Abstract

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.

Highlights

  • Extracellular vesicles (EVs) are a heterogeneous group of cellderived membranous structures mainly comprising exosomes and microvesicles, which originate from the endosomal system and are shed from the plasma membrane, respectively.[1]

  • epidermal growth factor receptor (EGFR)-HA was co-localizated with EEA1, an early endosome marker, but neither LAMP1, a lysosomal marker, nor GFP-tagged LC3 puncta, an indicator of the autophagosomes induced by serum starvation (Supplementary information, Fig. S2a–c), suggesting that serum starvation does not affect the endocytosis of EGFR and this endocytic EGFR is localized to CD63positive multivesicular endosomes (MVEs) driven by RAB31Q65L rather than transported to lysosomes

  • Active RAB31 engages flotillin proteins (FLOTs) to drive EGFR-containing intraluminal vesicles (ILVs) formation depending on cholesterol and ceramide in lipid raft microdomains we further showed that ESCRT components Hrs and Tsg[101], as well as Alix[1,10,23] were not involved in the production of EGFR-containing exosomes driven by RAB31Q65L, baucase the EGFR protein level in the concentrated conditional media was not affected by knocking down of these molecules using two short hairpin RNAs (Supplementary information, Fig. S4a, b), suggesting that the formation of exosomes driven by RAB31Q65L is separated from that of exosomes driven by ESCRT

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Summary

Introduction

Extracellular vesicles (EVs) are a heterogeneous group of cellderived membranous structures mainly comprising exosomes and microvesicles, which originate from the endosomal system and are shed from the plasma membrane, respectively.[1] Exosomes are present in biological fluids and function in intercellular communication, allowing cells to exchange proteins, lipids, genetic materials, amino acids and metabolites.[2,3,4,5,6,7] Exosomes are generated as intraluminal vesicles (ILVs) within the lumen of endosomes during their maturation into multivesicular endosomes (MVEs) and secreted by the fusion of MVEs with the cell surface.[1,8] The formation of ILVs by the inward budding of MVEs is mostly mediated by the ESCRT (endosomal sorting complex required for transport) machinery,[1,8,9] as many cargoes, including currently well-known syndecan, tetraspanin CD63, and Toll-like receptor trafficking chaperone UNC93B1 etc., recruit Syntenin-AlixESCRT-III pathway by the cytoplasmic tails to mediate their ILV formation.[1,10,11,12,13] ESCRTIII is always considered to be required for the scission of the ILVs into the MVE lumen,[1] ILVs within the lumen of MVEs are still formed in the ESCRT-depleted cells, indicating that the ESCRT-independent pathways for ILV formation exist.[9] the first ESCRT-independent mechanism for ILV biogenesis was shown to require sphingolipid ceramide, which may allow the generation of raft-based microdomains inducing a spontaneous negative curvature on the membranes.[14]

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