Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro

• Published in: The Plant journal : for cell and molecular biology 1996-12, Vol.10 (6), p.1045-1053
• Main Authors: Caspari, T, Robl, I, Stotz, J, Tanner, W
• Format: Article
• Language: English
• Subjects: active transport; Amino Acid Sequence; amino acid sequences; binding proteins; Biological and medical sciences; Biological Transport; Biotin; Carrier Proteins - genetics; Carrier Proteins - isolation & purification; Carrier Proteins - metabolism; Cell physiology; Chlorella; Chlorella - chemistry; Chlorella - enzymology; Chlorella - metabolism; cytochrome-c oxidase; Electron Transport Complex IV - metabolism; Fundamental and applied biological sciences. Psychology; gene expression; gene transfer; glucose; Glucose - metabolism; hexoses; liposomes; Membrane Proteins - genetics; Membrane Proteins - isolation & purification; Membrane Proteins - metabolism; Molecular Sequence Data; Monosaccharide Transport Proteins; Plant physiology and development; Plant Proteins - genetics; Plant Proteins - isolation & purification; Plant Proteins - metabolism; plasma membrane; Plasma membrane and permeation; Proteolipids - metabolism; Proton Pumps - metabolism; Proton-Motive Force; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Schizosaccharomyces - genetics; Schizosaccharomyces pombe; Solubility; Symporters; uptake
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1046/j.1365-313x.1996.10061045.x

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.