THE SMALL GTP-BINDING PROTEINS RAB4 AND ARF ARE ASSOCIATED WITH RELEASED EXOSOMES DURING RETICULOCYTE MATURATION

During maturation of reticulocytes to erythrocytes, small vesicles, te rmed exosomes, are released into the extracellular medium. GTP-binding proteins associated with exosomes were identified by separating prote ins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, tran sferring the proteins to nitrocellulose, and probing the blots with [a lpha-P-32]GTP. At least three GTP-binding proteins were detected with a molecular mass of 27, 26, and 20 kDa. Binding of GTP by exosomes was resistant to trypsin in the absence, but not in the presence of deter gent. This indicates that the GTP-binding proteins are within the lume n of exosomes. During reticulocyte maturation, the amount of GTP-bindi ng proteins released from reticulocytes was proportional to the amount of exosomes released. Western blot analysis demonstrated that the 27 kDa, 26 kDa, and 20 kDa proteins were Rab5p, Rab4p and ADP-ribosylatio n factor (ARF), respectively. In reticulocytes, Rab4p was highly enric hed in exosomes and endosomes compared to plasma membrane and cytosol. Although mainly cytosolic, ARF was also found associated with endosom es and exosomes but not with plasma membrane. In contrast to Rab4p and ARF, Rab5p was enriched in the plasma membrane compared to cytosol, e xosomes and endosomes. As exosomes are believed to derive from endosom es, Rab4p and ARF may be involved in the mechanism of exosome formatio n.