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Purification of the Chlorella HUP1 hexose—proton symporter to homogeneity and its reconstitution in vitro
Author(s) -
Caspari Thomas,
Robl Ingrid,
Stolz Jürgen,
Tanner Widmar
Publication year - 1996
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1996.10061045.x
Subject(s) - symporter , biochemistry , chemistry , biotinylation , transporter , gene
Summary A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose—proton sym‐ porter. 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‐β‐ d ‐glucoside in the presence of Escherichia coli l ‐α‐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.