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

Nature Cell Biology volume 22, pages 187–199 (2020)Cite this article

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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.

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Fig. 1: Identification of proteins secreted via autophagy-dependent pathways using LC3 proximity-dependent biotinylation and quantitative secretomics.
Fig. 2: LC3-II and BirA*–LC3 biotinylated targets are secreted within EVs.
Fig. 3: Endogenous LC3 localizes with endosomes and EV-associated tetraspanins.
Fig. 4: TMT quantitative secretomics identifies EV proteins secreted via the LC3-conjugation machinery.
Fig. 5: LC3-conjugation machinery is required for EV loading and secretion of SAFB and HNRNPK.
Fig. 6: LDELS regulates the small non-coding RNA composition of EVs.
Fig. 7: LDELS requires nSMase2 and FAN.

Data availability

The MS proteomics data associated with this study have been deposited to the ProteomeXchange Consortium via the PRIDE80 partner repository with the dataset identifier PXD015479. RNA-seq data have been deposited in GEO: GSE137618. Furthermore, the data and/or reagents that support the findings of this study are available from the corresponding author, J.D., upon reasonable request. Source data for Figs. 1–7 and Extended Data Figs. 1–7 are provided online.

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Acknowledgements

We thank M. Krummel (UCSF) for generously providing reagents, members of the Debnath laboratory for helpful discussions and R. Perera (UCSF) for critical reading of the manuscript. We also thank S. Kilinc and A. Goga (UCSF) for assistance with EV concentration and size analysis, and A. Navickas for advice on RNA-seq library preparation. Grant support includes the NIH (CA201849, CA126792, CA201849 and CA213775 to J.D.; R00CA194077 and R01CA240984 to H.G.; K08CA184116 to A.P.W.; and AG057462 to J.D. and E.J.H.), the DOD BCRP (W81XWH-11-1-0130 to J.D.), the Samuel Waxman Cancer Research Foundation (to J.D.), a UCSF QB3 Calico Longevity Fellowship (to J.D. and A.M.L.) and a Dale Frey Breakthrough Award from the Damon Runyon Cancer Research Foundation (DFS 14-15 to A.P.W.). Fellowship support includes a Banting Postdoctoral Fellowship from the Government of Canada (201409BPF-335868) and a Cancer Research Society Scholarship for Next Generation of Scientists to A.M.L., NSF Graduate Student Fellowships (1650113 to T.S. and 1144247 to J.G.), a Canadian Institutes of Health Research Post-doctoral Fellowship to F.K., NRSA awards from the NCI (F31CA217015 to T. Marsh, F30CA224693 to J.Y.L.), a UCSF IRACDA Postdoctoral Fellowship (K12GM081266) to T. Monkkonen and an NCI T32 training grant (T32CA108462) to A.V.

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Authors and Affiliations

  1. Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, CA, USA

    Andrew M. Leidal, Timothy Marsh, Tina Solvik, Jordan Ye, Juliet Goldsmith, Jennifer Y. Liu, Yu-Hsin Huang, Teresa Monkkonen, Ariadne Vlahakis, Eric J. Huang & Jayanta Debnath

  2. Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA

    Hector H. Huang & Arun P. Wiita

  3. State Key Laboratory of Membrane Biology, Tsinghua University-Peking University Joint Center for Life Sciences, School of Life Sciences, Tsinghua University, Beijing, China

    Dachuan Zhang & Li Yu

  4. Department of Surgery, University of California San Francisco, San Francisco, CA, USA

    FuiBoon Kai

  5. Department of Biochemistry and Biophysics, Department of Urology, and Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA

    Hani Goodarzi

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  1. Andrew M. Leidal
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Contributions

J.D. and A.M.L. conceived the study and designed the experiments. A.M.L., H.H.H., A.P.W. and J.D. designed and optimized the quantitative proteomics workflows. A.M.L., J.D. and H.G. designed, performed and analysed the extracellular and intracellular RNA-seq experiments. A.M.L., T.S., J.Y., F.K., J.Y.L., T. Monkkonen and A.V. performed biochemical and cell biological experiments. H.H.H. and A.P.W. performed MS and analysed the resulting liquid chromatography (LC)–MS/MS data with A.M.L. A.M.L. and J.G. performed bioinformatics analysis of the BirA*–LC3-labelled secretome. A.M.L., T. Marsh, Y.-H.H. and E.J.H. performed mouse experiments and primary astrocyte isolation. D.Z. and L.Y. performed APEX staining and TEM. A.M.L. and J.D. analysed the biochemical and cell biological data. A.M.L. and J.D. wrote the paper, with input from all other authors.

Corresponding author

Correspondence to Jayanta Debnath.

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Competing interests

J.D. is a Scientific Advisory Board Member for Vescor Therapeutics, LLC.

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Extended data

Extended Data Fig. 1 Functional validation of the BirA*-LC3 recombinant probe.

a, Cells stably expressing myc-BirA*-LC3, myc-BirA* or vector control were incubated in either full (F) or serum free media (S) for 4h in the absence or presence of 50 µM chloroquine (CQ) for the last 1h. Cells were lysed and subject to immunoblotting for indicated proteins (n=2 biologically independent experiments). b, Representative images of cells stably expressing GFP-LC3 and myc-BirA*-LC3 or myc-BirA* and immunostained with anti-myc antibody (n=3 biologically independent samples). c, Representative images of cells stably expressing myc-BirA*-LC3 or myc-BirA* and co-immunostained with anti-LC3 (green) and anti-myc (magenta) antibody (n=3 biologically independent samples). d, Biotinylation blots reproduced from Fig. 1b with accompanying Ponceau S stained membranes of whole cell lysate (WCL) and conditioned media (CM) (n=3 biologically independent experiments). e, Schematic of experiment to test for intracellular versus extracellular origin of BirA* and BirA*-LC3-mediated biotinylated targets isolated from CM. f, Representative Strep-HRP blot for biotinylated proteins in the precipitated CM from myc-BirA*-LC3 or myc-BirA* cells co-incubated (Co) or post-incubated (Post) with 50 µM biotin for 24h and negative control (Neg). CM was probed to validate expression and secretion of the myc-tagged recombinant proteins (n=2 biologically independent experiments). Unprocessed blots available in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 BirA*-LC3B-labelled secretome is enriched in RBPs.

a, Volcano plot of BirAf*-LC3-labeled secretome quantified by mass spectrometry. SILAC labelled biotin-tagged proteins plotted according to -log10 p-values as determined by two-tailed t-test and log2 fold enrichment (BirA*-LC3/BirA*) (n=3 biologically independent samples). Grey horizontal dotted line: significance cut-off with p-value of 0.05. Log2 fold change reflects LC3-BirA* to BirA* alone ratio. Grey vertical dotted line: 2-fold enriched and de-enriched cut-off. Pink: significantly enriched proteins relative to BirA* alone. Red: Class I enriched proteins represented in heat map in Fig. 1. Inset: Expanded view of significantly enriched proteins. b, Venn diagram showing overlap of secretory autophagy candidates (Class I and II hits) with the LC3B intracellular interactome defined in Behrends et al. 2010. c, Venn diagram showing the overlap of secretory autophagy candidates (Class I and II hits) with the entire ATG8 intracellular interactome defined in Behrends et al. 2010. d, Ranked list of proteins with greatest connectivity to secretory autophagy candidates as determined by the Enrichr gene enrichment analysis tool (n=3 biologically independent samples; 200 enriched proteins in Class I + II datasets). Statistical significance calculated by one-way Fisher’s exact test and adjusted using the Benjamini–Hochberg method. LC3 family members highlighted in red. e, Network map of autophagy-dependent secretion candidates. Class I and II secretory autophagy candidates mapped to zero-order protein interaction network using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and proteins associated with extracellular exosomes or with RNA-binding functions coloured in red and blue, respectively. f, Pie chart plotting percentage of Class I and II secretory autophagy candidates assigned to Gene Ontology (GO) term extracellular exosome by PANTHER. g, Venn diagram showing overlap of class I and II secretory autophagy candidates with the mRNA binding proteins from Castello et al. 2012. Data available in Source Data Extended Data Fig. 2.

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Extended Data Fig. 3 Endogenous LC3-II is secreted within EVs isolated from cultured cells and murine plasma.

a, Whole cell lysate (WCL) and extracellular vesicle lysates (EVs) from murine RAW264.7 macrophages treated with 100 ng/ml LPS for 24h and 20 μM nigericin for 1 h, murine B16F10 melanoma cells, and murine LLC1 cells were immunoblotted for LC3, SAFB, HNRNPK and extracellular vesicle marker proteins (n=2 biologically independent experiments). b, Workflow employed to obtain plasma and tissue from CAG-CreER;Atg12flox/flox mice in which Atg12 was systemically deleted via tamoxifen treatment. c, Extracellular vesicles (EVs) isolated from the plasma of naïve wild-type mice (CAG-CreER or Atg12flox/flox) and mice in which Atg12 was systemically deleted by 4-OHT treatment (CAG-CreER; Atg12flox/flox) were lysed and immunoblotted for LC3 and the indicated extracellular vesicle marker proteins (n=2 biologically independent experiments). d, Whole cell lysates (WCL) derived from the renal tissue of mice in Panel c were immunoblotted for LC3 and the indicated marker proteins (n=2 biologically independent experiments). e, Workflow employed to obtain CM from murine astrocytes (CAG-CreER;Atg5flox/flox) in which Atg5 was deleted ex vivo via 4-OHT treatment. f, Extracellular vesicles (EVs) isolated from the conditioned media of naive wild-type (Atg5flox/flox) primary astrocytes and astrocyte cultures in which Atg5 was deleted ex vivo (CAG-CreER; Atg5flox/flox) by 4-OHT treatment were lysed and immunoblotted for LC3 and CD9 (n=2 biologically independent experiments). g, Whole cell lysates (WCL) primary astrocyte cultures in Panel d were immunoblotted for LC3 and CD9 (n=2 biologically independent experiments). h, Representative fluorescence micrographs from wild-type, ATG7-/- and ATG14-/- HEK293T cells transfected with mCherry-Rab5Q79L (yellow). Cells were immunostained for endogenous LC3 (green) and CD63 (magenta) (n=3 biologically independent samples). Scale bar=10μm. Unprocessed blots available in Source Data Extended Data Fig. 3.

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Extended Data Fig. 4 Components of stress granules and P-bodies secreted in EVs through mechanisms requiring the LC3-conjugation machinery.

a, Proportion of BirA*-LC3B- labelled secretome (Class I, II candidates) detected in the total extracellular vesicle (EV) proteome defined by TMT quantitative mass spectrometry. b, Venn diagram showing overlap of EV components requiring ATG7 and ATG12 for secretion with the fixed and unfixed stress granule proteome from Jain et al., 2016. c, Venn diagram showing the overlap of EV components requiring ATG7 and ATG12 for secretion with the P-body proteome from Hubstenberger et al., 2017. d, Representative fluorescence micrographs from wild-type HEK293T cells transfected with mCherry-Rab5Q79L (blue) and immunostained for endogenous LC3 (green) and SAFB or HNRNPK (magenta) (n=3 biologically independent samples). Scale bar=10μm. e, Whole cell (WCL) and EV lysates harvested from equal numbers of cells stably expressing non-targeting (NT) or ATG3 shRNA were immunoblotted for indicated proteins (n=3 biologically independent samples). f, Quantification of indicated protein levels in EVs from cells stably expressing shRNAs targeting ATG3 relative to non-targeting shRNA (mean ±s.e.m.; n=3 biologically independent samples). g, Quantification of Lactate Dehydrogenase (LDH) in EV-depleted conditioned media from wild-type (WT) HEK293T cells treated 100 μM Etoposide (Etop) for 24h or WT and ATG knockout cells serum starved for 24h (mean ±s.e.m.; n=3 biologically independent experiments). h, Cell death in wild-type (WT) and ATG knockout cells (KO) after 24h in full serum media (FM) or serum starved media (SS) quantified using Calcein-AM and ethidium bromide staining (mean ±s.e.m.; n=3 biologically independent experiments). i, Whole cell and EV lysates from wild-type cells grown in EV-depleted full serum media (FM) or EV-depleted FM with 100 nM Rapamycin (Rap) for 24h. Immunoblots probed against the indicated proteins (n=2 biologically independent experiments). j, Quantification of the relative levels of indicated proteins in EVs from Rap-treated cells in Panel d (line=mean; n=2 biologically independent experiments). Data and unprocessed blots available in Source Data Extended Data Fig. 4.

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Extended Data Fig. 5 LC3-conjugation machinery controls EV-mediated secretion of diverse RBPs.

a, EVs from WT and ATG deficient cells normalized for protein concentration and immunoblotted to detect endogenous LC3A, LC3B, LC3C, GABARAP (GR), GABARAPL1 (GRL1), GABARAPL2 (GRL2), and indicated marker proteins (n=2 biologically independent experiments). b, Whole cell (WCL) and EV lysates from WT and ATG7-/- cells were normalized for protein concentration and immunoblotted for indicated proteins (n=3 biologically independent experiments). c, HEK293T cells co-transfected with FLAG-tagged G3BP1, LARP1 or SF3A1, and myc-tagged LC3B, GABARAP (GR), LC3C respectively, or myc-BirA* were lysed, immunoprecipitated (IP) with anti-myc antibody and immunoblotted (WB) with indicated antibodies (n=3 biologically independent experiments). d, Diagram mapping the domains and primary LC3-interaction region (LIR) in SAFB. e, Volcano plot of mRNA and long non-coding RNA (large RNA) detected in EVs from WT and ATG7-/- cells. Results plotted according to -log10 p-values as determined by DESeq2 and log2 fold enrichment (n=3 biologically independent samples; WT/ATG7-/-). Grey dots: RNAs not enriched in EVs from WT or ATG7-/- cells identified with a p-value >0.05 and/or log2 fold change between -0.5 and 0.5 (-0.5<log2FC<0.5). Black dots: Large RNAs enriched in EVs from WT cells or ATG7-/- cells. f, Volcano plot of mRNA and long non-coding RNA (large RNA) detected in EVs from WT and ATG12-/- cells. Results plotted according to -log10 p-values as determined by DESeq2 and log2 fold enrichment (n=3 biologically independent samples; WT/ATG12-/-). Grey dots: RNAs not enriched in EVs from WT or ATG12-/- cells identified with a p-value >0.05 and/or log2 fold change between -0.5 and 0.5 (-0.5<log2FC<0.5). Black dots: Large RNAs enriched in EVs from WT or ATG12-/- cells. g, Venn diagram showing the overlap of mRNA and long non-coding RNAs (large RNAs) enriched in EVs from WT relative to ATG7-/- cells and EVs from WT relative to ATG12-/- cells. Data and unprocessed blots available in Source Data Extended Data Fig. 5.

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Extended Data Fig. 6 LC3 delivery into ILVs of Rab5Q79L endosomes requires CHMP4b and nSMase2, but is independent of other ESCRT machinery components.

a, Representative fluorescence micrographs from wild-type HEK293T cells co-transfected with mCherry-Rab5Q79L (magenta) and non-targeting (NT) control siRNA or siRNAs that deplete ATG7, ALIX, TSG101, VPS4a/b, CHMP3, CHMP4b and nSMase2. Cells were immunostained for endogenous LC3 (green) (n=2 biologically independent experiments). Scale bar=10μm. b, Scatter plot of the proportion of mCherry-Rab5Q79L endosomes that overlap with LC3 in immuno-stained cells in Panel a (mean ± s.e.m.; n=23 biologically independent samples). Statistical significance calculated by one-way ANOVA coupled with Fisher’s least significant difference test. c, Lysates from cells in Panel a were immunoblotted with antibodies the various siRNA targets and GAPDH as a loading control. Representative blots are shown (n=2 biologically independent experiments). Non-specific bands are indicated with an asterisk (*). d, Quantitative PCR (QPCR) measurement of nSMase2 mRNA in HEK293T cells transfected with siRNAs targeting nSMase2, nSMase2 (nSM2) relative to non-targeting siRNA (NT) control cells (line=mean; n=1, 2 technical replicates). Data and unprocessed blots available in Source Data Extended Data Fig. 6.

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Extended Data Fig. 7 LC3-dependent EV loading and secretion (LDELS) requires FAN and nSMase2.

a, Whole cell lysate harvested from equal numbers of HEK293Ts stably expressing non-targeting (NT) shRNA or shRNAs that deplete ATG7 or nSMase2(nSM2) immunoblotted for indicated proteins (n=2 biologically independent experiments). b, Quantitative PCR (QPCR) for nSMase2 mRNA in HEK293Ts stably expressing shRNAs targeting ATG7, nSMase2 (nSM2) relative to non-targeting shRNA (NT) control cells (line=mean; n=1, 2 technical replicates). c, Whole cell lysates from HEK293Ts stably expressing non-targeting shRNA (NT) or shRNAs that deplete ATG7 or FAN were immunoblotted with antibodies for the indicated proteins (n=2 biologically independent experiments). d, HEK293Ts were EBSS starved for the indicated times, treated with DMSO or 50nM Bafilomycin A1 (Baf A1) for 1 h prior to lysis, lysed and immunoblotted for FAN and the indicated proteins (n=2 biologically independent experiments). e, HEK293Ts expressing non-targeting (NT) shRNA or shRNA that depletes FAN were starved in EBSS for 4h, treated with DMSO or 50nM Bafilomycin A1 (Baf A1) for 1h prior to lysis, lysed and immunoblotted for the indicated proteins (n=2 biologically independent experiments). f, Representative fluorescence micrographs from wild-type cells co-transfected with mCherry-Rab5Q79L (magenta) and non-targeting (NT) control siRNA or siRNAs that deplete FAN. Cells were immunostained for endogenous LC3 (green)(n=2 biologically experiments). Scale bar=10μm. g, Lysates from cells in Panel f were immunoblotted with antibodies against FAN and GAPDH as a loading control (n=2 biologically independent experiments). h, Scatter plot of the proportion of mCherry-Rab5Q79L endosomes overlapping with LC3 in immuno-stained cells in Panel f (mean ±s.e.m.; n=22 biologically independent samples). Statistical significance calculated by unpaired two-tailed t-test. i, Whole cell lysate from HEK293Ts analysed in Fig. 7j that were co-expressing non-targeting (NT) shRNA or shRNA that depletes endogenous FAN along with FLAG-tagged wild-type FAN (WT) or mutant FAN (F199A) were immunoblotted for the indicated proteins (n=2 biologically independent experiments). j, Proposed model for LC3-dependent EV loading and secretion (LDELS) in comparison to classical autophagy. Data and unprocessed blots available in Source Data Extended Data Fig. 7.

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Leidal, A.M., Huang, H.H., Marsh, T. et al. The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles. Nat Cell Biol 22, 187–199 (2020). https://doi.org/10.1038/s41556-019-0450-y

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