Functional reconstitution of the Trypanosoma brucei plasma-membrane D-glucose transporter.
The D-glucose transporter of Trypanosoma brucei was solubilized from the plasma membrane and reconstituted into proteoliposomes. Using the reconstitution of D-glucose transport as the assay and non-specific L-glucose uptake as control, we have purified a membrane protein fraction from T. brucei bloodstream-form ghosts by EDTA/alkali treatment and solubilization with the detergents octylglucoside or octylthioglucoside. Upon removal of the detergent by dialysis, the solubilized protein fraction was reconstituted in sonicated liposomes by a freeze/thaw-sonication step. The reconstituted transporter catalyzed specific D-glucose uptake and was compared in several characteristics with the native facilitated-diffusion transporter as present in live trypanosomes [Seyfang, A. & Duszenko, M. (1991) Eur. J. Biochem. 202, 191-196]. As in vivo, the uptake is time dependent and Na+ independent. Transporter substrate affinity and inhibitor specificity are completely retained and it is inhibited by mercuric ions, phloretin and cytochalasin B, but only partially inhibited by phlorizin. The reconstituted transporter also demonstrates trans-stimulation properties indicative of the carrier-mediated transport of D-glucose. In contrast to the human erythrocyte-type glucose transporter, in T. brucei D-fructose uptake was also catalyzed by the same reconstituted protein fraction and specific D-glucose or D-fructose transport were mutually competitive. Both the inhibitor studies and the fructose transport capacity in the reconstituted system are in good agreement with the native transport in live trypanosomes. The specific activity of D-glucose transport was 1.9 +/- 0.3 nmol.min-1.mg protein-1 at 0.2 mM D-glucose and the yield was about 0.8% of total ghost protein after removal of the variant-surface-glycoprotein coat. The successful functional reconstitution of a protozoan glucose transporter represents an important step towards its purification and detailed characterization. This is especially interesting since bloodstream-form trypanosomes depend entirely upon glycolysis for their ATP production.
Seyfang
A
A
Physiologisch-chemisches Institut, Universität Tübingen, Germany.
Duszenko
M
M
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D003571:Cytochalasin B / Q000494:pharmacology; D003902:Detergents; D003956:Dialysis; D004492:Edetic Acid; D005947:Glucose / Q000378:metabolism; D005960:Glucosides; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism*; D008628:Mercury / Q000494:pharmacology; D009004:Monosaccharide Transport Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010693:Phloretin / Q000494:pharmacology; D012995:Solubility; D013010:Sonication; D013863:Thioglucosides; D014346:Trypanosoma brucei brucei / Q000737:chemistry*
D003902:Detergents; D005960:Glucosides; D008081:Liposomes; D009004:Monosaccharide Transport Proteins; D013863:Thioglucosides; C018619:octyl-beta-D-glucoside; D003571:Cytochalasin B; C042989:n-octyl-beta-D-thioglucopyranoside; D004492:Edetic Acid; D008628:Mercury; D005947:Glucose; D010693:Phloretin