 mM. Cells preloaded with D-xylose showed exchange transport on subsequent addition of glucosamine. 3. Transport of glucosamine was energized by the membrane potential. Uncoupling agents such as carbonyl cyanide m-chlorophenyl-hydrazone and the lipophilic cation TPP+ (tetraphenylphosphonium ion) at concentrations that depolarized the membrane potential inhibited the uptake of glucosamine. Conversely the transport of glucosamine partly dissipated the membrane potential, which was monitored by radioactively labelled lipophilic cations. 4. The translocated charges were electrically compensated by the extrusion of protons and K+ (1 glucosamine molecule/0.85 H+ + 0.15 K+). 5. An increase of the pH in the range 4.75-8.75 lead to a decrease of the half-saturation constant from 5 mM to 1 mM and to an optimum of the maximal velocity at pH 6.75. We suggest that this fair constancy is due to the carrier not distinguishing between the protonated form of glucosamine (pH less than 7.75) and the deprotonated form (pH greater than 7.75). The increase of V(T) (maximal transport velocity) between pH 4.75 and 6.75 is due to the increase of the membrane potential: the decrease between pH 6.75 and 8.75 is due to the deprotonization of the carrier.