title: Interactions between ferroportin and hephaestin in rat enterocytes are reduced after iron ingestion.
abstract: OBJECTIVE
  Ferroportin (Fpn) is a multiple transmembrane protein required for iron export into the systemic circulation, in cooperation with hephaestin (Heph). Despite the importance of Fpn in iron transport, there is controversy about its topology and functional state upon interaction with Heph.
  
  
  METHODS
  The topology of Fpn was determined using monospecific antisera against its different epitopes, in sheets of cells from duodenum that were or were not permeabilized with detergent. Immunoprecipitation and blue native polyacrylamide gel electrophoresis, followed by immunoblot analysis, were used to determine the extent of interactions between Fpn and Heph. Antisera against the intracellular, C-termini of divalent metal transporter (Dmt1) and Heph served as controls.
  
  
  RESULTS
  Immunofluorescence analysis with antisera against amino acids 172-193 of Fpn (anti-Fpn 172) detected Fpn only in permeabilized cells, whereas anti-Fpn 232 (amino acids 232-249), anti-Fpn 370 (amino acids 370-420), and anti-Fpn C (the C-terminus) detected Fpn in nonpermeabilized and permeabilized cells. Immunoprecipitation studies showed that Fpn and Heph coprecipitated with either anti-Fpn or anti-Heph. Blue native polyacrylamide gel electrophoresis studies revealed that a fraction of Fpn comigrates with Heph; the apparent interaction decreases after iron ingestion.
  
  
  CONCLUSIONS
  Studies with antisera to different epitopes of Fpn indicate that the topology of Fpn is consistent with an 11-transmembrane model, with the C-terminus exposed on the cell surface. Reduced interactions between Fpn and Heph after iron ingestion indicate that this is a regulatory mechanism for limiting further iron absorption.
authors.affiliation: Department of Medicine, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA.
authors.forename: Kwo-Yih
authors.initials: KY
authors.lastname: Yeh
authors.forename: Mary
authors.initials: M
authors.lastname: Yeh
authors.forename: Jonathan
authors.initials: J
authors.lastname: Glass
mesh_terms: D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D027682:Cation Transport Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002570:Ceruloplasmin / Q000378:metabolism; D004386:Duodenum / Q000166:cytology / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D020895:Enterocytes / Q000378:metabolism*; D018604:Epitope Mapping; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D007408:Intestinal Absorption; D019266:Iron, Dietary / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D051381:Rats; D017207:Rats, Sprague-Dawley; D013329:Structure-Activity Relationship; D013997:Time Factors; D000098742:Ferroportin
chemical_list: D027682:Cation Transport Proteins; C492163:Heph, protein, rat; D019266:Iron, Dietary; D008565:Membrane Proteins; D000098742:Ferroportin; D002570:Ceruloplasmin