Wild-type and mutant ferroportins do not form oligomers in transfected cells.
Ferroportin [FPN; Slc40a1 (solute carrier family 40, member 1)] is a transmembrane iron export protein expressed in macrophages and duodenal enterocytes. Heterozygous mutations in the FPN gene result in an autosomal dominant form of iron overload disorder, type-4 haemochromatosis. FPN mutants either have a normal iron export activity but have lost their ability to bind hepcidin, or are defective in their iron export function. The mutant protein has been suggested to act as a dominant negative over the wt (wild-type) protein by multimer formation. Using transiently transfected human epithelial cell lines expressing mouse FPN modified by the addition of a haemagglutinin or c-Myc epitope at the C-terminus, we show that the wtFPN is found at the plasma membrane and in Rab5-containing endosomes, as are the D157G and Q182H mutants. However, the delV162 mutant is mostly intracellular in HK2 cells (human kidney-2 cells) and partially addressed at the cell surface in HEK-293 cells (human embryonic kidney 293 cells). In both cell types, it is partially associated with the endoplasmic reticulum and with Rab5-positive vesicles. However, this mutant is complex-glycosylated like the wt protein. D157G and G323V mutants have a defective iron export capacity as judged by their inability to deplete the intracellular ferritin content, whereas Q182H and delV162 have normal iron export function and probably have lost their capacity to bind hepcidin. In co-transfection experiments, the delV162 mutant does not co-localize with the wtFPN, does not prevent its normal targeting to the plasma membrane and cannot be immunoprecipitated in the same complex, arguing against the formation of FPN hetero-oligomers.
Gonçalves
Ana Sofia
AS
INSERM, U773, 16 rue Henri Huchard, 75018 Paris, France.
Muzeau
Françoise
F
Blaybel
Rand
R
Hetet
Gilles
G
Driss
Fathi
F
Delaby
Constance
C
Canonne-Hergaux
François
F
Beaumont
Carole
C
D000818:Animals; D000906:Antibodies / Q000378:metabolism; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D005293:Ferritins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007501:Iron / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D020125:Mutation, Missense; D014162:Transfection; D000098742:Ferroportin
D000906:Antibodies; D027682:Cation Transport Proteins; D000098742:Ferroportin; D005293:Ferritins; D007501:Iron