Robl, Ingrid and Grassl, Renate and Tanner, Widmar and Opekarova, Miroslava (2000) Properties of a reconstituted eukaryotic hexose/proton symporter solubilized by structurally related non-ionic detergents: specific requirement of phosphatidylcholine for permease stability. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1463 (2). pp. 407-418. ISSN 0005-2736, 0006-3002
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Overexpression of the hexose/proton symporter HUP1 from Chlorella kessleri in S. cerevisiae permits a one-step purification via a biotinylation domain. Milligram amounts of the protein are obtained starting from 21 of yeast culture. The HUP1 protein is used as a model eukaryotic membrane protein of the 'major facilitator superfamily' (MFS) to study specific lipid requirements for activity and stability. Testing two series of detergents revealed that n-nonyl-beta-D-glucoside (NG) and n-octyl-beta-D-glucoside (OG) solubilize the HUP1 protein efficiently. Only the use of NG resulted in long-term stabilization of the HUP1 protein in the absence of external lipids. When affinity purified protein was extracted with organic solvents, a stoichiometric amount of phosphatidyl choline, phosphatidyl ethanolamine and ergosterol in the ratio of close to 2:1 was detected. These lipids were only observed, however, when the protein purification was carried out in the presence of NG; no lipids were copurified with the HUP1 protein in the presence of OG. Of the three lipids copurified, phosphatidyl choline showed a crucial role in ensuring maximal HUP1 permease activity and stability when added back to the OG-protein. The requirement of phosphatidylcholine documents a specific effect of lipids on vectorial transport mediated by a eukaryotic membrane protein of the MFS family. (C) 2000 Elsevier Science B.V. All rights reserved.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | THIN-LAYER CHROMATOGRAPHY; CYTOCHROME-C-OXIDASE; SACCHAROMYCES-CEREVISIAE; PLASMA-MEMBRANE; P-GLYCOPROTEIN; IN-VITRO; LIPIDS; ATPASE; PHOSPHATIDYLINOSITOL; IDENTIFICATION; hexose/proton cotransporter; phosphatidylcholine; nonyl-glucoside; Chlorella kessleri |
| Subjects: | 500 Science > 590 Zoological sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Pflanzenwissenschaften > Lehrstuhl für Zellbiologie und Pflanzenphysiologie (Prof. Dr. Klaus Grasser) |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 07 Jun 2022 06:23 |
| Last Modified: | 07 Jun 2022 06:23 |
| URI: | https://pred.uni-regensburg.de/id/eprint/42823 |
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