Over-expression of wild-type and mutant HFE in a human melanocytic cell line reveals an intracellular bridge between MHC class I pathway and transferrin iron uptake.
Hereditary hemochromatosis (HH) is a frequent recessive disorder of iron metabolism characterised by systemic iron overload. In Northern Europe, more than 90% of HH patients are homozygous for a mis-sense mutation (C282Y) in the HFE1 gene product. The HFE protein is the heavy chain of a MHC class I-related molecule and associates with beta2 microglobulin and the transferrin receptor. Its precise roles in iron metabolism and in the pathophysiology of HH are still unclear. In order to identify the cellular processing of HFE, an important step towards the understanding of the function of the protein, we stably over-expressed the wild type and mutated forms fused to the Green Fluorescent Protein in a melanocytic MHC class I expressing cell line, the Mel Juso cell line. In wild type and mutant clones, the fusion proteins were not detected at the cell surface but only in the cytoplasm. Their sub-cellular localisation was determined by co-labelling of cells with organite-specific antibodies and confocal microscopy. HFE-GFP followed initially HLA class I intracellular processing but co-localised with transferrin in early endosomes without recycling at the cell surface. The C282Y-GFP fusion protein followed a different folding pathway to exit endoplasmic reticulum. Over-expression of the wild-type protein lead to a decrease in diferric transferrin uptake. Our model will be of use in the elucidation of the functional interaction between intracellular HFE and iron transporters transferrin/transferrin receptor complexes and Slc11A2 (also named N-Ramp2 or DMT1) in different endosomal compartments.
Fergelot
Patricia
P
UMR 6061 CNRS "Génétique et développement", Faculté de Médecine, 2, avenue du Pr Léon-Bernard CS 34317, 35043 Rennes cedex, France. patricia.fergelot@univ-rennes1.fr
Orhant
Magali
M
Thénié
Agnès
A
Loyer
Pascal
P
Ropert-Bouchet
Martine
M
Lohyer
Stéphanie
S
Le Gall
Jean-Yves
JY
Mosser
Jean
J
D015703:Antigens, CD / Q000378:metabolism; D015152:Blotting, Northern; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D037282:Calreticulin / Q000378:metabolism; D045744:Cell Line, Tumor; D018699:Coated Vesicles / Q000502:physiology; D020756:Coatomer Protein / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D004721:Endoplasmic Reticulum / Q000502:physiology; D011992:Endosomes / Q000502:physiology; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006056:Golgi Apparatus / Q000502:physiology; D049452:Green Fluorescent Proteins; D006367:HeLa Cells / Q000378:metabolism; D006432:Hemochromatosis / Q000378:metabolism; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008545:Melanoma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000502:physiology; D009154:Mutation / Q000235:genetics; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D021381:Protein Transport; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D060149:Tetraspanin 30; D014162:Transfection; D014168:Transferrin / Q000378:metabolism*; D001613:beta 2-Microglobulin / Q000378:metabolism
D015703:Antigens, CD; C554586:CD63 protein, human; D037282:Calreticulin; D020756:Coatomer Protein; C103673:HFE protein, human; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D060149:Tetraspanin 30; D014168:Transferrin; D001613:beta 2-Microglobulin; C028283:diferric transferrin; D049452:Green Fluorescent Proteins; D020126:Brefeldin A