Membrane topology mapping of vitamin K epoxide reductase by in vitro translation/cotranslocation.
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.
Tie
Jian-Ke
JK
Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Nicchitta
Christopher
C
von Heijne
Gunnar
G
Stafford
Darrel W
DW
D004721:Endoplasmic Reticulum / Q000201:enzymology*; D017930:Genes, Reporter; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases
D011993:Recombinant Fusion Proteins; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases