Membrane topology of human VKOR. (A) The proposed 3-TM (based on Tie et al15 ) and 4-TM (based on Li et al16 ) topology models of human VKOR. Cysteine residues (C43, C51, C85, and C96) in the disputed loop sequence are marked in red. Cysteine residues (C16, C132, and C135) located within the TM helices are not shown. The loop sequence (A34-W59) specifically recognized by a VKOR antibody is marked green in the 3-TM model and blue in the 4-TM model. (B) Localization of the disputed loop sequence of human VKOR by immunofluorescence confocal microscopy in live mammalian cells. COS-7 cells expressing wild-type human VKOR were either selectively or fully permeabilized with digitonin or Triton X-100, respectively. Cytoplasmic proteins are accessible to antibody staining after selective permeabilization with digitonin, whereas ER lumen proteins are accessible for staining after full permeabilization with Triton X-100. Cells were coimmunostained with anti-VKOR (red) and anti-PDI (green) antibodies (top panel) or with anti-VKOR (red) and anti-tubulin (green) antibodies (bottom panel). The cell nucleus was stained by Hoechst 33342 (blue). (C) PEGylation of the cytoplasmic-accessible cysteines of VKOR with a membrane-impermeable maleimide polyethylene glycol (mPEG-MAL-5000). C-terminal FLAG-tagged VKOR or its C43/51A mutant was stably expressed in HEK293 cells. Intact microsomes were prepared and labeled with mPEG-MAL-5000 with/without Triton X-100 permeabilization. VKOR bands were visualized by western blot analysis. (D) Cell-based activity assay of VKOR and its C43/51A mutant. Data are presented as mean ± standard deviation (SD) (n = 3).
