The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles

Nat Cell Biol. 2020 Feb;22(2):187-199. doi: 10.1038/s41556-019-0450-y. Epub 2020 Jan 13.

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

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adaptor Proteins, Vesicular Transport / deficiency
  • Adaptor Proteins, Vesicular Transport / genetics
  • Animals
  • Autophagosomes / chemistry
  • Autophagosomes / metabolism*
  • Autophagy / genetics*
  • Autophagy-Related Protein 7 / deficiency
  • Autophagy-Related Protein 7 / genetics
  • Autophagy-Related Proteins / deficiency
  • Autophagy-Related Proteins / genetics
  • Biological Transport
  • Biotinylation
  • Extracellular Vesicles / chemistry
  • Extracellular Vesicles / metabolism*
  • Gene Expression Profiling
  • Gene Expression Regulation
  • HEK293 Cells
  • Heterogeneous-Nuclear Ribonucleoprotein K / genetics
  • Heterogeneous-Nuclear Ribonucleoprotein K / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Lysosomes / chemistry
  • Lysosomes / metabolism
  • Matrix Attachment Region Binding Proteins / genetics
  • Matrix Attachment Region Binding Proteins / metabolism
  • Mice
  • Microtubule-Associated Proteins / genetics*
  • Microtubule-Associated Proteins / metabolism
  • Nuclear Matrix-Associated Proteins / genetics
  • Nuclear Matrix-Associated Proteins / metabolism
  • Proteomics / methods
  • RAW 264.7 Cells
  • RNA, Small Untranslated / genetics
  • RNA, Small Untranslated / metabolism
  • RNA-Binding Proteins / classification
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / metabolism
  • Sphingomyelin Phosphodiesterase / genetics
  • Sphingomyelin Phosphodiesterase / metabolism

Substances

  • ATG14 protein, human
  • Adaptor Proteins, Vesicular Transport
  • Autophagy-Related Proteins
  • Heterogeneous-Nuclear Ribonucleoprotein K
  • Intracellular Signaling Peptides and Proteins
  • MAP1LC3A protein, human
  • Matrix Attachment Region Binding Proteins
  • Microtubule-Associated Proteins
  • NSMAF protein, human
  • Nuclear Matrix-Associated Proteins
  • RNA, Small Untranslated
  • RNA-Binding Proteins
  • Receptors, Estrogen
  • SAFB protein, human
  • HNRNPK protein, human
  • SMPD3 protein, human
  • Sphingomyelin Phosphodiesterase
  • Atg7 protein, human
  • Autophagy-Related Protein 7