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.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Adaptor Proteins, Vesicular Transport / deficiency
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Adaptor Proteins, Vesicular Transport / genetics
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Animals
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Autophagosomes / chemistry
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Autophagosomes / metabolism*
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Autophagy / genetics*
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Autophagy-Related Protein 7 / deficiency
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Autophagy-Related Protein 7 / genetics
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Autophagy-Related Proteins / deficiency
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Autophagy-Related Proteins / genetics
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Biological Transport
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Biotinylation
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Extracellular Vesicles / chemistry
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Extracellular Vesicles / metabolism*
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Gene Expression Profiling
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Gene Expression Regulation
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HEK293 Cells
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Heterogeneous-Nuclear Ribonucleoprotein K / genetics
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Heterogeneous-Nuclear Ribonucleoprotein K / metabolism
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Humans
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Intracellular Signaling Peptides and Proteins / genetics
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Intracellular Signaling Peptides and Proteins / metabolism
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Lysosomes / chemistry
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Lysosomes / metabolism
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Matrix Attachment Region Binding Proteins / genetics
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Matrix Attachment Region Binding Proteins / metabolism
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Mice
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Microtubule-Associated Proteins / genetics*
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Microtubule-Associated Proteins / metabolism
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Nuclear Matrix-Associated Proteins / genetics
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Nuclear Matrix-Associated Proteins / metabolism
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Proteomics / methods
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RAW 264.7 Cells
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RNA, Small Untranslated / genetics
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RNA, Small Untranslated / metabolism
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RNA-Binding Proteins / classification
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RNA-Binding Proteins / genetics*
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RNA-Binding Proteins / metabolism
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Receptors, Estrogen / genetics
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Receptors, Estrogen / metabolism
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Sphingomyelin Phosphodiesterase / genetics
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Sphingomyelin Phosphodiesterase / metabolism
Substances
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ATG14 protein, human
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Adaptor Proteins, Vesicular Transport
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Autophagy-Related Proteins
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Heterogeneous-Nuclear Ribonucleoprotein K
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Intracellular Signaling Peptides and Proteins
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MAP1LC3A protein, human
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Matrix Attachment Region Binding Proteins
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Microtubule-Associated Proteins
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NSMAF protein, human
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Nuclear Matrix-Associated Proteins
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RNA, Small Untranslated
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RNA-Binding Proteins
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Receptors, Estrogen
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SAFB protein, human
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HNRNPK protein, human
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SMPD3 protein, human
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Sphingomyelin Phosphodiesterase
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ATG7 protein, human
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Autophagy-Related Protein 7