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Interactions of ferroportin with hephaestin on the basolateral membrane of human intestinal cells
Author(s) -
Kim EunYoung,
Ham SooKyung,
Becker Erin,
Han Okhee
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.229.3
Subject(s) - chemistry , enterocyte , ferroportin , förster resonance energy transfer , yellow fluorescent protein , fluorescence , biophysics , microbiology and biotechnology , biochemistry , biology , small intestine , metabolism , gene , physics , quantum mechanics , iron homeostasis
To investigate interactions between ferroportin (FPN) an iron exporter and hephaestin (Heph) a copper‐bound ferroxidase, we performed experiments using fluorescence energy transfer (FRET). We prepared appropriate fluorescent probes by attaching cyan fluorescent protein (CFP) to the N‐terminus of Heph and yellow fluorescent protein (YFP) to the N‐terminus of FPN. In cells stably expressing Heph‐CFP, we first assessed CFP fluorescence, pixel by pixel. In cells stably expressing both Heph‐CFP and FPN‐YFP, we then reassessed fluorescence of each pixel at the CFP donor emission wavelength. An YFP‐induced decrease in CFP emission suggests that the two proteins are associated. We found that an FPN‐YFP induced the decrease of Heph‐CFP emission suggesting the association of these two proteins on the basolateral membrane of human intestinal Caco‐2 cells. Our FRET experiments show that the distance of FPN‐YFP from Heph‐CFP is ~ 5 nm. Our FRET studies also indicate that neither FPN nor Heph is associated with TfR1 on the basolateral membrane of the enterocyte, although we showed the colocalization of these two proteins with TfR1 on the basolateral membrane of the enterocyte.