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.

外泌体作为腔内囊泡 (ILV) 在多泡内体 (MVE) 内产生，并在 MVE 与细胞膜融合期间分泌。外泌体生物发生的机制仍然缺乏探索。在这里，我们确定 RAB31 标记并控制了一个独立于 ESCRT 的外泌体途径。被表皮生长因子受体 (EGFR) 磷酸化的活性 RAB31 与脂筏微域中的 flotillin 蛋白结合，以驱动 EGFR 进入 MVE 以形成 ILV，这与 ESCRT（运输所需的内体分选复合物）机制无关。活性 RAB31 与 SPFH 结构域相互作用并通过 flotillin 蛋白的 Flotillin 结构域驱动 ILV 形成。同时，RAB31 招募 GTPase 激活蛋白 TBC1D2B 来灭活 RAB7，从而防止 MVE 与溶酶体融合，并使 ILV 作为外泌体分泌。这些发现表明，RAB31 在外泌体的生物发生中具有双重功能：驱动 ILV 形成和抑制 MVE 降解，为更好地理解外泌体生物发生提供了一个精致的框架。