Characterization of coated vesicles that participate in endocytic recycling

While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding the transport carriers that mediate this pathway. In this study, we overexpressed a point mutant o f ADP-ribosylation factor 6 (ARF6), that perturbs its GTPase cycle, to accu mulate endosome-derived coated vesicles. Characterization by their purifica tion revealed that, upon cell homogenization, these vesicles were mostly ag gregated with larger noncoated membranes, and could be released with high-s alt treatment. Equilibrium centrifugation revealed that these vesicles had buoyant density similar to the COP-coated vesicles. To purify the ARF6-regu lated vesicles to homogeneity, enriched fractions from equilibrium centrifu gation were subjected to immunoisolation through the hemagglutinin (HA) epi tope of the mutant ARF6, by using a newly developed, high-affinity, anti-HA monoclonal antibody. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunit s of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, b ecause they are enriched for transferrin receptor and also the v-SNARE cell ubrevin that functions in transport from the recycling endosome to the plas ma membrane. Thus, we have characterized transport vesicles that participat e in endocytic recycling.