PURIFICATION OF THE CHLORELLA HUP1 HEXOSE-PROTON SYMPORTER TO HOMOGENEITY AND ITS RECONSTITUTION IN-VITRO

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose-proton symporter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes acc umulation of D-glucose analogs and restores growth of a glucose-uptake -deficient yeast strain. Crude membranes were solubilized with octyl-b eta-D-glucoside in the presence of Escherichia coil L-alpha-phosphatid ylethanolamine. Biotinylated symporter was purified to homogeneity by biotin avidin affinity chromatography The symporter protein was recons tituted together with cytochrome-c oxidase prepared from beef heart mi tochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driv en Hf-pump generating a proton motive force (inside negative and alkal ine) while transferring electrons from cytochrome-c to oxygen; this en ergy is used by the symporter to accumulate D-glucose at least 30-fold . In the absence of the driving force the transport protein facilitate s diffusion of D-glucose until the concentration equilibrium is reache d. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.