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

gamma-Carboxylation of vitamin K-dependent proteins is dependent on formation of reduced vitamin K-1(Vit. K1H2) in the endoplasmic reticulum (ER), where it works as an essential cofactor for gamma-carboxylase in post-translational gamma-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 thiore-doxin-like CXXC center involved in reduction of vitamin K-1 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 gamma-carboxylation of the synthetic gamma-carboxylase peptide substrate FLEEL. In liver microsomes, reduced RNase-triggered gamma-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 gamma-carboxylation of proteins is provided by dithiol-dependent oxidative protein folding in the ER and thus is linked to de novo protein synthesis.