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Abstract
Starvation-induced autophagy requires activation of the ULK complex at the phagophore. Two Golgi proteins, WAC and GM130, regulate autophagy, however their mechanism of regulation is unknown. In search of novel interaction partners of WAC, we found that GM130 directly interacts with WAC, and this interaction is required for autophagy. WAC is bound to the Golgi by GM130. WAC and GM130 interact with the Atg8 homolog GABARAP and regulate its subcellular localization. GABARAP is on the pericentriolar matrix, and this dynamic pool contributes to autophagosome formation. Tethering of GABARAP to the Golgi by GM130 inhibits autophagy, demonstrating an unexpected role for a golgin. WAC suppresses GM130 binding to GABARAP, regulating starvation-induced centrosomal GABARAP delivery to the phagophore. GABARAP, unlipidated and lipidated, but not LC3B, GABARAPL1, and GATE-16, specifically promotes ULK kinase activation dependent on the ULK1 LIR motif, elucidating a unique non-hierarchical role for GABARAP in starvation-induced activation of autophagy.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Figures
WAC Promotes Starvation-Induced Autophagy and ULK1 Kinase Activation (A) siRNA-resistant EGFP-WAC or vector was expressed in HEK293A cells treated for 72 hr with either RISC-free (RF) or WAC siRNA. After 2 hr starvation with EBSS and BafA1, cells were analyzed by immunoblot. (B) HEK293A cells treated with RF or WAC siRNA for 72 hr were incubated in full medium (FM) or EBSS (ES) for 15, 30, and 60 min. (C) Quantification of (B); statistical analysis using unpaired Student’s t test, mean ± SEM, n = 3 experiments, ∗p ≤ 0.05. (D) HEK293A cells were treated with RF or WAC siRNA for 72 hr then starved 2 hr, fixed, and labeled with WIPI2. Scale bars, 50 μm. (E) WIPI2 puncta in (D) were counted. Mean ± SEM from n = 2 experiments, >150 cells counted per condition, unpaired Student’s t test, ∗∗∗p ≤ 0.001. (F) HEK293A cells treated with RF or WAC siRNA for 72 hr before immunoblotting. (G) HEK293A cells were treated with RF, ULK1, or WAC siRNA for 72 hr, incubated in fed or 2 hr EBSS (Starved), and labeled for Atg9 and GM130. Scale bars, 50 μm. (H) Venn diagram of number of genes significantly (p < 0.05) up- or downregulated in HEK293A cells treated with WAC, RNF20, or RNF40 siRNA for 72 hr versus RF control. See also Figure S1 and Table S1. Excised lanes are indicated by a gap, and remaining lanes are from the same gel.
The WAC-GM130 Interaction Is Direct and Independent of RNF40 (A) Anti-WAC and anti-GFP immunoprecipitates analyzed by immunoblotting. HC, IgG heavy chain. (B) HEK293A cells in full medium (FM) or EBSS (ES) for 2 hr prior to lysis, followed by treatment as in (A). (C) Quantification of GM130 as in (B) after normalization to immunoprecipitated WAC. Statistical analysis using unpaired Student’s t test, mean ± SEM, n = 4, ∗∗p ≤ 0.01. (D) Quantification of RNF40 as in (B) after normalization to immunoprecipitated WAC. Mean ± SEM, n = 2. (E) GFP-TRAP pull down of HeLa WAC-FLAP cell followed by immunoblot. Cells in FM or EBSS (ES) for 2 hr prior to lysis. (F and G) WAC in FM or EBSS for 30 min in HEK293 (F) or 30 and 120 min in MEFS (G) were analyzed by Phos-tag SDS-PAGE. (H) GFP-TRAPs of GFP-WAC full length (FL) or aa1–319 (NT), salt washed, treated or not with lambda phosphatase, incubated with HEK293 lysate, and immunoblotted with anti-GM130. (I) Human WAC isoform 1 and deletion mutants used here: WW domain, putative nuclear export signal (NES), and C-terminal coiled-coil (CC) domain, including the GM130 binding region. GM130 binding ability is indicated. Black square, 10aa or 7aa heptad deletions. Grey circles, ISLS mutations. (J) HEK293A cells co-transfected with 3xHA-GM130 and EGFP-WAC FL, NT, aa320–647 (CT), aa163–647 (ΔN), or aa1–610 (ΔCC) immunoprecipitated with anti-HA and immunoblotted. (K) GFP-TRAP of cells expressing EGFP or EGFP-WAC FL, ISLS, ΔCC, 1–620, or 1–630 and immunoblot of GM130 and RNF40. (L) GST-WAC FL or GST-WAC ΔCC incubated with purified Strep-Tag II-GM130. GST-WAC monitored by Ponceau S staining. Right, colloidal Coomassie staining of Strep-Tag II-GM130. (M) Human GM130 isoform 1, boxes 1–6, coiled-coil domains. USO1 (p115), Rab1b, Grasp65, and WAC binding regions are indicated. (N) HEK293A cell lysates containing EGFP-WAC FL or ΔCC were mixed with HA-GM130 aa692–1,002 or HA-GM130 aa774–1,002 lysates, immunoprecipitated with anti-HA, and analyzed by immunoblotting. See also Figure S2. Excised lanes are indicated by a gap, and remaining lanes are from the same gel.
GM130 Tethers WAC to the Golgi, and the Interaction Is Required for Autophagosome Formation (A and B) WAC localized with βCOP, GM130, or ERGIC-53 (A) or p230 and TGN46 (B). Scale bars, 10 μm. (C) WAC, TGN46, and GM130 in GM130-depleted cells. (∗) cell depleted of GM130. Scale bars, 25 μm. (D) EGFP-WAC FL or ΔCC expressed after WAC depletion, labeled with indicated antibodies for epifluorescence microscopy. (E) EGFP-WAC (FL) or aa1–319 (NT) co-expressed with GM130-ΔCterm-HA-MAO labeled with anti-HA. Scale bars, 10 μm. (F) RF or WAC (siW) siRNA-treated cells for 72 hr transfected with vector (Vec), Myc-WAC (WT), or Myc-WAC aa1–610 (ΔC), starved for 2 hr with EBSS. LC3B puncta were analyzed by confocal microscopy. (G) LC3B puncta from (F), Mean ± SEM of n = 3, >400 cells counted per condition, unpaired Student’s t test, ∗∗∗∗p ≤ 0.0001. See also Figure S3.
GM130 Is a Negative Regulator of the Early Stages of Autophagy and Interacts with GABARAP (A) HEK293A cells treated with RF or GM130 siRNA-01 or -03 incubated in full medium (FM), or EBSS (ES) with or without BafA for 2 hr. (B) LC3-II levels from (A). Mean ± SEM of n = 4, Mann-Whitney test, ∗p ≤ 0.05. (C) HEK293A cells treated with RF or GM130 siRNA incubated in full medium (FM) or EBSS (ES) for 2 hr. Statistical analysis using unpaired Student’s t test, mean ± SEM, n = 3, ∗∗p ≤ 0.01. 30 fields of cells were analyzed per condition. (D) HEK293A cells treated with RF or GM130 siRNA-01 or -03 incubated in full medium (FM) or EBSS (ES) for 2 hr and immunoblot. (E) Quantification of (D). Statistical analysis using unpaired Student’s t test, mean ± SEM, n = 5, ∗∗p ≤ 0.01. (F) p62 degradation from Figure S4A, mean ± SEM of n = 5, Mann-Whitney test, ∗p ≤ 0.05. (G) HEK293A cells in FM or EBSS for 2 hr. Arrows indicate colocalization. Scale bars, 20 μm. (H) Myc-WAC and EGFP, EGFP-LC3A, LC3B, LC3C, GABARAP, GABARAPL1, or GATE-16 co-expressed, followed by GFP-TRAP and immunoblot. (I) Immunoprecipitation of GABARAP from fed or starved cells and immunoblot analysis. B, beads; Ctrl, Anti-GFP; GABA, Anti-GABARAP. (J) Purified Strep II-GM130 was incubated with recombinant WAC 320–647 before binding to recombinant GST or GST-GABARAP beads and immunoblot. Statistical analysis using unpaired Student’s t test, mean ± SEM, n = 3. ∗∗p ≤ 0.01. (K) Co-expressed EGFP-GABARAP and GM130-ΔCterm-HA-MAO labeled with anti-HA. Scale bars, 10 μm. See also Figure S4. Excised lanes are indicated by a gap, and remaining lanes are from the same gel.
Non-lipidated GABARAP Localization at the Centrosome Is Regulated by Starvation and Microtubules (A) HEK293A, HeLa, U2OS, RPE-1, HCT116, and MEF cells were labeled as indicated. Scale bars, 5 μm. (B) HEK293A cells labeled as indicated. Scale bars, 10 μm. (C) HEK293A cells were treated for 72 hr with RF or GABARAP siRNAs and labeled as in (A). Scale bars, 50 μm. Inset, line scan. (D) Line scans of (C). (E) GFP-GABARAP expression in HEK293 Flp-In T-Rex cells after tetracycline for 24 hr. Scale bars, 20 μm. (F) HEK293A cells incubated in FM, EBSS (ES), or EBSS with wortmannin for 2 hr. Scale bars, 20 μm. (G) HEK293A cells expressing Myc-GABARAP or G116A mutant labeled as indicated. Scale bars, 20 μm. (H) HEK293A cells in FM or FM plus nocodazole (FM + N) for 5 hr. Scale bars, 20 μm. (I) Quantification of centrosomal GABARAP as in (H). Mean ± SEM of n = 3, >300 cells counted per condition, unpaired Student’s t test, ∗∗∗∗p ≤ 0.0001. (J) Quantification of centrosomal GABARAP after 2 hr with FM or EBSS. Mean ± SEM of n = 3, >420 cells counted per condition, unpaired Student’s t test, ∗∗∗∗p ≤ 0.0001. See also Figure S5.
Centrosomal GABARAP Contributes to Autophagosome Formation (A) EosFP-GABARAP in starved cells was imaged every 30 s using a swept field confocal microscope. 5 × 1 s images were captured prior to photoconversion (PC). PC moment is set to 0 s. Yellow arrow, photoconverted region. Magenta arrowhead, distal region. White and blue arrows track defined puncta. Scale bars, 10 μm. (B) Quantification of fluorescence intensity from video in (A). PC region marked by yellow arrow and distal region marked by magenta arrowhead in (A). Intensity 5 s prior to PC moment is set to 1 for normalization. (C) Confocal microscopy performed on cell from video in (A). After imaging, cells were fixed and stained for LC3 and γ-tubulin. Arrows and arrowheads correspond to structures shown in (A). Scale bars, 10 μm. (D) EosFP-GABARAP as in (A). PC moment is set to 0 s. Yellow box (top), photoconverted region. Yellow box (bottom), distal region. Scale bar, 10 μm. (E) Quantification of intensities from boxed regions in (D). Intensity 60 s prior to PC moment is set to 1 for normalization. (F) EosFP-GABARAP expressing cells in FM with nocodazole for 2 hr, then incubated in EBSS with nocodazole and imaged. Images captured every 3 s. Blue arrows track defined puncta. Scale bars, 20 μm. (G) Confocal microscopy performed on cells from video in (F). After imaging, cells were fixed and stained for GM130. Arrows correspond to structures shown in (F). Scale bars, 20 μm. (H) HEK293A cells were treated with RF, WAC, or GM130 siRNAs for 72 hr and incubated with full medium (Fed) or EBSS (Starved) for 2 hr. Scale bars, 50 μm. See also Figure S6 and Movies S1 and S2.
WAC Inhibits GM130 Binding of GABARAP and GABARAP-Mediated ULK1 Activation (A) EosFP-GABARAP expressed in HEK293A cells treated with WAC siRNA for 72 hr. Live-cell imaging under starvation conditions. During photoconversion (PC), images from a single z slice were acquired every 0.4 s. After PC, z stacks were acquired every 13 s. Yellow arrows denote PC regions. Scale bars, 20 μm. (B) After time-lapse imaging, cells shown in (A) were fixed and stained for GM130. Scale bars, 20 μm. (C) HEK293A cells were treated with RF or WAC siRNA for 72 hr and incubated with full medium (F) or EBSS (S) for 2 hr followed by GABARAP immunoprecipitation and immunoblotting. B, beads; Ab, GABARAP antibody; Lys, lysate. (D) Quantification of (C), Student’s t test, ∗∗p ≤ 0.01. Mean ± SEM of n = 3. (E) HEK293A cells treated with RF or GABARAP siRNA for 72 hr, incubated in EBSS for 2 hr followed by immunoblot. p-Atg13, pSer318. (F) Quantification of (E), Student’s t test, ∗∗∗p ≤ 0.001. Mean ± SEM of n = 3. (G) HEK293A cells treated with RF, LC3B, GABARAP, GABARAPL1, or GATE-16 siRNAs for 72 hr before 2 hr incubation in EBSS and immunoblot. p-Atg13, pSer318. (H) Quantification of (G), Student’s t test, ∗∗∗∗p ≤ 0.0001. Mean ± SEM of n = 3. (I) HEK293A cells expressing GFP, GFP-GABARAP, or GFP-GABARAP G116A were incubated in FM or ES for 2 hr prior to GFP-TRAP and immunoblot. (J) HEK293A cells expressing GFP or GFP-GABARAP G116A were incubated in EBSS for 2 hr prior to subcellular fractionation, GFP-TRAP, and immunoblot. Atg9 marks the membrane fraction and SOD1 the cytosol. (K) HEK293 cells stably expressing GFP-DFCP1 transfected with Myc-GABARAP or G116A mutant were starved for 2 hr in EBSS, and the GFP-DFCP1 compartment was immunoisolated prior to immunoblot. Flag M2 antibody was used as a control. (L) The GFP-DFCP1 compartment was isolated from HEK293 cells co-expressing the indicated constructs and starved in EBSS for 2 hr. Solubilised membranes were subjected to immunoprecipitation with anti-Myc or anti-FLAG M2 control. (M) HEK293A cells expressing indicated proteins analyzed by immunoblot. ∗, non-specific band. KI, kinase-inactive ULK1. Quantification shown in Figure S7F. See also Figure S7 and Movie S3. Excised lanes are indicated by a gap, and remaining lanes are from the same gel.
References
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- Alemu E.A., Lamark T., Torgersen K.M., Birgisdottir A.B., Larsen K.B., Jain A., Olsvik H., Øvervatn A., Kirkin V., Johansen T. ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs. J. Biol. Chem. 2012;287:39275–39290. - PMC - PubMed
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