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. Here we 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.

STING, an endoplasmic reticulum (ER) transmembrane protein, mediates innate immune activation upon cGAMP stimulation and is degraded through autophagy. Here, we report that activated STING could be transferred between cells to promote antitumor immunity, a process triggered by RAB22A-mediated non-canonical autophagy. Mechanistically, RAB22A engages PI4K2A to generate PI4P that recruits the Atg12–Atg5–Atg16L1 complex, inducing the formation of ER-derived RAB22A-mediated non-canonical autophagosome, in which STING activated by agonists or chemoradiotherapy is packaged. This RAB22A-induced autophagosome fuses with RAB22A-positive early endosome, generating a new organelle that we name Rafeesome (RAB22A-mediated non-canonical autophagosome fused with early endosome). Meanwhile, RAB22A inactivates RAB7 to suppress the fusion of Rafeesome with lysosome, thereby enabling the secretion of the inner vesicle of the autophagosome bearing activated STING as a new type of extracellular vesicle that we define as R-EV (RAB22A-induced extracellular vesicle). Activated STING-containing R-EVs induce IFNβ release from recipient cells to the tumor microenvironment, promoting antitumor immunity. Consistently, RAB22A enhances the antitumor effect of the STING agonist diABZI in mice, and a high RAB22A level predicts good survival in nasopharyngeal cancer patients treated with chemoradiotherapy. Our findings reveal that Rafeesome regulates the intercellular transfer of activated STING to trigger and spread antitumor immunity, and that the inner vesicle of non-canonical autophagosome originated from ER is secreted as R-EV, providing a new perspective for understanding the intercellular communication of organelle membrane proteins.