Caspari, Thomas and Robl, Ingrid and Stolz, Jürgen and Tanner, Widmar (1996) Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro. PLANT JOURNAL, 10 (6). pp. 1045-1053. ISSN 0960-7412, 1365-313X
Full text not available from this repository.Abstract
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 biotin avidin 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 Hf-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.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | YEAST SCHIZOSACCHAROMYCES-POMBE; MONOSACCHARIDE-H+ SYMPORTER; HISTIDINE-TAGGED PROTEIN; PLASMA-MEMBRANE ATPASE; ESCHERICHIA-COLI; SACCHAROMYCES-CEREVISIAE; AFFINITY-CHROMATOGRAPHY; FUNCTIONAL EXPRESSION; ARABIDOPSIS-THALIANA; GLUCOSE-TRANSPORTER |
| Subjects: | 500 Science > 570 Life sciences 500 Science > 580 Botanical sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Pflanzenwissenschaften > Lehrstuhl für Zellbiologie und Pflanzenphysiologie (Prof. Dr. Klaus Grasser) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 09 Jun 2023 12:37 |
| Last Modified: | 09 Jun 2023 12:37 |
| URI: | https://pred.uni-regensburg.de/id/eprint/51327 |
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