Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.