title: Degradation of AP2 during reticulocyte maturation enhances binding of hsc70 and Alix to a common site on TFR for sorting into exosomes.
abstract: Reticulocytes release small membrane vesicles termed exosomes during their maturation in erythrocytes. The transferrin receptor (TfR) is completely lost from the red cell surface by its segregation in the secreted vesicles where it interacts with the heat shock cognate 70 kDa protein (hsc70). We have now determined a region of the TfR that can potentially interact with hsc70. The peptide P1 (YTRFSLARQV) from the TfR cytosolic domain: (i). binds to hsc70 (ii). with an increased affinity in oxidative conditions, (iii). competes for binding of an unfolded protein to hsc70, and (iv). inhibits the interaction of hsc70 with a recombinant protein corresponding to the cytosolic domain of the receptor. This peptide encompasses the internalization motif (YTRF) of the receptor, and accordingly an affinity column made with the immobilized peptide retains hsc70 and also the AP2 adaptor complex. On the other hand, we show that AP2 is degraded by the proteasome system during reticulocyte maturation and that the presence of the proteasome inhibitor during in vitro red cell maturation inhibits AP2 degradation and specifically decreases TfR secretion via exosomes. Finally, coimmunoprecipitation of Alix with the exosomal TfR, and binding of P1 peptide to the Alix homolog PalA suggest that Alix also interacts with the YTRF motif and contributes to exosomal TfR sorting.
authors.affiliation: UMR CNRS 5539, Universitè Montpellier II, cc107, 34095 Montpellier, France.
authors.forename: Charles
authors.initials: C
authors.lastname: Géminard
authors.forename: Aude
authors.initials: A
authors.lastname: De Gassart
authors.forename: Lionel
authors.initials: L
authors.lastname: Blanc
authors.forename: Michel
authors.initials: M
authors.lastname: Vidal
mesh_terms: D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D033965:Adaptor Protein Complex alpha Subunits / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019921:Biotinylation; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D019281:Dimerization; D004912:Erythrocytes / Q000166:cytology / Q000378:metabolism; D005089:Exocytosis; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D051381:Rats; D011990:Receptors, Transferrin / Q000737:chemistry / Q000378:metabolism*; D012156:Reticulocytes / Q000166:cytology* / Q000378:metabolism*; D013696:Temperature
chemical_list: D033962:Adaptor Protein Complex 2; D033965:Adaptor Protein Complex alpha Subunits; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; C494373:Hspa8 protein, rat; D007976:Leupeptins; C492143:Pdcd6ip protein, rat; D010446:Peptide Fragments; D011990:Receptors, Transferrin; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde