title: The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme.
abstract: The membrane topology of vitamin K epoxide reductase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and its recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive green fluorescent protein (GFP) fused to the N- or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin-like transmembrane protein (TMX) to demonstrate ER localization of the major loop. The topology is identical with the bacterial homologue from Synechococcussp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase (PDI) family in recycling VKOR.
authors.affiliation: The Institute of Molecular, Cell and Systems Biology, CMVLS, University of Glasgow, Glasgow G12 8QQ, U.K.
authors.forename: Zhenbo
authors.initials: Z
authors.lastname: Cao
authors.affiliation: The Institute of Molecular, Cell and Systems Biology, CMVLS, University of Glasgow, Glasgow G12 8QQ, U.K.
authors.forename: Marcel
authors.initials: M
authors.lastname: van Lith
authors.affiliation: The Institute of Molecular, Cell and Systems Biology, CMVLS, University of Glasgow, Glasgow G12 8QQ, U.K.
authors.forename: Lorna J
authors.initials: LJ
authors.lastname: Mitchell
authors.affiliation: The Institute of Molecular, Cell and Systems Biology, CMVLS, University of Glasgow, Glasgow G12 8QQ, U.K.
authors.forename: Marie Anne
authors.initials: MA
authors.lastname: Pringle
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Inaba
authors.affiliation: The Institute of Molecular, Cell and Systems Biology, CMVLS, University of Glasgow, Glasgow G12 8QQ, U.K. neil.bulleid@glasgow.ac.uk.
authors.forename: Neil J
authors.initials: NJ
authors.lastname: Bulleid
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046940:Synechococcus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C578523:VKORC1 protein, human; C531327:VKORC1L1 protein, human; D064417:Vitamin K Epoxide Reductases
keywords: endoplasmic reticulum; membrane protein; redox-sensitive green fluorescent protein (GFP); thioredoxin (TMX); vitamin K; vitamin K epoxide reductase