Characteristics of the interaction between Hsc70 and the transferrin receptor in exosomes released during reticulocyte maturation.
The transferrin receptor (TfR) of reticulocytes is released in vesicular form (exosomes) during their maturation to erythrocytes. The heat shock cognate 70-kDa protein (Hsc70) has been demonstrated to interact with the cytosolic domain of the TfR and could thus trigger the receptor toward this secretion pathway. We investigated the characteristics of the interaction between Hsc70 and the TfR in exosomes with an in vitro binding assay using TfR immobilized on Sepharose beads and purified Hsc70. The results show that Hsc70 binds to exosomal TfR with characteristics expected of a chaperone/peptide interaction. We demonstrated that heat-denatured luciferase competed for in vitro binding, dependent on the nucleotide bound to Hsc70, and that this interaction activates the ATPase activity of Hsc70. Moreover, we used immunosuppressive agents that interact with Hsc70, thus decreasing Hsc70 binding to TfR in our in vitro binding assay and enabling us to assess the role of this interaction in vivo during reticulocyte maturation.
Géminard
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UMR CNRS 5539, Université Montpellier II, cc107, 34095 Montpellier, France.
Nault
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Johnstone
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RM
Vidal
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002847:Chromatography, Agarose; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006358:Hot Temperature; D015151:Immunoblotting; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011990:Receptors, Transferrin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism*; D012756:Sheep; D013696:Temperature; D013997:Time Factors
D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D007166:Immunosuppressive Agents; D018832:Molecular Chaperones; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D008156:Luciferases; D000251:Adenosine Triphosphatases