VESICULATION AND SORTING FROM PC12-DERIVED ENDOSOMES IN-VITRO

Formation of small vesicles resembling synaptic vesicles can be recons tituted in vitro by incubating labeled homogenates of PC12 cells with ATP and two cytoplasmic proteins, AP3 and ARF1 [Faundez, V., Horng, J. -T. Sr Kelly, R. B. (1998) Cell 93, 423-432], To determine whether AP3 was mediating budding from plasma membranes or endosomes the organell e that generated the synaptic vesicles was characterized. The budding activity was enriched in organelles that labeled at 15 degrees C, but not at 4 degrees C, that excluded a marker of plasma membranes and tha t contained internalized transferrin, indicating that the precursor wa s an endosome. Vesicles formed from the endosomal precursor in vitro e xcluded transferrin, We conclude that ARF-mediated vesiculation into s ynaptic vesicle-sized organelles uses an endosomal precursor and occur s simultaneously in vitro with sorting of synaptic vesicle proteins fr om other membrane protein constituents of the endosome.