Abstract
Vitamin K epoxide reductase (VKOR) is essential for the production of reduced vitamin K that is required for modification of vitamin K-dependent proteins. Three- and four-transmembrane domain (TMD) topology models have been proposed for VKOR. They are based on in vitro glycosylation mapping of the human enzyme and the crystal structure of a bacterial (Synechococcus) homologue, respectively. These two models place the functionally disputed conserved loop cysteines, Cys-43 and Cys-51, on different sides of the endoplasmic reticulum (ER) membrane. In this study, we fused green fluorescent protein to the N or C terminus of human VKOR, expressed these fusions in HEK293 cells, and examined their topologies by fluorescence protease protection assays. Our results show that the N terminus of VKOR resides in the ER lumen, whereas its C terminus is in the cytoplasm. Selective modification of cysteines by polyethylene glycol maleimide confirms the cytoplasmic location of the conserved loop cysteines. Both results support a three-TMD model of VKOR. Interestingly, human VKOR can be changed to a four-TMD molecule by mutating the charged residues flanking the first TMD. Cell-based activity assays show that this four-TMD molecule is fully active. Furthermore, the conserved loop cysteines, which are essential for intramolecular electron transfer in the bacterial VKOR homologue, are not required for human VKOR whether they are located in the cytoplasm (three-TMD molecule) or the ER lumen (four-TMD molecule). Our results confirm that human VKOR is a three-TMD protein. Moreover, the conserved loop cysteines apparently play different roles in human VKOR and in its bacterial homologues.
Highlights
The membrane topology and the role of certain cysteines in human vitamin K epoxide reductase (VKOR) are disputed
Results show that when green fluorescence protein (GFP) is fused to the N terminus of Vitamin K epoxide reductase (VKOR) (GFP-VKOR), it is protected from trypsin digestion (Fig. 2A)
The C-terminal GFP fusion of VKOR (VKOR-GFP) is susceptible to protease digestion in digitoninpermeabilized cells. This result suggests that the N terminus of VKOR is located in the endoplasmic reticulum (ER) lumen, and the C terminus is located in the cytoplasm
Summary
The membrane topology and the role of certain cysteines in human vitamin K epoxide reductase (VKOR) are disputed. Our results suggest that VKOR is a three-transmembrane domain (TMD) protein with its N terminus located in the ER lumen and C terminus in the cytoplasm (Fig. 1A) In this model, the active site cysteines (Cys-132 and Cys-135) are located at the N terminus of the third TMD facing the ER lumen. An intramolecular electron transfer pathway between the two pairs of conserved cysteines, similar to that of DsbB, has been proposed for the bacterial VKORHs as well as for mammalian VKOR [26, 27] These results raise the question as to whether the bacterial enzymes and the mammalian ones have the same membrane topology and whether they employ different mechanisms to regenerate the active site cysteines. Our results support the three-TMD model of human VKOR and suggest that intramolecular electron transfer between the loop cysteines and the active site cysteines of VKOR is not necessary for its function
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