The LC3-conjugation Machinery Specifies the Loading of RNA-binding Proteins into Extracellular Vesicles

Overview

Journal Nat Cell Biol

Specialty Cell Biology

Date 2020 Jan 15

PMID 31932738

Citations 241

Authors

Andrew M Leidal

Hector H Huang

Timothy Marsh

Tina Solvik

Dachuan Zhang

Jordan Ye

FuiBoon Kai

Juliet Goldsmith

Jennifer Y Liu

Yu-Hsin Huang

Teresa Monkkonen

Ariadne Vlahakis

Eric J Huang

Hani Goodarzi

Li Yu

Arun P Wiita

Jayanta Debnath

Affiliations

Soon will be listed here.

Abstract

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

Citing Articles

Oncogenic RAS induces a distinctive form of non-canonical autophagy mediated by the P38-ULK1-PI4KB axis.

Wang X, Li S, Lin S, Han Y, Zhan T, Huang Z Cell Res. 2025; .

PMID: 40055523 DOI: 10.1038/s41422-025-01085-9.

Engineered Extracellular Vesicles as a New Class of Nanomedicine.

Wen X, Hao Z, Yin H, Min J, Wang X, Sun S Chem Bio Eng. 2025; 2(1):3-22.

PMID: 39975802 PMC: 11835263. DOI: 10.1021/cbe.4c00122.

Aging, cancer, and autophagy: connections and therapeutic perspectives.

Zapateria B, Arias E Front Mol Biosci. 2025; 11:1516789.

PMID: 39935707 PMC: 11811537. DOI: 10.3389/fmolb.2024.1516789.

A 'torn bag mechanism' of small extracellular vesicle release via limiting membrane rupture of en bloc released amphisomes (amphiectosomes).

Visnovitz T, Lenzinger D, Koncz A, Vizi P, Barkai T, Vukman K Elife. 2025; 13.

PMID: 39918406 PMC: 11805505. DOI: 10.7554/eLife.95828.

Targeting secretory autophagy in solid cancers: mechanisms, immune regulation and clinical insights.

Li X, Zhao H Exp Hematol Oncol. 2025; 14(1):12.

PMID: 39893499 PMC: 11786567. DOI: 10.1186/s40164-025-00603-0.

References

Kaur J, Debnath J . Autophagy at the crossroads of catabolism and anabolism. Nat Rev Mol Cell Biol. 2015; 16(8):461-72. DOI: 10.1038/nrm4024. View

Lock R, Kenific C, Leidal A, Salas E, Debnath J . Autophagy-dependent production of secreted factors facilitates oncogenic RAS-driven invasion. Cancer Discov. 2014; 4(4):466-79. PMC: 3980002. DOI: 10.1158/2159-8290.CD-13-0841. View

Bel S, Pendse M, Wang Y, Li Y, Ruhn K, Hassell B . Paneth cells secrete lysozyme via secretory autophagy during bacterial infection of the intestine. Science. 2017; 357(6355):1047-1052. PMC: 5702267. DOI: 10.1126/science.aal4677. View

DeSelm C, Miller B, Zou W, Beatty W, van Meel E, Takahata Y . Autophagy proteins regulate the secretory component of osteoclastic bone resorption. Dev Cell. 2011; 21(5):966-74. PMC: 3244473. DOI: 10.1016/j.devcel.2011.08.016. View

Guo H, Chitiprolu M, Roncevic L, Javalet C, Hemming F, Trung M . Atg5 Disassociates the VV-ATPase to Promote Exosome Production and Tumor Metastasis Independent of Canonical Macroautophagy. Dev Cell. 2017; 43(6):716-730.e7. DOI: 10.1016/j.devcel.2017.11.018. View

Murrow L, Malhotra R, Debnath J . ATG12-ATG3 interacts with Alix to promote basal autophagic flux and late endosome function. Nat Cell Biol. 2015; 17(3):300-10. PMC: 4344874. DOI: 10.1038/ncb3112. View

Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V . Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β. EMBO J. 2011; 30(23):4701-11. PMC: 3243609. DOI: 10.1038/emboj.2011.398. View

Duran J, Anjard C, Stefan C, Loomis W, Malhotra V . Unconventional secretion of Acb1 is mediated by autophagosomes. J Cell Biol. 2010; 188(4):527-36. PMC: 2828925. DOI: 10.1083/jcb.200911154. View

Manjithaya R, Anjard C, Loomis W, Subramani S . Unconventional secretion of Pichia pastoris Acb1 is dependent on GRASP protein, peroxisomal functions, and autophagosome formation. J Cell Biol. 2010; 188(4):537-46. PMC: 2828923. DOI: 10.1083/jcb.200911149. View

10.

Torisu T, Torisu K, Lee I, Liu J, Malide D, Combs C . Autophagy regulates endothelial cell processing, maturation and secretion of von Willebrand factor. Nat Med. 2013; 19(10):1281-7. PMC: 3795899. DOI: 10.1038/nm.3288. View

11.

Cadwell K, Debnath J . Beyond self-eating: The control of nonautophagic functions and signaling pathways by autophagy-related proteins. J Cell Biol. 2017; 217(3):813-822. PMC: 5839790. DOI: 10.1083/jcb.201706157. View

12.

Malhotra V . Unconventional protein secretion: an evolving mechanism. EMBO J. 2013; 32(12):1660-4. PMC: 3680731. DOI: 10.1038/emboj.2013.104. View

13.

Ponpuak M, Mandell M, Kimura T, Chauhan S, Cleyrat C, Deretic V . Secretory autophagy. Curr Opin Cell Biol. 2015; 35:106-16. PMC: 4529791. DOI: 10.1016/j.ceb.2015.04.016. View

14.

Roux K, Kim D, Raida M, Burke B . A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 2012; 196(6):801-10. PMC: 3308701. DOI: 10.1083/jcb.201112098. View

15.

Behrends C, Sowa M, Gygi S, Harper J . Network organization of the human autophagy system. Nature. 2010; 466(7302):68-76. PMC: 2901998. DOI: 10.1038/nature09204. View

16.

Nanjappa V, Thomas J, Marimuthu A, Muthusamy B, Radhakrishnan A, Sharma R . Plasma Proteome Database as a resource for proteomics research: 2014 update. Nucleic Acids Res. 2013; 42(Database issue):D959-65. PMC: 3965042. DOI: 10.1093/nar/gkt1251. View

17.

Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J . STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res. 2014; 43(Database issue):D447-52. PMC: 4383874. DOI: 10.1093/nar/gku1003. View

18.

Castello A, Fischer B, Eichelbaum K, Horos R, Beckmann B, Strein C . Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell. 2012; 149(6):1393-406. DOI: 10.1016/j.cell.2012.04.031. View

19.

Maas S, Breakefield X, Weaver A . Extracellular Vesicles: Unique Intercellular Delivery Vehicles. Trends Cell Biol. 2016; 27(3):172-188. PMC: 5318253. DOI: 10.1016/j.tcb.2016.11.003. View

20.

Mathieu M, Martin-Jaular L, Lavieu G, Thery C . Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nat Cell Biol. 2019; 21(1):9-17. DOI: 10.1038/s41556-018-0250-9. View