TY - JOUR

T1 - Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro

AU - Caspari, T

AU - Robl, Ingrid

AU - Stolz, J

AU - Tanner, Widmar

PY - 1996/12

Y1 - 1996/12

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Biological Transport

KW - Biotin

KW - Carrier Proteins

KW - Chlorella

KW - Electron Transport Complex IV

KW - Glucose

KW - Membrane Proteins

KW - Molecular Sequence Data

KW - Monosaccharide Transport Proteins

KW - Plant Proteins

KW - Proteolipids

KW - Proton Pumps

KW - Proton-Motive Force

KW - Recombinant Fusion Proteins

KW - Schizosaccharomyces

KW - Solubility

KW - Symporters

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1046/j.1365-313X.1996.10061045.x

DO - 10.1046/j.1365-313X.1996.10061045.x

M3 - Article

C2 - 9011086

VL - 10

SP - 1045

EP - 1053

JO - Plant Journal

JF - Plant Journal

SN - 0960-7412

IS - 6

ER -