Critical parameters for functional reconstitution of glucose transport in Trypanosoma brucei membrane vesicles.
The glucose transporter of Trypanosoma brucei was reconstituted by incorporating Escherichia coli phospholipid liposomes into detergent-solubilised trypanosome membranes. Proteoliposome vesicles were formed by detergent dilution and used in glucose-uptake assays. The minima for functional reconstitution of the glucose transporter were established and used to probe the mechanism of glucose transport. The uptake pattern of radiolabelled glucose showed a counterflow transient at about 3 s, after which the sugar equilibrated across the proteoliposomal membrane. This observation is consistent with a facilitated transporter. There was a six-fold increase in the initial rate of glucose uptake compared to non-reconstituted or native membranes. In addition, the transporter exhibited stereospecificity to D-glucose but poorly transported L-glucose. Directionality, stereoselectivity or substrate specificity and cis-inhibition by phloridzin were therefore the main criteria for validation of glucose transport. The observed counterflow transient also provided further evidence for a facilitated glucose transporter within the trypanosome plasma membrane, and was the single most important criterion for this assertion. A stoichiometry of 0.78 mol of glucose per mol of transporter was estimated.
Bayele
H K
HK
Department of Biochemistry, University of Bath, Bath, UK. h.bayele@rfc.ucl.ac.uk
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D005947:Glucose