Membrane Topology Mapping of Vitamin K Epoxide Reductase by in Vitro Translation/Cotranslocation

Jian-Ke Tie,Christopher Nicchitta,Gunnar Von Heijne,Darrel W Stafford

doi:10.1074/jbc.m500765200

Abstract

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle. Recently, the gene encoding the catalytic subunit of VKOR was identified as a 163-amino acid integral membrane protein. In this study we report the experimentally derived membrane topology of VKOR. Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro. However, in the intact enzyme there are only three transmembrane domains, residues 10-29, 101-123, and 127-149, and membrane-integration of residues 75-97 appears to be suppressed by the surrounding sequence. Results of N-linked glycosylation-tagged full-length VKOR shows that the N terminus of VKOR is located in the endoplasmic reticulum lumen, and the C terminus is located in the cytoplasm. Further evidence for this topological model of VKOR was obtained with freshly prepared intact microsomes from insect cells expressing HPC4-tagged full-length VKOR. In these experiments an HPC4 tag at the N terminus was protected from proteinase K digestion, whereas an HPC4 tag at the C terminus was susceptible. Altogether, our results suggest that VKOR is a type III membrane protein with three transmembrane domains, which agrees well with the prediction by the topology prediction program TMHMM.

Highlights

Vitamin K epoxide reductase (VKOR) catalyzes the conversion of vitamin K 2,3-epoxide into vitamin K in the vitamin K redox cycle
Our results show that four hydrophobic regions predicted as the potential transmembrane domains in VKOR can individually insert across the endoplasmic reticulum membrane in vitro
The five programs that predict the location of the C terminus all agree that the C terminus of VKOR is located in the cytoplasm

Summary

The abbreviations used are

Gla, ␥-carboxyglutamic acid; VKOR, vitamin K epoxide reductase; VKORC1, vitamin K epoxide reductase complex subunit 1; TM, transmembrane; HPC4, peptide epitope comprising residues; EDQVDPRLIDGK, ER endoplasmic reticulum; RM, canine rough microsomes; MES, 2-(N-morpholino)ethanesulfonic acid, hemisodium salt; CHAPS, (3-[(3-cholamidopropyl)di-methylammonio]1-propane sulfonate); Endo H, endoglycosidase H; PVDF, polyvinylidene difluoride. Concomitant with ␥-glutamyl carboxylation, the reduced form of vitamin K (vitamin K hydroquinone) is converted to vitamin K 2,3-epoxide, which must be converted back to vitamin K hydroquinone for the reaction to continue because of limited vitamin K amounts in vivo [4] This cyclic conversion of vitamin K establishes a redox cycle known as the vitamin K cycle [5]. Several computer programs to predict the topology of membrane proteins are available (18 –21). We first determined the topology of VKOR using multiple prediction programs These predictions were tested by in vitro translation/cotranslocation of a series of VKOR truncations containing an N-linked glycosylation reporter tag.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION