Exosomes are generated within the multivesicular endosomes (MVEs) as intraluminal vesicles (ILVs) and secreted during the fusion of MVEs with the cell membrane. The mechanisms of exosome biogenesis remain poorly explored. Researchers from the Sun Yat-sen University Cancer Center identify that RAB31 marks and controls an ESCRT-independent exosome pathway. Active RAB31, phosphorylated by epidermal growth factor receptor (EGFR), engages flotillin proteins in lipid raft microdomains to drive EGFR entry into MVEs to form ILVs, which is independent of the ESCRT (endosomal sorting complex required for transport) machinery. Active RAB31 interacts with the SPFH domain and drives ILV formation via the Flotillin domain of flotillin proteins. Meanwhile, RAB31 recruits GTPase-activating protein TBC1D2B to inactivate RAB7, thereby preventing the fusion of MVEs with lysosomes and enabling the secretion of ILVs as exosomes. These findings establish that RAB31 has dual functions in the biogenesis of exosomes: driving ILVs formation and suppressing MVEs degradation, providing an exquisite framework to better understand exosome biogenesis.
The proposed model for the functions of RAB31 in exosome pathway
EGFR are endocytosed into cells to form signaling endosomes (SE) and early endosomes (EE) regulated by RAB5, and then are transported from early to late endosomes (LE) regulated by transition from RAB5 to RAB7. a At this time, ESCRT machinery sorts the ubiquitylated EGFR into intraluminal vesicles (ILVs) that are destined to lysosomes for degradation by the fusion of multivesicular endosomes (MVEs) with lysosomes regulated by RAB7. b However, high RAB31, guarding on the late endosomes, encounters active EGFR and can be activated via tyrosine phosphorylation by EGFR, and then active RAB31 engages FLOTs in lipid rafts to drive EGFR entry into MVEs to form ILVs. Meanwhile, RAB31 recruits TBC1D2B to inactivate RAB7 preventing the fusion of MVEs with lysosomes, thereby enabling that the sequestered EGFR ILVs are secreted as exosomes. c Representative image of MVE membrane budding to form ILVs driven by the active RAB31 in NCI-H1975 cells. The white triangles indicate the budding moments of MVE membrane. Immunofluorescence of endogenous RAB31 (green) and CD63 (red) in NCI-H1975 cells. Scale bar, 5 μm.