AGGREGATION REROUTES MOLECULES FROM A RECYCLING TO A VESICLE-MEDIATEDSECRETION PATHWAY DURING RETICULOCYTE MATURATION

Endocytosis of the Tf/TfR complex is essentially the only pathway acti ve in maturing reticulocytes, while exosomes, formed by invagination o f the endosomal membrane, pro,ide a mechanism to eliminate seemingly o bsolescent proteins, including the TfR, when their function is complet ed, In this study, we examined molecular trafficking in the recycling and exosome-directed pathways during endocytosis in maturing reticuloc ytes. To this end, the dow of two exogenously inserted fluorescent lip id analogs, 2-oxa-1,3-diazol-4-yl)amino]caproyl])sphingomyelin (C6-NBD -SM) and N-(lissamine rhodamine B sulfonyl) phosphatidyl ethanolamine (N-Rh-PE) was monitored and compared to that of the transferrin (Tf)/T f receptor (TfR) complex, Prior to elimination via exosomes, the TfR a ctively recycles with a half-time of approx. 2 minutes, The recycling kinetics of C6-NBD-SM, as bulk plasma membrane marker, are identical t o those of the apoTf/TfR complex, as shown by fluorescence microscopy and biochemical analysis. By contrast, although efficiently internaliz ed along the same pathway, N-Rh-PE does not return to the cell surface , More specifically, sucrose gradient analysis and immunoisolation exp eriments demonstrated that N-Rh-PE accumulates in exosomes, which are eventually released into the extracellular medium, Fluorometric measur ements showed that exogenously inserted N-Rh-PE is present in the reti culocyte plasma membrane as small molecular clusters, Moreover, a clos e correlation was observed between the fate of crosslinked proteins, i ncluding the TfR and acetylcholinesterase (AChE), and the fate of the clustered lipid N-Rh-PE, Thus antibody-induced aggregation of specific proteins like the TfR and AChE, which are normally sorted into exosom es during reticulocyte maturation, enhances their shedding by the exos omal pathway. Taken together, the results support the hypothesis that aggregation of either proteins or lipids act as a general sorting sign al for exosomal processing, thereby inhibiting reentry in a recycling pathway and providing an effective means for clearing molecules from t he cell surface and their eventual elimination from the cells.