LC3-dependent EV loading and secretion (LDELS) promotes TFRC (transferrin receptor) secretion via extracellular vesicles

Abstract

LC3-dependent EV loading and secretion (LDELS) is a secretory autophagy pathway in which the macroautophagy/autophagy machinery facilitates the packaging of cytosolic cargos, such as RNA-binding proteins, into extracellular vesicles (EVs) for secretion outside of the cell. Here, we identify TFRC (transferrin receptor), one of the first proteins found to be secreted via EVs, as a transmembrane cargo of the LDELS pathway. Similar to other LDELS targets, TFRC secretion via EVs genetically requires components of the MAP1LC3/LC3-conjugation machinery but is independent of other ATGs involved in classical autophagosome formation. Furthermore, the packaging and secretion of this transmembrane protein into EVs depends on multiple ESCRT pathway components and the small GTPase RAB27A. Based on these results, we propose that the LDELS pathway promotes TFRC incorporation into EVs and its secretion outside the cell.Abbreviations: ATG: autophagy related; ESCRT: endosomal sorting complexes required for transport; EV: extracellular vesicle; EVP: extracellular vesicle and particle; ILV: intralumenal vesicle; LDELS: LC3-dependent EV loading and secretion; LIR: LC3-interacting region; MVE: multivesicular endosome; RBP: RNA-binding protein; TMT: tandem mass tag; TFRC: transferrin receptor.

Keywords: ATG7; ATG8; LC3-conjugation; extracellular vesicles; secretory autophagy; transferrin receptor.

Figure 1.

ATG7 is genetically required for the secretion of transmembrane proteins via extracellular vesicles. (A) Volcano plot of the proteins identified in 100,000 g EVP-enriched fractions for WT and ATG7^−/− HEK293T cells quantified by TMT mass spectrometry in Leidal et al. 2020 [8]. TMT-labeled proteins are plotted according to their -log10 P values as determined by two-tailed t-test and log2 fold enrichment (WT/ATG7^−/−; n = 4). Gray dots: proteins not relatively enriched in EVPs from WT or ATG7^−/− cells identified with P > 0.05 and/or log2 fold change between −0.5 and 0.5. Green dots: proteins significantly enriched in EVPs from WT cells relative to ATG7^−/− cells. Red dots: proteins significantly enriched in EVPs from ATG7^−/− cells relative to WT cells. Yellow dots: membrane proteins identified by gene ontology (GO) analysis. (B) 100,000 g EVP fractions (100 K) from WT and ATG7^−/− cells were collected, normalized for protein concentration, and immunoblotted to detect the endogenous levels of the indicated proteins (n = 3). (C) Quantification of TFRC levels in EVPs from ATG7^−/− (cyan) cells relative to WT (gray) (mean ± s.e.m.; n = 3). Statistical significance calculated by one-way ANOVA coupled with Tukey’s post hoc test. (D) Schematic detailing the differential centrifugation protocol employed to isolate small EVP-enriched fractions from cell cultures. (E) Whole cell lysate (WCL) and fractionated conditioned media (CM) collected from serum-starved HEK293Ts. CM was subjected to serial differential ultracentrifugation to recover large EVs (10,000 g; 10 K) and small EVPs (100,000 g; 100 K). Equal volumes of fractionated CM were probed for the indicated proteins alongside WCL normalized to protein content of the 100 K fraction (n = 3). (F) Quantification of TFRC in the indicated fractions of CM from serum-starved cells relative to WCL (mean ± s.e.m.; n = 3). Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test. (G) Representative immunoblots of EVs from CM separated via linear sucrose density gradient ultracentrifugation, fractionated and immunoblotted to detect TFRC, LC3, and CD63 (n = 3). (H) Percent of total secreted TFRC, LC3, and CD63 detected in individual linear sucrose gradient fractions (mean ± s.e.m.; n = 3). (I) Representative immunoblots of EVs immunopurified from concentrated CM fractions using a normal mouse IgG isotype control or an antibody against TFRC (n = 3).

Figure 2.

TFRC (transferrin receptor) secretion in EVs requires the LC3-conjugation pathway but is independent of other components mediating classical autophagosome formation. (A) Whole cell lysate (WCL; top) and 100,000 g EV fractions (100 K; bottom) from the indicated cell types were collected, normalized for protein concentration and immunoblotted to detect the endogenous levels of the indicated proteins (n = 3). Individual lanes shown are from the same representative immunoblot. (B) Quantification of TFRC and LC3-II levels in EVs from the indicated ATG-deficient cell lines relative to WT (mean ± s.e.m.; n = 3). Statistical significance calculated by one-way ANOVA coupled with Tukey’s post hoc test. (C) WCL (top) and 100 K fractions (bottom) from equal numbers of wild-type HEK293T cells transfected with non-targeting (NT) control siRNA or siRNAs targeting ATG3 and ATG5 and immunoblotted for the indicated proteins (n = 3). (D) Quantification of TFRC and LC3-II levels in EV fractions from cells treated with siRNAs targeting the indicated proteins relative to cells treated with NT control siRNA (mean ± s.e.m.; n = 3). Statistical significance calculated by one-way ANOVA coupled with Tukey’s post hoc test. (E) HEK293Ts cultured in serum-starved or complete media and treated with DMSO or 20 nM bafilomycin A₁ (Baf A1) for 18 h were then lysed and immunoblotted for TFRC and the indicated proteins (n = 3). Quantifications of TFRC, SQSTM1/p62, and LC3-II relative protein levels (values normalized to GAPDH) in WCLs from cells cultured in the indicated conditions relative to complete, vehicle-treated media are shown. (F) WCL (top) and 100 K fractions (bottom) from serum-starved HEK293Ts treated with vehicle or 20 nM BafA1 for 16 h were collected and immunoblotted to detect the indicated proteins (n = 3). (G) Quantification of TFRC in EV fractions from BafA1-treated cells relative to vehicle control (mean ± s.e.m.; n = 3). Statistical significance calculated by an unpaired two-tailed t-test.

Figure 3.

Atg8-family proteins bind TFRC via a cytoplasmic domain LIR motif. (A) HEK293T cells co-transfected with HA-tagged TFRC and MYC-tagged LC3A, LC3B, LC3C, GABARAP (GR), GABARAPL1 (GRL1), GABARAPL2 (GRL2) were lysed, immunoprecipitated with anti-MYC antibody and immunoblotted with the indicated antibodies (n = 3). (B) Domain map and the putative LIRs of TFRC highlighted in yellow. (C) Cells co-transfected with luciferase-tagged cytoplasmic domain from WT TFRC(1–63) or mutant TFRC(1–63)^Y20AF23A (AA) along with MYC-tagged LC3B or MYC-tagged GABARAP as indicated. Cells were lysed, immunoprecipitated with anti-MYC antibody and immunoblotted with the indicated antibodies (n = 3). (D) WCL (top) and 100 K fractions (bottom) from cells stably expressing HA-tagged WT TFRC, mutant TFRC^Y20AF23A (AA), or truncated TFRC lacking the cytoplasmic domain (TFRC∆3-59; ∆CD) were immunoblotted for the indicated markers (n = 3). (E) Quantification of TFRC-Ha in EVs from equal numbers of HEK293T cells expressing HA-tagged WT TFRC, mutant TFRC^Y20AF23A (AA), or truncated TFRC lacking the cytoplasmic domain (TFRC∆3-59; ∆CD) (mean ± s.e.m.; n = 3). Statistical significance calculated by one-way ANOVA coupled with Tukey’s post hoc test. (F) Representative immunoblots of the indicated proteins from untreated EVs and EVs incubated with 100 µg ml⁻¹ trypsin and/or 1% Triton X-100 (TX-100) for 30 min at 4°C (n = 3).

Figure 4.

ESCRT machinery and RAB27A promotes TFRC loading and secretion via EVs. (A) Whole cells lysates (WCL; left) and EV lysates (100 K; right) from cells treated in the absence or presence of 5 µM GW4869 for 24 h and immunoblotted for the indicated proteins (n = 3). (B) Quantification of TFRC levels in EV fractions from GW4869-treated cells relative to vehicle control (mean ± s.e.m.; n = 3). Statistical significance calculated by an unpaired two-tailed t-test. (C) WCL (top) and 100 K fractions (bottom) from equal numbers of wild-type HEK293T cells transfected with non-targeting (NT) control siRNA or siRNAs targeting ATG7, PDCD6IP, HGS, and TSG101 and immunoblotted for the indicated proteins (n = 3). (D) Quantification of TFRC and LC3-II levels in EV fractions from cells treated with siRNAs targeting the indicated proteins relative to cells treated with NT control siRNA (mean ± s.e.m.; n = 3). Statistical significance calculated by one-way ANOVA coupled with Tukey’s post hoc test. (E) WCL (top) and 100 K fractions (bottom) from equal numbers of cells stably expressing non-targeting (NT) shRNA or shRNAs that deplete RAB27A immunoblotted for indicated proteins (n = 3). (F) Quantification of TFRC and LC3-II levels in EV fractions from RAB27A depleted cells (shRAB27A) relative to controls expressing NT shRNA. (mean ± s.e.m.; n = 3).