Disulfide-dependent Protein Folding Is Linked to Operation of the Vitamin K Cycle in the Endoplasmic Reticulum: A PROTEIN DISULFIDE ISOMERASE-VKORC1 REDOX ENZYME COMPLEX APPEARS TO BE RESPONSIBLE FOR VITAMIN K₁ 2,3-EPOXIDE REDUCTION

γ-Carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K₁ (Vit.K₁H₂) in the endoplasmic reticulum (ER), where it works as an essential cofactor for γ-carboxylase in post-translational γ-carboxylation of vitamin K-dependent proteins. Vit.K₁H₂ is produced by the warfarin-sensitive enzyme vitamin K 2,3-epoxide reductase (VKOR) of the vitamin K cycle that has been shown to harbor a thioredoxin-like CXXC center involved in reduction of vitamin K₁ 2,3-epoxide (Vit.K>O). However, the cellular system providing electrons to the center is unknown. Here data are presented that demonstrate that reduction is linked to dithiol-dependent oxidative folding of proteins in the ER by protein disulfide isomerase (PDI). Oxidative folding of reduced RNase is shown to trigger reduction of Vit.K>O and γ-carboxylation of the synthetic γ-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered γ-carboxylation is inhibited by the PDI inhibitor bacitracin and also by small interfering RNA silencing of PDI in HEK 293 cells. Immunoprecipitation and two-dimensional SDS-PAGE of microsomal membrane proteins demonstrate the existence of a VKOR enzyme complex where PDI and VKORC1 appear to be tightly associated subunits. We propose that the PDI subunit of the complex provides electrons for reduction of the thioredoxin-like CXXC center in VKORC1. We can conclude that the energy required for γ-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.