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Journal of Cell Science, Vol 110, Issue 16 1867-1877, Copyright © 1997 by Company of Biologists
JOURNAL ARTICLES |
M Vidal, P Mangeat and D Hoekstra
UMR 5539 CNRS, Universite Montpellier II, France. mvidal@univ-montp2.fr
Endocytosis of the Tf/TfR complex is essentially the only pathway active in maturing reticulocytes, while exosomes, formed by invagination of the endosomal membrane, provide a mechanism to eliminate seemingly obsolescent proteins, including the TfR, when their function is completed. In this study, we examined molecular trafficking in the recycling and exosome-directed pathways during endocytosis in maturing reticulocytes. To this end, the flow of two exogenously inserted fluorescent lipid analogs, N-(N-[6-[(7-nitrobenz-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)/Tf receptor (TfR) complex. Prior to elimination via exosomes, the TfR actively recycles with a half-time of approx. 2 minutes. The recycling kinetics of C6-NBD-SM, as bulk plasma membrane marker, are identical to those of the apoTf/TfR complex, as shown by fluorescence microscopy and biochemical analysis. By contrast, although efficiently internalized along the same pathway, N-Rh-PE does not return to the cell surface. More specifically, sucrose gradient analysis and immunoisolation experiments demonstrated that N-Rh-PE accumulates in exosomes, which are eventually released into the extracellular medium. Fluorometric measurements showed that exogenously inserted N-Rh-PE is present in the reticulocyte plasma membrane as small molecular clusters. Moreover, a close correlation was observed between the fate of crosslinked proteins, including 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 exosomes during reticulocyte maturation, enhances their shedding by the exosomal pathway. Taken together, the results support the hypothesis that aggregation of either proteins or lipids act as a general sorting signal for exosomal processing, thereby inhibiting reentry in a recycling pathway and providing an effective means for clearing molecules from the cell surface and their eventual elimination from the cells.
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