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 accumulation of D-glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-beta-D-glucoside in the presence of Escherichia coli L-alpha-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H(+)-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate D-glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of D-glucose until the concentration equilibrium is reached. 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.
Caspari
T
T
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, Germany. thomas.caspari@biologie.uni-regensburg.de
Robl
I
I
Stolz
J
J
Tanner
W
W
D000595:Amino Acid Sequence; D001692:Biological Transport; D001710:Biotin; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002708:Chlorella / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism; D005947:Glucose / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D017494:Proton Pumps / Q000378:metabolism*; D018892:Proton-Motive Force; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D012995:Solubility; D027981:Symporters
D002352:Carrier Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010940:Plant Proteins; D011510:Proteolipids; D017494:Proton Pumps; D011993:Recombinant Fusion Proteins; D027981:Symporters; C089820:monosaccharide-hydrogen cotransporter; C020485:proteoliposomes; D001710:Biotin; D003576:Electron Transport Complex IV; D005947:Glucose