The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme (original) (raw)

Cao, Zhenbo, Van Lith, Marcel, Mitchell, Lorna J., Pringle, Marie Anne ORCID: https://orcid.org/0000-0002-1358-4610, Inaba, Kenji and Bulleid, Neil J. ORCID: https://orcid.org/0000-0002-9839-5279(2016) The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme.Biochemical Journal, 473(7), pp. 851-858. (doi: 10.1042/bj20151223) (PMID:26772871)

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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 Synechococcus sp., 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.

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Funder and Project Information

1

Identifying the reductive pathway in the mammalian endoplasmic reticulum.

Neil Bulleid

BB/L00593X/1

RI MOLECULAR CELL & SYSTEMS BIOLOGY

1

Protein Folding and Thiol Modification in the Mammalian Endoplasmic Reticulum

Neil Bulleid

103720/Z/14/Z

RI MOLECULAR CELL & SYSTEMS BIOLOGY

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