title: Direct isolation of a functional violaxanthin cycle domain from thylakoid membranes of higher plants.
abstract: A special domain of the thylakoid membrane of higher plants has been isolated which carries out the de-epoxidation of the xanthophyll cycle pigment violaxanthin to zeaxanthin. Recent models indicate that in the chloroplast of higher plants, the violaxanthin (V) cycle takes place within specialized domains in the thylakoid membrane. Here, we describe a new procedure to directly isolate such a domain in functional state. The procedure consists of a thylakoid membrane isolation at a pH value of 5.2 which realizes the binding of the enzyme V de-epoxidase (VDE) to the membrane throughout the preparation process. Isolated thylakoid membranes are then solubilized with the very mild detergent n-dodecyl α-D-maltoside and the pigment-protein complexes are separated by sucrose gradient ultracentrifugation. The upper main fraction of the sucrose gradient represents a V cycle domain which consists of the major light-harvesting complex of photosystem II (LHCII), a special lipid composition with an enrichment of the galactolipid monogalactosyldiacylglycerol (MGDG) and the VDE. The domain is isolated in functional state as evidenced by the ability to convert the LHCII-associated V to zeaxanthin. The direct isolation of a V cycle domain proves the most important hypotheses concerning the de-epoxidation reaction in intact thylakoid membranes. It shows that the VDE binds to the thylakoid membrane at low pH values of the thylakoid lumen, that it binds to membrane regions enriched in LHCII, and that the domain contains high amounts of MGDG. The last point is in line with the importance of the galactolipid for V solubilisation and, by providing inverted hexagonal lipid structures, for VDE activity.
authors.affiliation: Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103, Leipzig, Germany. rgoss@rz.uni-leipzig.de.
authors.forename: Reimund
authors.identifier: http://orcid.org/0000-0001-9347-8158
authors.initials: R
authors.lastname: Goss
authors.affiliation: Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103, Leipzig, Germany.
authors.forename: Anne
authors.initials: A
authors.lastname: Greifenhagen
authors.affiliation: Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103, Leipzig, Germany.
authors.forename: Juliane
authors.initials: J
authors.lastname: Bergner
authors.affiliation: Institute for Bioanalytical Chemistry, Centre for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany.
authors.forename: Daniela
authors.initials: D
authors.lastname: Volke
authors.affiliation: Institute for Bioanalytical Chemistry, Centre for Biotechnology and Biomedicine, University of Leipzig, Deutscher Platz 5, 04103, Leipzig, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
authors.affiliation: Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103, Leipzig, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Wilhelm
authors.affiliation: Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103, Leipzig, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Schaller-Laudel
mesh_terms: D015153:Blotting, Western; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D010088:Oxidoreductases / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea / Q000378:metabolism; D020524:Thylakoids / Q000737:chemistry / Q000502:physiology*; D024341:Xanthophylls / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D065146:Zeaxanthins / Q000378:metabolism
chemical_list: D024341:Xanthophylls; D065146:Zeaxanthins; C005613:violaxanthin; D010088:Oxidoreductases; C023111:epoxidase
keywords: LHCII; MGDG; Thylakoid membrane domain; Violaxanthin de-epoxidase; Xanthophyll cycle; Zeaxanthin