A new type of ERGIC-ERES membrane contact mediated by TMED9 and SEC12 is required for autophagosome biogenesis

Abstract

Under stress, the endomembrane system undergoes reorganization to support autophagosome biogenesis, which is a central step in autophagy. How the endomembrane system remodels has been poorly understood. Here we identify a new type of membrane contact formed between the ER-Golgi intermediate compartment (ERGIC) and the ER-exit site (ERES) in the ER-Golgi system, which is essential for promoting autophagosome biogenesis induced by different stress stimuli. The ERGIC-ERES contact is established by the interaction between TMED9 and SEC12 which generates a short distance opposition (as close as 2-5 nm) between the two compartments. The tight membrane contact allows the ERES-located SEC12 to transactivate COPII assembly on the ERGIC. In addition, a portion of SEC12 also relocates to the ERGIC. Through both mechanisms, the ERGIC-ERES contact promotes formation of the ERGIC-derived COPII vesicle, a membrane precursor of the autophagosome. The ERGIC-ERES contact is physically and functionally different from the TFG-mediated ERGIC-ERES adjunction involved in secretory protein transport, and therefore defines a unique endomembrane structure generated upon stress conditions for autophagic membrane formation.

Fig. 1. The ERGIC membrane protein TMED9 regulates autophagosome biogenesis.

a The ERGIC fraction from Atg5 KO MEF cells was collected and digested with the indicated concentrations of trypsin with or without Na₂CO₃ (0.25 M, pH 11) on ice for 20 min. PMSF (1 mM) was incubated to quench trypsin digestion, and the digested membrane was harvested by 100,000× g centrifugation. Cell-free lipidation was performed with the digested membranes and cytosols prepared from starved HEK293T cells. b The LC3-lipidated ERGIC was immunoisolated and mass spectrometry was employed to identify proteins enriched in lipidated ERGIC. c Venn diagram showing membrane-anchored proteins enriched in the ERGIC. d Immunofluorescence of HeLa cells stably expressing TMEDs-V5 after Earle’s Balanced Salf Solution (EBSS) starvation (1 h) with anti-V5 and anti-LC3 antibodies. e Immunoblots showing expression of the TMEDs indicated in d. f Quantification of the LC3 puncta area in d. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P values were obtained from two-tailed t-test. g LC3 lipidation in HeLa cells transfected with control or siRNAs against TMED9 with or without TMED9-V5 re-expression. The cells were incubated in nutrient-rich medium or starved in EBSS in the absence or presence of 500 nM bafilomycin A1 for 1 h. Immunoblots were performed to determine the levels of indicated proteins. Quantification was based on the ratio of lipidated LC3 to tubulin with the control set as 1.00 (control siRNA with starvation and bafilomycin A1). The blots are representative of at least three independent experiments. h Immunofluorescence of HeLa cells (control, TMED9 knockdown (KD) or TMED9 KD with TMED9-V5 expression) with anti-V5 and anti-LC3 antibodies. The cells were starved in EBSS for 1 h. Asterisks indicate cells with TMED9-V5 expression. i Quantification of the LC3 puncta area (μm²/100 μm² cell area) analyzed in h. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. j EM images showing the TMED9-Apex2-labeled membrane and the adjacent autophagosomes. The cells were starved in EBSS for 1 h. Scale bar sizes are indicated in the picture.

Fig. 2. TMED9 is required for multiple types of autophagy in response to different stimuli.

a–c LC3 lipidation in HeLa cells transfected with control or siRNAs against TMED9. The cells were incubated in nutrient-rich medium, low glucose medium (a), medium without serum (b), or treated with 1 μM rapamycin for 8 h (c) in the absence or presence of 500 nM bafilomycin A1. Immunoblots were performed to determine the levels of indicated proteins. Quantification was performed similarly to Fig.1g. The blots are representative of at least three independent experiments. L, low exposure; H, high exposure. d Quantification of the ratio of lipidated LC3 to tubulin with the control set as 1.00 (control siRNA with starvation and bafilomycin A1) analyzed in a–c. Error bars represent standard deviations of > 3 independent experiments. P value was obtained from two-tailed t-test. e HeLa cells stably expressing the mCherry-pHluorin-LC3B were transfected with control or siRNAs against TMED9 and were incubated in nutrient-rich medium, EBSS (1 h), low glucose medium (8 h), medium without serum (8 h), or treated with 1 μM Rapamycin for 8 h. Confocal microscopy was performed. f, g Quantification of autolysosome (red, f) and autophagosome (yellow, g) numbers per cell as shown in e. Error bars represent standard deviations of > 300 cells from three independent experiments (> 100 cells per experiment). P value was obtained from two-tailed t-test. h Control and TMED9 KD HeLa cells were co-transfected with mitoKeima and Parkin and treated with or without 20 µM CCCP for 4 h. Confocal microscopy detection was performed. The results are representative of at least three independent experiments. i The ratio of 561 nm (mitophagy)/440 nm (non-mitophagy) signals was calculated for h as relative mt-Keima signal. Error bars represent standard deviations of > 300 cells from three independent experiments (> 100 cells per experiment). P value was obtained from two-tailed t-test. j Control and TMED9 KD HeLa cells co-expressing mt-Keima and Parkin were treated with or without 20 µM CCCP for 4 h. Cells were analyzed by FACS using V610 and Y610-mCherry detectors (Beckman CytoFLEX LX). The FACS results are representative of at least three independent experiments. k The percentage of cells with mitophagy (P9) based on Y610-mCherry/V610 calculated for j. Error bars represent standard deviations of three experiments. P value was obtained from two-tailed t-test. l Lysosome-dependent turnover of SOD1(G93A) in CHX chase assay in control or TMED9 KD HeLa cells. Quantification was based on the ratio of SOD1(G93A) to RPN1 with the control set as 1.00 (control siRNA at time 0). The blots are representative of at least three independent experiments.

Fig. 3. TMED9 modulates ERES–ERGIC contact.

a 3D-STORM analysis of HeLa cells (control and TMED9 KD) labeled with anti-ERGIC-53 and anti-SEC12 antibodies. The cells were starved in EBSS for 1 h. b Quantification of contact events per 100 structures as shown in a. Error bars represent standard deviations of > 20 cells from three independent experiments (> 6 cells per experiment). P value was obtained from two-tailed t-test. c 3D-STORM analysis of HeLa cells expressing TMED9-V5 labeled with anti-ERGIC-53, anti-SEC12, and anti-V5 antibodies. The cells were starved in EBSS for 1 h. Arrows point to the TMED9-enriched ERGIC-ERES-associated region. d, f SD-SIM analysis of HeLa cells stably expressing EGFP-ERGIC-53 and mCherry-SEC12 incubated in nutrient-rich medium (NR) or starved (ST) in EBSS (d) or HeLa cells transfected with control or siRNAs against TMED9 starved in EBSS (f). Live imaging was performed starting from 30 min after the start of each treatment and pictures of each of the time points were shown. Contact analysis (white puncta) was performed using Imaris. e, g Quantification of contact area (μm²/100 μm² ERGIC area) and contact duration in d, f. Error bars represent standard deviations of > 30 cells from three independent experiments (> 10 cells per experiment). P value was obtained from two-tailed t-test. h 3D-tomography of ERGIC–ERES in HeLa cells (control and TMED9 KD) starved in EBSS for 1 h. The values shown in the insets represent two tight contact sites between the ERES and ERGIC. Scale bar sizes are indicated in the picture. Red, ERES; green, ERGIC; cyan, transport vesicles; blue, autophagosome; orange, yellow, and magenta, autophagic cargos. i Heatmap showing the distances between the ERES and ERGIC in h. The results were calculated and plotted using MATLAB. The x, y, and z axis show the dimensions of ERES (nm) in the tomogram. The scale bar represents the contact distance (nm).

Fig. 4. The interaction between TMED9 and SEC12 drives ERGIC–ERES contact formation.

a Co-IP analysis of TMED9-V5 with FLAG-SEC12, TMED10, and TMED2 in HEK293T cells using anti-V5 agarose. b Co-IP analysis of TMED9 with SEC12 in HEK293T cells starved in EBSS for 1 h using anti-SEC12 antibody. c Co-IP analysis of TMED9-V5 with FLAG-SEC12 in HEK293T cells incubated in nutrient-rich medium or starved in EBSS for 1 h using anti-V5 agarose. d Co-IP analysis of FLAG-SEC12 variants (1–386 (cy); 1–239 (cyN); and 240–386 (cyC)) with TMED9-V5 in HEK293T cells starved in EBSS for 1 h using anti-V5 agarose. e Co-IP analysis of TMED9-V5 variants (FL, full-length TMED9; ΔGOLD, GOLD domain-deleted TMED9; ΔCC, CC domain-deleted TMED9; ΔCT, C-terminal tail-deleted TMED9) with FLAG-SEC12 in HEK293T cells starved in EBSS for 1 h using anti-V5 agarose. f Co-IP analysis of TMED7-CT, TMED9-CT, and TMED10-CT (triple repeats of CT with GFP in the N-terminus and V5 tag in the C-terminus) with FLAG-SEC12-cy in HEK293T cells starved in EBSS for 1 h using anti-V5 agarose. g Co-IP analysis of TMED9-V5 variants (WT, full-length TMED9; m1, R223A/H224A/L225A; m2, L225A/K226A/S227A; m3, S227A/F228A/F229A; m4, F229A/E230A; m5, K232A/K233A; m6, K233A/L234A/V235A) with FLAG-SEC12-cy in HEK293T cells starved in EBSS for 1 h using anti-V5 agarose. h Peptides (9-CT, CT of TMED9; 9-CT-m4, CT of TMED9-m4 in g) were immobilized on agarose beads followed by analysis of FLAG-SEC12 interaction in an in vitro pull-down assay. i Immunofluorescence of HeLa cells treated with 100 μM H89 for 20 min with anti-ERGIC-53 antibody. j Co-IP analysis of TMED9-V5 with FLAG-SEC12 in HEK293T cells treated with 100 μM H89 for 20 min using anti-V5 agarose. k Immunofluorescence and 3D-STORM of HeLa cells (control, TMED9 KD, TMED9 KD with TMED9-V5 or TMED9-ΔCT-V5 expression) with anti-ERGIC-53 and anti-SEC12 antibodies. The cells were starved in EBSS for 1 h. l Quantification of contact events per 100 structures as shown in k. Error bars represent standard deviations of > 20 cells from three independent experiments (> 6 cells per experiment). P value was obtained from two-tailed t-test. m Fluorescence images of HeLa cells stably expressing ddGFP(A)-SEC12 and TMED9-ddGFP(B) incubated in nutrient-rich medium or starved in EBSS for 1 h with and without 100 μM H89 treatment for 20 min. n Quantification of contact area (μm²/100 μm² cell area) as shown in m. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. o Co-IP analysis of FLAG-SEC12 with TMED9 in HEK293T cells treated with Tat-TM9CT peptides (50 μM, 3 h) and starved in EBSS for 1 h using anti-FLAG agarose. p Fluorescence images of HeLa cells stably expressing ddGFP(A)-SEC12 and TMED9-ddGFP(B) treated with control or Tat-TM9CT peptides (50 μM, 3 h) and starved in EBSS for 1 h. q Quantification of contact area (μm²/100 μm² cell area) as shown in p. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. r Fluorescence images of contact formation between “ERES-GUVs” and “ERGIC-GUVs” with indicated proteins or peptides. s, t Quantification of tether/contact ratio (ratio of ERES-GUV in contact with ERGIC-GUV, s) and overlap ratio (overlap area to ERES-GUV area, t) as shown in r. Error bars represent standard deviations of > 150 GUVs from three independent experiments (> 50 GUVs per experiment). P value was obtained from two-tailed t-test.

Fig. 5. The interaction of TMED9 and SEC12 regulates autophagosome biogenesis.

a Immunofluorescence of HeLa cells (control, TMED9 KD, TMED9 KD with TMED9-V5, TMED9-ΔCT-V5, or TMED9-m4-V5 expression) with anti-V5 and anti-LC3 antibodies. The cells were starved in EBSS for 1 h. Asterisks indicate cells with indicated TMED9 variant expression. b Quantification of the LC3 puncta area (μm²/100 μm² cell area) analyzed in a. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value obtained from two-tailed t-test. c LC3 lipidation in HeLa cells transfected with control or siRNAs against TMED9 with or without TMED9-V5, TMED9-ΔCT-V5, or TMED9-m4-V5 re-expression. The cells were incubated in nutrient-rich medium or starved in EBSS in the absence or presence of 500 nM bafilomycin A1 for 1 h. Immunoblots were performed to determine the levels of indicated proteins. Quantification was performed similarly to Fig. 1g. The blots are representative of seven independent experiments. d Quantification of the ratio of lipidated LC3 to tubulin with the control set as 1.00 (control siRNA with starvation and bafilomycin A1) analyzed in c. Error bars represent standard deviations of seven independent experiments. P value was obtained from two-tailed t-test. e Co-IP analysis of FLAG-SEC12 with TMED9 in HEK293T cells transfected with or without GFP-tagged triple CT of TMED9 using anti-FLAG agarose. The cells were starved in EBSS for 1 h. f Immunofluorescence of HeLa cells transfected with GFP-tagged triple CT of TMED9 after EBSS starvation (1 h) with anti-LC3 antibody. Asterisks indicate cells with indicated protein expression. g Quantification of the LC3 puncta area (μm²/100 μm² cell area) analyzed in f. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. h LC3 lipidation in HeLa cells transfected with or without GFP-tagged triple CT of TMED9. The cells were incubated in nutrient-rich medium or starved in EBSS in the absence or presence of 500 nM bafilomycin A1 for 1 h. Immunoblots were performed to determine the levels of indicated proteins. Quantification was performed similarly to Fig. 1g. The blots are representative of at least three independent experiments. i Quantification of the ratio of lipidated LC3 to tubulin with the control set as 1.00 (control siRNA with starvation and bafilomycin A1) analyzed in h. Error bars represent standard deviations of > 3 independent experiments. P value was obtained from two-tailed t-test. j HeLa cells stably expressing mCherry-pHluorin-LC3B were treated with control or Tat-TM9CT peptides (50 μM, 4 h) and incubated in nutrient-rich medium, EBSS, low glucose medium, or medium without serum. Confocal microscopy was performed. k, l Quantification of autolysosome (red, k) and autophagosome (yellow, l) numbers per cell as shown in j. Error bars represent standard deviations of > 300 cells from three independent experiments (> 100 cells per experiment). P value was obtained from two-tailed t-test.

Fig. 6. The function of ERGIC–ERES contact in ERGIC–COPII formation.

a HEK293T cells were transfected with control or siRNAs against TMED9. After 72 h, the cells were incubated in nutrient-rich medium or starved in EBSS for 1 h. The cell lysates and the ERGIC membrane fractions were analyzed by immunoblot to determine the levels of indicated proteins. Quantification shows relative SEC12 relocation to the ERGIC under the indicated conditions. The control siRNA transfection group with nutrient-rich medium treatment was set as 1.00. The blots are representative of at least three independent experiments. b SIM analysis of ERGIC and COPII in HeLa cells (control and TMED9 KD) with anti-ERGIC-53 and anti-SEC31A antibodies. The cells were starved in EBSS for 1 h. c Quantification of COPII overlap area with the ERGIC (μm²/100 μm² ERGIC area) as shown in b. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. d The structure of the SEC12 cytoplasmic domain (PDB 5tf2). The values shown in the figure represent the dimension of SEC12. Structure model was created by PyMOL. e HEK293T cells were infected with RUSH-SEC12 lentivirus. The cells were transfected with siRNA against SEC12. After 72 h, the cells were treated with or without 40 μM biotin for 1 h. The cell lysates and the ERGIC membrane fractions were analyzed by immunoblot to determine the levels of indicated proteins in left panel. Quantification shows the percentage of SEC12 relocation to the ERGIC under the indicated conditions. The efficiency of SEC12 knockdown was shown in right panel. FLAG-Str, FLAG-Streptavidin. f SIM analysis of ERGIC and COPII in HeLa cells stably expressing RUSH-SEC12. The cells were transfected with siRNAs against SEC12 and TMED9, incubated in nutrient-rich medium or EBSS for 1 h with or without 40 μM biotin for 1 h, and labeled with anti-ERGIC-53 and anti-SEC31A antibodies. g Quantification of COPII overlap area with the ERGIC (μm²/100 μm² ERGIC area) as shown in f. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. h Immunoblot showing the transactivation of SAR1 on the liposome via SEC12-bound beads. Beads with or without SEC12 were incubated with liposomes with or without TMED9-CT, together with indicated variants of SAR1 in the presence or absence of GTP. After reaction, the liposomes were isolated followed by immunoblot. Quantification was based on the ratio of SAR1 with the group of highest transactivation (with SEC12, TM9-CT, SAR1, and 0.15 nM GTP set as 1.00). The blots are representative of at least three independent experiments. i Fluorescence imaging showing the recruitment of SAR1-BFP to the TMED9-CT-labeled liposomes attached to streptavidin agarose beads. Beads with or without SEC12 were incubated with beads coated with control liposomes or TMED9-CT. Indicated SAR1-BFP variants with GTP were incubated with the indicated combination of beads. Confocal imaging was performed to analyze the recruitment of SAR1-BFP to the liposomes on the beads in contact with beads with SEC12. j Quantification of SAR1 recruitment ratio as shown in i. Error bars represent standard deviations of > 150 beads with liposomes from three independent experiments (> 50 beads with liposomes per experiment). P value obtained from two-tailed t-test.

Fig. 7. The requirement of ERES enlargement in ERGIC–ERES contact formation.

a Immunofluorescence of HeLa cells transfected with control and siRNAs against TMED9, CTAGE5 or FIP200 with the anti-SEC12 antibody. The cells were incubated in nutrient-rich medium or starved in EBSS for 1 h. b Quantification of the SEC12 puncta area (ratio of puncta > 0.1 μm²) analyzed in a. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. c, d Co-IP analysis of TMED9-V5 with FLAG-SEC12 in HEK293T cells transfected with control or siRNAs against CTAGE5 (c) or FIP200 (d) using anti-V5 agarose. Quantification was based on the ratio of FLAG-SEC12 to TMED9-V5 of IP fraction with the control setting as 1.00 (control siRNA). The blots are representative of at least three independent experiments. e 3D-STORM analysis of HeLa cells transfected with control and siRNAs against FIP200 with anti-ERGIC-53 and anti-SEC12 antibodies. The cells were starved in EBSS for 1 h. f Quantification of contact events per 100 structures as shown in e. Error bars represent standard deviations of > 20 cells from three independent experiments (> 6 cells per experiment). P value obtained from two-tailed t-test. g Fluorescence images of HeLa cells stably expressing ddGFP(A)-SEC12 and TMED9-ddGFP(B) transfected with control and siRNAs against CTAGE5 or FIP200. The cells were starved in EBSS for 1 h. h Quantification of contact area (μm²/100 μm² cell area) as shown in g. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value obtained from two-tailed t-test. i Co-IP analysis of FLAG-SEC12 with FIP200 and CTAGE5 in HEK293T cells transfected with control and siRNAs against TMED9 using anti-FLAG agarose under steady state. Quantification was based on the ratio of FIP200 to FLAG-SEC12 of IP fraction with a control setting of 1.00 (control siRNA). The blots are representative of at least three independent experiments.

Fig. 8. ERGIC–ERES contact is distinct from TFG-mediated ERES–ERGIC tethering.

a Secretion of ssGFP in HEK293T cells transfected with control and siRNAs against TFG or TMED9. The blots are representative of at least three independent experiments. b LC3 lipidation in HeLa cells transfected with control or siRNAs against TMED9 or TFG. The cells were incubated in nutrient-rich medium or EBSS for 1 h in the absence or presence of 500 nM bafilomycin A1. Immunoblots were performed to determine the levels of indicated proteins. Quantification was performed similarly to Fig. 1g. The blots are representative of at least three independent experiments. c Quantification of the ratio of lipidated LC3 to tubulin with the control set as 1.00 (control siRNA with starvation and bafilomycin A1) analyzed in b. Error bars represent standard deviations of > 3 independent experiments. P value was obtained from two-tailed t-test. d HeLa cells stably expressing mCherry-pHluorin-LC3B were transfected with control or siRNAs against TFG and incubated in nutrient-rich medium or starved in EBSS for 1 h. Confocal microscopy was performed. e, f Quantification of autolysosome (red, e) and autophagosome (yellow, f) numbers per cell as shown in d. Error bars represent standard deviations of > 300 cells from three independent experiments (> 100 cells per experiment). P values were obtained from two-tailed t-test. g Immunofluorescence of HeLa cells transfected with control or siRNAs against TMED9 or TFG with anti-SEC12 antibody. The cells were incubated in nutrient-rich medium or starved in EBSS for 1 h. h Quantification of the SEC12 puncta area (ratio of puncta > 0.1 μm²) analyzed in g. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. i Co-IP analysis of TMED9-V5 with FLAG-SEC12 in HEK293T cells transfected with control and siRNA against TFG using anti-V5 agarose. The cells were starved in EBSS for 1 h. The blots are representative of at least three independent experiments j 3D-STORM analysis of HeLa cells transfected with control or siRNAs against TFG with anti-ERGIC-53 and anti-SEC12 antibodies. The cells were starved in EBSS for 1 h. k Quantification of contact events per 100 structures as shown in j. Error bars represent standard deviations of > 20 cells from three independent experiments (> 6 cells per experiment). P value was obtained from two-tailed t-test. l Fluorescence images of HeLa cells stably expressing ddGFP(A)-SEC12 and TMED9-ddGFP(B) transfected with control and siRNAs against TFG. The cells were starved in EBSS for 1 h. m Quantification of contact area (μm²/100 μm² cell area) as shown in l. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment). P value was obtained from two-tailed t-test. n Immunofluorescence and 3D-STORM of HeLa cells transfected with TMED9-V5 with anti-V5, anti-ERGIC-53 and anti-TFG antibodies. The cells were starved in EBSS for 1 h. o SIM analysis of HeLa cells stably expressing ddGFP(A)-SEC12 and TMED9-ddGFP(B) with anti-TFG antibody. The cells were starved in EBSS for 1 h. p Quantification of overlap area ratio (relative to ddGFP signal area) as shown in o. Error bars represent standard deviations of > 150 cells from three independent experiments (> 50 cells per experiment).

Fig. 9. A model for ERES–ERGIC contact formation and autophagosome biogenesis.

a In steady-state conditions, SEC12 (dark red oval) associates with CTAGE5 (purple oval) and localizes to the ERES. TFG (orange oval) tethers ERES and COPII vesicles for protein cargo transport to the ERGIC and Golgi. b Upon starvation, SEC12-ERES is enlarged and surrounds the ERGIC, the process of which is dependent on FIP200 (green oval) and CTAGE5. The remodeled ERES forms a contact with the ERGIC via TMED9–SEC12 interaction, which is independent of TFG and leads to the relocation of SEC12 to the ERGIC (i) and/or transactivation of COPII vesicle formation on the ERGIC by ERES-localized SEC12 (ii). c The ERES–ERGIC contact triggers the assembly of ERGIC–COPII vesicles as a membrane template for LC3 lipidation, a potential vesicular pool for the assembly of the pre-autophagosomal membrane.