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TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy

TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy Scientific Report TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy Doris Popovic & Ivan Dikic Abstract induction [14]. Dispersal of ATG9 during autophagy is dependent on ULK1 [14] and contributes to the formation of autophagosome The RabGAP protein TBC1D5 controls cellular endomembrane traf- precursors initiated by DFCP1 [15]. However, the nature of ATG9 ficking processes and binds the retromer subunit VPS29 and the spatio-temporal redistribution remains unclear. Moreover, recent ubiquitin-like protein ATG8 (LC3). Here, we describe that TBC1D5 studies suggest that ATG9 traffic carriers may not incorporate into also associates with ATG9 and the active ULK1 complex during autophagosomes, but only transiently associate with them [15]. autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and Previously, we proposed that proteins known to accelerate the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 hydrolysis of GTP on small Rab GTPases (RabGAP) act as autopha- to late endosomes upon activation of autophagy, whereas inhibi- gic adaptors that regulate autophagy-endocytosis crosstalk via direct tion of clathrin-mediated endocytosis or AP2 depletion alters ATG9 interaction with the autophagy ubiquitin-like protein LC3 [16]. trafficking and its association with TBC1D5. Taken together, our TBC1D14 and TBC1D25 play a role in early and late stages of auto- data show that TBC1D5 and the AP2 complex are important novel phagosome maturation, respectively [17,18]. TBC1D5 binds directly regulators of the rerouting of ATG9-containing vesicular carriers to LC3 and retromer subunit VPS29, via LC3 interacting region 1 toward sites of autophagosome formation. (LIR1), and regulates internalization and TGN retrieval of CI-M6PR. During autophagy, mutually exclusive binding to LC3 mediates its Keywords AP2; ATG9; autophagy; clathrin endocytosis; TBC1D5 transfer from retromer to the autophagosomal membrane [16]. Subject Categories Autophagy & Cell Death; Membrane & Intracellular Although retromer complex associates with the autophagic machinery Transport [19], its role in autophagy is unclear [20]. ATG9 is the only autophagic DOI 10.1002/embr.201337995 | Received 13 September 2013 | Revised 6 protein reported to co-fractionate with retromer, TGN46, and February 2014 | Accepted 10 February 2014 | Published online 6 March 2014 CI-M6PR-containing compartments [14]. We therefore hypothesized EMBO Reports (2014) 15, 392–401 that TBC1D5 may impact on ATG9 trafficking within the endosomal system and we aimed to define the link between coordinated trafficking patterns of ATG9, retromer, and TBC1D5. Introduction Here, we report the functional interaction between TBC1D5, ATG9, and AP2 complex. During autophagy, TBC1D5 dissociates from Macroautophagy (autophagy) is a membrane-dependent process, retromer and associates with ATG9 and active ULK1. Additionally, critical for delivery of various cytoplasmic cargo (e.g., protein aggre- we show that ATG9 trafficking is regulated by AP2 and TBC1D5. gates, damaged organelles, and pathogens) to the lysosomes (for We propose that during autophagy, the efficient rerouting of ATG9 degradation) [1] and secretion of molecules that mediate immune carriers toward PAS requires the dynamic transition of TBC1D5 to responses [2]. Numerous membrane sources are implicated in the autophagosomes and that AP2 complex mediates such trafficking formation of autophagosomes: endoplasmic reticulum [3], mito- reorganization. chondria [4,5], plasma membrane [6], and Golgi complex [7–10]. This suggests that autophagy closely intersects with and depends on various membrane trafficking events inside cell [11]. More than 30 Results and Discussion autophagy-related genes take part in phagophore formation [12] including the essential ULK1/ATG13/FIP200 kinase complex and TBC1D5 associates with ATG9 and ULK1 ATG9. While the trafficking of yeast ATG9 appears better under- stood, knowledge on its mammalian homologue is far more elusive ATG9 has 6 transmembrane regions and localizes to TGN and Rab7-, [13]. Mammalian ATG9 localizes to the trans-Golgi network and to Rab9-, and CI-M6PR-containing endosomes [14]. Upon autophagy late endosomes under basal conditions and cycles between its stimuli, ATG9 relocates to peripheral sites driving the growth of reservoirs and phagophore assembly site (PAS) upon autophagy the phagophore. In U2OS cells stably expressing mCherry-ATG9, Buchmann Institute for Molecular Life Sciences (BMLS) and Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt, Germany *Corresponding author. Tel: +49 69 6301 5964; Fax: +49 69 6301 5577; E-mail: [email protected] 392 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A B D E F G H ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 393 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic Figure 1. TBC1D5 and retromer associate with ATG9. A Co-localization of endogenous TBC1D5 and VPS35 with mCherry-ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h). B, C Quantification of co-localization experiments presented in (A). D Co-immunoprecipitation of HA-Flag-TBC1D5 with myc-VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated. E, F Co-immunoprecipitation of endogenous TBC1D5 and ATG9 with myc-VPS29 (E) transiently overexpressed in 293T cells or with HA-Flag-VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h). G Co-immunoprecipitation of endogenous VPS29 and ATG9 with HA-Flag-TBC1D5 stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h). H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment. I Magnified still images extracted from Supplementary Movie S2. mCherry-TBC1D5 co-localizes with GFP-ATG9 upon autophagy induction by mTOR inhibitor, KU0063794. Data information: Bar graphs (B, C), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. endogenous TBC1D5, retromer subunit VPS35, and mCherry-ATG9 TBC1D5 is required for ATG9 trafficking co-localized in steady state as well as during autophagy induced by the mTOR inhibitor KU0063974 (Fig 1A). Closer inspection Recently, it has been suggested that retromer is both required and of ATG9-containing vesicles showed that ATG9 does not enter dispensable for autophagy [15,19]. To test whether retromer retromer-associated endosomal tubules (Supplementary Fig S1A). contributes to ATG9 trafficking, we stably depleted U2OS cells of Co-localization of TBC1D5 with ATG9 was steadily high in both TBC1D5 or retromer subunit VPS29 and compared them with tested conditions (Fig 1B), while retromer co-localization decreased control shRNA-expressing cells (Fig 2A). In VPS29-deficient cells, upon autophagy induction (Fig 1C). Endogenous ATG9, LC3, and we observed destabilization of all retromer subunits, but no appar- TBC1D5 also co-localized in cells (Supplementary Fig S1B). Next, ent defect in autophagy flux upon autophagy stimulation (Fig 2A, we tested whether TBC1D5 and retromer associate with ATG9. Supplementary Fig S4A). Depletion of TBC1D5 severely affected TBC1D5 and ATG9 co-precipitated with retromer subunit VPS29 the LC3 level and stabilized ATG9, suggesting that TBC1D5 acts (Fig 1D and E) or VPS35 (Fig 1F) only in non-stimulated conditions. also independently of retromer (Fig 2A). Separate regulation of Upon autophagy induction, HA-Flag-TBC1D5 bound less endoge- retromer and ATG9 by TBC1D5 is possible because neither retro- nous VPS29, but interacted stronger with ATG9 (Fig 1G). mer co-localizes with LC3 (Supplementary Fig S1C) nor ATG9 TBC1D5 association with autophagosomes is regulated via bind- associates with retromer during autophagy. TBC1D5-deficient cells ing to LC3 [16]. As expected from our previous study, autophago- showed decreased number of ATG9 vesicular structures in fed somes containing TBC1D5 were negative for retromer staining conditions (Fig 2B and C) while, upon autophagy induction, ATG9 was enriched in late endosomes as indicated by LAMP1 staining (Supplementary Fig S1C). The early autophagosomal marker DFCP1 partially co-localized with TBC1D5 (Supplementary Fig S2B), while (Fig 2B and D). Importantly, retromer depletion did not lead to ATG16 did not (Supplementary Fig S2B), indicating that TBC1D5 similar defect in ATG9 localization, indicating that retromer is specifically associates with ATG9 labeled early autophagosomes. dispensable for ATG9 trafficking during autophagy. We rescued Live-cell imaging of U2OS cells stably expressing GFP-ATG9 and the effect on ATG9 by introducing shRNA-resistant TBC1D5 to mCherry-TBC1D5 revealed co-localization of ATG9- and TBC1D5- assure that observed phenotype is specific for TBC1D5 depletion containing vesicles at steady state (Supplementary Movie S1) and (Fig 2F and G). during autophagy (Fig 1I, Supplementary Movies S2 and S3). In addition, the major upstream kinase ULK1 co-localized with TBC1D5 and ATG9A interact with AP2 complex TBC1D5 (Supplementary Fig S3A) and together with the phosphory- lated form of ATG13 co-precipitated with TBC1D5 (Fig 1H), suggest- As ATG9 accumulated in late endosomes and lysosomes upon ing that TBC1D5 associates with the active kinase as well. TBC1D5 depletion, we hypothesized that TBC1D5 regulates sorting Figure 2. TBC1D5 is required for ATG9 trafficking. A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS–PAGE. BU2OS cells stably expressing mCherry-ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti-LAMP1 antibody. C Quantification of ATG9 vesicular structures in cells from (B) under non-stimulated conditions. D Quantification of ATG9 and LAMP1 co-localization in cells presented in (B). E Quantification of ATG9 and LAMP1 co-localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc-TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post-transfection, fixed, and immunostained with anti-LAMP1 and anti-myc antibodies. F Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA-resistant myc-TBC1D5. G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA-resistant myc-TBC1D5, treated with KU0063794 (6 h). 20 h post-transfection cells were treated, subsequently fixed, and immunostained with anti-myc and anti-LAMP1 antibodies. Data information: Bar graphs (C, E), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. Bar graph (D), mean  s.d. n = 3, one-way ANOVA with Bonferroni s multiple comparison test, ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. 394 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A B D EF ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 395 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic or recycling of ATG9. Interestingly, our published mass spectrometry Next, we treated cells with the Dynamin 2 inhibitor Dynasore interactome of TBC1D5 contained two subunits of AP2 complex, but [25], which led to a significant increase in ATG9 and TBC1D5 co- no other endosome-TGN regulatory proteins such as TIP47 or localization with AP2 (Fig 5A-C), similar to the effect of starva- PACS1 [21]. Available PPI (protein-protein interaction) database tion (Fig 5D, Supplementary Fig S8A). This co-localization (Biogrid) reports TBC1D5 as an AP2M1 interactor [22]. We purified became even more pronounced when cells were pretreated with AP2M1 subunit and confirmed its binding to overexpressed Dynasore and subsequently starved (Fig 5D, Supplementary HA-Flag-TBC1D5 expressed in cells (Fig 3A), as well as purified Fig S8A). However, the plasma membrane regions enriched with GST-TBC1D5 (Supplementary Fig S5A). TBC1D5 interaction with AP2/ATG9/TBC1D5 excluded LC3 (Supplementary Fig S8B) and AP2 complex was independent on LIR1 or GAP activity (Supple- the number of LC3-positive puncta was reduced (Fig 5E), indicat- mentary Fig S5B). TBC1D5 depletion led to the destabilization ing that Dynasore delayed autophagosome formation. Inspection of AP2 complex (Fig 3B) and AP2 redistribution to the perinuclear of live cells showed that Dynasore treatment led to co-localization area (Fig 3C). Moreover, TBC1D5 and ATG9 co-localized with the of ATG9 and TBC1D5 in static vesicles (Supplementary Movie AP2 complex (Fig 3D, Supplementary Fig S6A). Importantly, co- S5). Notably, depletion of AP2 complex abolished the TBC1D5 localization of ATG9 with AP2 was enhanced during autophagy interaction with ATG9 (Fig 5F), suggesting that TBC1D5-ATG9 (Fig 3E, Supplementary Fig S6B) and readily disturbed upon association is dynamic and is restricted to the AP2-clathrin- the depletion of TBC1D5 (Supplementary Fig S6B). Imaging of associated fraction of ATG9. GFP-ATG9 stably expressing U2OS cells (Supplementary Movie S4) Direct TBC1D5 interaction with LC3 [16] and AP2 complex revealed numerous highly mobile vesicles. Based on this notion, we prompted us to hypothesize that TBC1D5 regulates the recruitment hypothesized that ATG9 is incorporated into AP2-clathrin-coated of ATG9-AP2-containing vesicles to the autophagic membranes vesicles (CCVs) of plasma membrane origin. Diameter of ATG9- (Fig 5G). Further experiments, however, are needed to gain containing vesicles has been estimated to be 30–60 nm [20], which mechanistic understanding of TBC1D5 connection to AP2, and is somewhat close to CCV size [23]. As proposed, AP2 complex and to pinpoint regulatory mechanisms underlying this interaction in clathrin readily co-precipitated with ATG9 (Fig 3F), prominently cells [26]. upon autophagy induction, while retromer dissociated from ATG9 Few reports agree on the relevance of the AP2 complex in (Fig 3F). Taken together, our data indicate that TBC1D5 regulates ATG16 trafficking and lysosome biogenesis [6,27]. Our findings ATG9 via AP2 complex. indicate that TBC1D5 and AP2 complex directly affect ATG9 traf- ficking. During the revision of our manuscript, Puri et al [28] have AP2 and clathrin-mediated endocytosis (CME) are required for also reported that ATG9 traffics from plasma membrane in the ATG9 sorting and autophagy clathrin-positive carriers that are routed differently from ATG16 carriers [6,27]. In that study, ATG9 has been co-localized with AP2 complex binds to clathrin and acts as a major hub for early endosomal GTPase Rab5, whereas ATG16 failed to do so. protein interactions in clathrin-coated pits. Its recruitment during However, both ATG9 and ATG16 would coalesce at stage of the last stage of CCV formation at the plasma membrane precedes recycling endosomes in dependence on SNARE protein VAMP1. Dynamin 2-dependent pinching-off [24]. To understand the role Interestingly, TBC1D5 associates with ATG9 but does not co-localize of ATG9 interaction with AP2 and clathrin, we silenced expres- with ATG16, while depletion of TBC1D5 enhances endosomal sion of AP2 or blocked maturation of CCVs by expressing a localization of ATG9. Our data support the idea that TBC1D5 dominant-negative form of Dynamin 2. In normal growing condi- might specifically regulate the recruitment of ATG9 from early tions, AP2 depletion enhanced ATG9 localization on the plasma stage of endocytosis to the site of forming autophagosomes. It membrane (Fig 4A), while upon autophagy induction, ATG9 accu- remains to be addressed by what mechanisms ATG9 and ATG16 mulated mostly in the TGN if compared to control siRNA-treated are targeted to a different population of clathrin-coated vesicles, cells (Fig 4A and B, Supplementary Fig S7A). Moreover, lack of and whether additional components of endocytic machinery AP2 induced accumulation of p62 (Fig 4C) and significantly participate in their segregation or collision. impaired LC3 lipidation (Supplementary Fig S7B) consistent with In conclusion, we propose a dual role for TBC1D5: it regulates reported data [6]. Differently, the dominant-negative form of membrane trafficking, upon autophagy induction, and it contributes Dynamin 2 (K44A) dispersed ATG9 toward the plasma membrane to different routes of endosomal sorting including clathrin-AP2- (Fig 4D). mediated sorting and retromer-Rab7-dependent retrograde transport. Figure 3. TBC1D5 and ATG9 bind to AP2 complex. A HA-Flag-TBC1D5 expression was induced in T-REx HeLa cells for 20 h, lysates were subjected to GST pull-down, using GST and GST-AP2M1. Co-precipitated TBC1D5 was detected with anti-Flag antibody. B Control cells and TBC1D5 (shRNA#1)U2OS cell lysates subjected to SDS–PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1. C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h). DU2OS cells stably expressing HA-Flag-ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co-localization are indicated with arrows. EU2OS cells stably expressing HA-Flag-ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti-AP2A1 antibody. FU2OS cells stably expressing mCherry-ATG9A were treated with KU0063794 (6 h), lysed in co-immunoprecipitation buffer. Lysates from DMSO- or KU0063794-treated cells were split and equal volumes were incubated with RFP-Trap beads or GFP-Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS–PAGE. 396 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A BC E F ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 397 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic B C Figure 4.AP2 complex is required for ATG9 trafficking. AU2OS cells stably expressing HA-Flag-ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post-transfection, fixed, and stained with anti-HA antibody. B Co-localization of HA-Flag-ATG9A with TGN46. C Lysates of U2OS cells transfected with siRNA targeting AP2A1, or siRNA control AllStars analyzed by SDS–PAGE. DU2OS cells stably expressing mCherry-ATG9 were transiently transfected with GFP-Dynamin 2 or GFP-Dynamin 2 (K44A), 20 h post-transfection cells were fixed and immunostained with anti-TBC1D5 antibody. ATG9A and Dynamin 2 co-localization is indicated by arrows. Data information: Bar graphs (B), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. 398 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A B E F ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 399 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic Figure 5. CME inhibition alters ATG9-TBC1D5 interaction. AU2OS cells stably expressing HA-Flag-ATG9A treated with DMSO or Dynasore (100 lM) 15 min, fixed and immunostained with anti-HA and anti-TBC1D5 antibodies. B Quantification of ATG9A and AP2 co-localization from experiment in (A), pictures from 3 independent experiments were analyzed and co-localization quantified, statistics is calculated as described in Supplementary Materials and Methods. C RGB-line profile across the vesicle indicated with arrow in (B); red—AP2; green—ATG9A; blue—TBC1D5. D Quantification of ATG9A and AP2 co-localization. Cells were starved in EBSS media (45 min) or pretreated with Dynasore (15 min) and subsequently starved for 45 min in combination with Dynasore. Control cells were treated with DMSO. E Quantification of LC3 puncta in cells stably expressing HA-Flag-ATG9A, treated and presented as in (D). 100 cells were quantified in 3 independent experiments. F T-REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo#1 targeting AP2A1 (40 nM). 72 h post-transfection, expression of HA-Flag-TBC1D5 was induced with doxycycline (50 ng/ml), and 96 h post-transfection cells were treated with Dynasore for 15 min, or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min. Cells were lysed in co-immunoprecipitation buffer, and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4°C. Beads were washed 3 times with incubation buffer and subjected to SDS–PAGE. G Proposed model for AP2 and TBC1D5 role in ATG9 trafficking toward autophagosomes. At steady state ATG9 traffics from Golgi to the endosomes and to the plasma membrane. Plasma membrane fraction gets internalized via AP2. During autophagy ATG9-AP2 vesicles redistribute toward autophagosomes via interaction of TBC1D5 with AP2 and LC3. Data information: Bar graphs (B, D, E), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. Materials and Methods and in vitro binding assays can be found in Supplementary Materials and Methods. Dynasore treatments Supplementary information for this article is available online: Dynasore (Sigma Aldrich Cat. Nr. D7693) was dissolved in DMSO http://embor.embopress.org according to the manufacturer, and used at final concentrations of 100 lM. Acknowledgements We thank Daniela Höller, David McEwan, Ivana Novak, Paolo Grumati, and Autophagy stimulation Aliaksandr Khaminets for critical reading and comments on the manuscript. The work was supported by the Cluster of Excellence “Macromolecular Autophagy was stimulated by starvation or chemical inhibition of Complexes” of the Goethe University Frankfurt (EXC115), LOEWE Oncology mTOR. First, cells were washed 2 times in starvation media (EBSS, Signaling Network, and the LineUB European Research Council Advanced Gibco Cell Culture) and subsequently incubated for 45 min or 4 h. Grant to ID. For mTOR inhibition, we used a KU0063794 inhibitor [29], dissolved in DMSO according to the manufacturer (Axon Medchem, Author contributions Cat. Nr. 1472), and used at a final concentration of 1 lM. Control D.P. conducted the experiments. D.P and I.D. designed the research, analyzed cells were treated with DMSO only. the data, and wrote the manuscript. Statistical analysis Conflict of interest The authors declare that they have no conflict of interest. Co-localization thresholded Pearson’s correlation coefficients were calculated in Volocity Demo software (Perkin Elmer). 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TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy

Popovic, Doris; Dikic, Ivan
EMBO Reports , Volume 15 (4) – Apr 1, 2014
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Copyright © The Authors 2014
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1469-221X
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10.1002/embr.201337995
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Abstract

Scientific Report TBC1D5 and the AP2 complex regulate ATG9 trafficking and initiation of autophagy Doris Popovic & Ivan Dikic Abstract induction [14]. Dispersal of ATG9 during autophagy is dependent on ULK1 [14] and contributes to the formation of autophagosome The RabGAP protein TBC1D5 controls cellular endomembrane traf- precursors initiated by DFCP1 [15]. However, the nature of ATG9 ficking processes and binds the retromer subunit VPS29 and the spatio-temporal redistribution remains unclear. Moreover, recent ubiquitin-like protein ATG8 (LC3). Here, we describe that TBC1D5 studies suggest that ATG9 traffic carriers may not incorporate into also associates with ATG9 and the active ULK1 complex during autophagosomes, but only transiently associate with them [15]. autophagy. Moreover, ATG9 and TBC1D5 interact with clathrin and Previously, we proposed that proteins known to accelerate the AP2 complex. Depletion of TBC1D5 leads to missorting of ATG9 hydrolysis of GTP on small Rab GTPases (RabGAP) act as autopha- to late endosomes upon activation of autophagy, whereas inhibi- gic adaptors that regulate autophagy-endocytosis crosstalk via direct tion of clathrin-mediated endocytosis or AP2 depletion alters ATG9 interaction with the autophagy ubiquitin-like protein LC3 [16]. trafficking and its association with TBC1D5. Taken together, our TBC1D14 and TBC1D25 play a role in early and late stages of auto- data show that TBC1D5 and the AP2 complex are important novel phagosome maturation, respectively [17,18]. TBC1D5 binds directly regulators of the rerouting of ATG9-containing vesicular carriers to LC3 and retromer subunit VPS29, via LC3 interacting region 1 toward sites of autophagosome formation. (LIR1), and regulates internalization and TGN retrieval of CI-M6PR. During autophagy, mutually exclusive binding to LC3 mediates its Keywords AP2; ATG9; autophagy; clathrin endocytosis; TBC1D5 transfer from retromer to the autophagosomal membrane [16]. Subject Categories Autophagy & Cell Death; Membrane & Intracellular Although retromer complex associates with the autophagic machinery Transport [19], its role in autophagy is unclear [20]. ATG9 is the only autophagic DOI 10.1002/embr.201337995 | Received 13 September 2013 | Revised 6 protein reported to co-fractionate with retromer, TGN46, and February 2014 | Accepted 10 February 2014 | Published online 6 March 2014 CI-M6PR-containing compartments [14]. We therefore hypothesized EMBO Reports (2014) 15, 392–401 that TBC1D5 may impact on ATG9 trafficking within the endosomal system and we aimed to define the link between coordinated trafficking patterns of ATG9, retromer, and TBC1D5. Introduction Here, we report the functional interaction between TBC1D5, ATG9, and AP2 complex. During autophagy, TBC1D5 dissociates from Macroautophagy (autophagy) is a membrane-dependent process, retromer and associates with ATG9 and active ULK1. Additionally, critical for delivery of various cytoplasmic cargo (e.g., protein aggre- we show that ATG9 trafficking is regulated by AP2 and TBC1D5. gates, damaged organelles, and pathogens) to the lysosomes (for We propose that during autophagy, the efficient rerouting of ATG9 degradation) [1] and secretion of molecules that mediate immune carriers toward PAS requires the dynamic transition of TBC1D5 to responses [2]. Numerous membrane sources are implicated in the autophagosomes and that AP2 complex mediates such trafficking formation of autophagosomes: endoplasmic reticulum [3], mito- reorganization. chondria [4,5], plasma membrane [6], and Golgi complex [7–10]. This suggests that autophagy closely intersects with and depends on various membrane trafficking events inside cell [11]. More than 30 Results and Discussion autophagy-related genes take part in phagophore formation [12] including the essential ULK1/ATG13/FIP200 kinase complex and TBC1D5 associates with ATG9 and ULK1 ATG9. While the trafficking of yeast ATG9 appears better under- stood, knowledge on its mammalian homologue is far more elusive ATG9 has 6 transmembrane regions and localizes to TGN and Rab7-, [13]. Mammalian ATG9 localizes to the trans-Golgi network and to Rab9-, and CI-M6PR-containing endosomes [14]. Upon autophagy late endosomes under basal conditions and cycles between its stimuli, ATG9 relocates to peripheral sites driving the growth of reservoirs and phagophore assembly site (PAS) upon autophagy the phagophore. In U2OS cells stably expressing mCherry-ATG9, Buchmann Institute for Molecular Life Sciences (BMLS) and Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt, Germany *Corresponding author. Tel: +49 69 6301 5964; Fax: +49 69 6301 5577; E-mail: [email protected] 392 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A B D E F G H ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 393 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic Figure 1. TBC1D5 and retromer associate with ATG9. A Co-localization of endogenous TBC1D5 and VPS35 with mCherry-ATG9A, in steady state and autophagy induced by mTOR inhibitor KU0063794 (6 h). B, C Quantification of co-localization experiments presented in (A). D Co-immunoprecipitation of HA-Flag-TBC1D5 with myc-VPS29, transiently overexpressed in 293T cells, DMSO or KU0063794 (6 h) treated. E, F Co-immunoprecipitation of endogenous TBC1D5 and ATG9 with myc-VPS29 (E) transiently overexpressed in 293T cells or with HA-Flag-VPS35 (F) transiently overexpressed in 293T cells, treated with DMSO or KU0063794 (6 h). G Co-immunoprecipitation of endogenous VPS29 and ATG9 with HA-Flag-TBC1D5 stably expressed in 293T cells, treated with DMSO or KU0063794 (6 h). H Co-immunoprecipitation of endogenous ULK1 and pATG13 (Ser318) with HA-Flag-TBC1D5, stably expressed in T-REx 293T cells, induced by doxycycline (50 ng/ml) for 20 h prior to DMSO or KU0063794 (6 h) treatment. I Magnified still images extracted from Supplementary Movie S2. mCherry-TBC1D5 co-localizes with GFP-ATG9 upon autophagy induction by mTOR inhibitor, KU0063794. Data information: Bar graphs (B, C), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. endogenous TBC1D5, retromer subunit VPS35, and mCherry-ATG9 TBC1D5 is required for ATG9 trafficking co-localized in steady state as well as during autophagy induced by the mTOR inhibitor KU0063974 (Fig 1A). Closer inspection Recently, it has been suggested that retromer is both required and of ATG9-containing vesicles showed that ATG9 does not enter dispensable for autophagy [15,19]. To test whether retromer retromer-associated endosomal tubules (Supplementary Fig S1A). contributes to ATG9 trafficking, we stably depleted U2OS cells of Co-localization of TBC1D5 with ATG9 was steadily high in both TBC1D5 or retromer subunit VPS29 and compared them with tested conditions (Fig 1B), while retromer co-localization decreased control shRNA-expressing cells (Fig 2A). In VPS29-deficient cells, upon autophagy induction (Fig 1C). Endogenous ATG9, LC3, and we observed destabilization of all retromer subunits, but no appar- TBC1D5 also co-localized in cells (Supplementary Fig S1B). Next, ent defect in autophagy flux upon autophagy stimulation (Fig 2A, we tested whether TBC1D5 and retromer associate with ATG9. Supplementary Fig S4A). Depletion of TBC1D5 severely affected TBC1D5 and ATG9 co-precipitated with retromer subunit VPS29 the LC3 level and stabilized ATG9, suggesting that TBC1D5 acts (Fig 1D and E) or VPS35 (Fig 1F) only in non-stimulated conditions. also independently of retromer (Fig 2A). Separate regulation of Upon autophagy induction, HA-Flag-TBC1D5 bound less endoge- retromer and ATG9 by TBC1D5 is possible because neither retro- nous VPS29, but interacted stronger with ATG9 (Fig 1G). mer co-localizes with LC3 (Supplementary Fig S1C) nor ATG9 TBC1D5 association with autophagosomes is regulated via bind- associates with retromer during autophagy. TBC1D5-deficient cells ing to LC3 [16]. As expected from our previous study, autophago- showed decreased number of ATG9 vesicular structures in fed somes containing TBC1D5 were negative for retromer staining conditions (Fig 2B and C) while, upon autophagy induction, ATG9 was enriched in late endosomes as indicated by LAMP1 staining (Supplementary Fig S1C). The early autophagosomal marker DFCP1 partially co-localized with TBC1D5 (Supplementary Fig S2B), while (Fig 2B and D). Importantly, retromer depletion did not lead to ATG16 did not (Supplementary Fig S2B), indicating that TBC1D5 similar defect in ATG9 localization, indicating that retromer is specifically associates with ATG9 labeled early autophagosomes. dispensable for ATG9 trafficking during autophagy. We rescued Live-cell imaging of U2OS cells stably expressing GFP-ATG9 and the effect on ATG9 by introducing shRNA-resistant TBC1D5 to mCherry-TBC1D5 revealed co-localization of ATG9- and TBC1D5- assure that observed phenotype is specific for TBC1D5 depletion containing vesicles at steady state (Supplementary Movie S1) and (Fig 2F and G). during autophagy (Fig 1I, Supplementary Movies S2 and S3). In addition, the major upstream kinase ULK1 co-localized with TBC1D5 and ATG9A interact with AP2 complex TBC1D5 (Supplementary Fig S3A) and together with the phosphory- lated form of ATG13 co-precipitated with TBC1D5 (Fig 1H), suggest- As ATG9 accumulated in late endosomes and lysosomes upon ing that TBC1D5 associates with the active kinase as well. TBC1D5 depletion, we hypothesized that TBC1D5 regulates sorting Figure 2. TBC1D5 is required for ATG9 trafficking. A Lysates of U2OS cells stably expressing control shRNA or shRNA targeting VPS29 or TBC1D5. Cells were treated with DMSO or KU0063794 (6 h), lysed in RIPA buffer (1% SDS) and lysates were subjected to SDS–PAGE. BU2OS cells stably expressing mCherry-ATG9 were depleted of VPS29 and TBC1D5 using shRNAs, treated with DMSO or KU0063794 (6 h), fixed with 2% PFA, and immunostained with anti-LAMP1 antibody. C Quantification of ATG9 vesicular structures in cells from (B) under non-stimulated conditions. D Quantification of ATG9 and LAMP1 co-localization in cells presented in (B). E Quantification of ATG9 and LAMP1 co-localization in cells stably depleted for TBC1D5 (shRNA#1) or VPS29 (shRNA#2), transiently transfected with myc-TBC1D5 plasmid resistant to TBC1D5 shRNA#1. Cells were treated with KU0063794 (6 h), 20 h post-transfection, fixed, and immunostained with anti-LAMP1 and anti-myc antibodies. F Control shRNA and U2OS cell lysates depleted for TBC1D5 (shRNA#1), transfected with empty plasmid or with shRNA-resistant myc-TBC1D5. G Immunofluorescence of TBC1D5 (shRNA#1) cells transiently transfected with shRNA-resistant myc-TBC1D5, treated with KU0063794 (6 h). 20 h post-transfection cells were treated, subsequently fixed, and immunostained with anti-myc and anti-LAMP1 antibodies. Data information: Bar graphs (C, E), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. Bar graph (D), mean  s.d. n = 3, one-way ANOVA with Bonferroni s multiple comparison test, ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. 394 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A B D EF ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 395 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic or recycling of ATG9. Interestingly, our published mass spectrometry Next, we treated cells with the Dynamin 2 inhibitor Dynasore interactome of TBC1D5 contained two subunits of AP2 complex, but [25], which led to a significant increase in ATG9 and TBC1D5 co- no other endosome-TGN regulatory proteins such as TIP47 or localization with AP2 (Fig 5A-C), similar to the effect of starva- PACS1 [21]. Available PPI (protein-protein interaction) database tion (Fig 5D, Supplementary Fig S8A). This co-localization (Biogrid) reports TBC1D5 as an AP2M1 interactor [22]. We purified became even more pronounced when cells were pretreated with AP2M1 subunit and confirmed its binding to overexpressed Dynasore and subsequently starved (Fig 5D, Supplementary HA-Flag-TBC1D5 expressed in cells (Fig 3A), as well as purified Fig S8A). However, the plasma membrane regions enriched with GST-TBC1D5 (Supplementary Fig S5A). TBC1D5 interaction with AP2/ATG9/TBC1D5 excluded LC3 (Supplementary Fig S8B) and AP2 complex was independent on LIR1 or GAP activity (Supple- the number of LC3-positive puncta was reduced (Fig 5E), indicat- mentary Fig S5B). TBC1D5 depletion led to the destabilization ing that Dynasore delayed autophagosome formation. Inspection of AP2 complex (Fig 3B) and AP2 redistribution to the perinuclear of live cells showed that Dynasore treatment led to co-localization area (Fig 3C). Moreover, TBC1D5 and ATG9 co-localized with the of ATG9 and TBC1D5 in static vesicles (Supplementary Movie AP2 complex (Fig 3D, Supplementary Fig S6A). Importantly, co- S5). Notably, depletion of AP2 complex abolished the TBC1D5 localization of ATG9 with AP2 was enhanced during autophagy interaction with ATG9 (Fig 5F), suggesting that TBC1D5-ATG9 (Fig 3E, Supplementary Fig S6B) and readily disturbed upon association is dynamic and is restricted to the AP2-clathrin- the depletion of TBC1D5 (Supplementary Fig S6B). Imaging of associated fraction of ATG9. GFP-ATG9 stably expressing U2OS cells (Supplementary Movie S4) Direct TBC1D5 interaction with LC3 [16] and AP2 complex revealed numerous highly mobile vesicles. Based on this notion, we prompted us to hypothesize that TBC1D5 regulates the recruitment hypothesized that ATG9 is incorporated into AP2-clathrin-coated of ATG9-AP2-containing vesicles to the autophagic membranes vesicles (CCVs) of plasma membrane origin. Diameter of ATG9- (Fig 5G). Further experiments, however, are needed to gain containing vesicles has been estimated to be 30–60 nm [20], which mechanistic understanding of TBC1D5 connection to AP2, and is somewhat close to CCV size [23]. As proposed, AP2 complex and to pinpoint regulatory mechanisms underlying this interaction in clathrin readily co-precipitated with ATG9 (Fig 3F), prominently cells [26]. upon autophagy induction, while retromer dissociated from ATG9 Few reports agree on the relevance of the AP2 complex in (Fig 3F). Taken together, our data indicate that TBC1D5 regulates ATG16 trafficking and lysosome biogenesis [6,27]. Our findings ATG9 via AP2 complex. indicate that TBC1D5 and AP2 complex directly affect ATG9 traf- ficking. During the revision of our manuscript, Puri et al [28] have AP2 and clathrin-mediated endocytosis (CME) are required for also reported that ATG9 traffics from plasma membrane in the ATG9 sorting and autophagy clathrin-positive carriers that are routed differently from ATG16 carriers [6,27]. In that study, ATG9 has been co-localized with AP2 complex binds to clathrin and acts as a major hub for early endosomal GTPase Rab5, whereas ATG16 failed to do so. protein interactions in clathrin-coated pits. Its recruitment during However, both ATG9 and ATG16 would coalesce at stage of the last stage of CCV formation at the plasma membrane precedes recycling endosomes in dependence on SNARE protein VAMP1. Dynamin 2-dependent pinching-off [24]. To understand the role Interestingly, TBC1D5 associates with ATG9 but does not co-localize of ATG9 interaction with AP2 and clathrin, we silenced expres- with ATG16, while depletion of TBC1D5 enhances endosomal sion of AP2 or blocked maturation of CCVs by expressing a localization of ATG9. Our data support the idea that TBC1D5 dominant-negative form of Dynamin 2. In normal growing condi- might specifically regulate the recruitment of ATG9 from early tions, AP2 depletion enhanced ATG9 localization on the plasma stage of endocytosis to the site of forming autophagosomes. It membrane (Fig 4A), while upon autophagy induction, ATG9 accu- remains to be addressed by what mechanisms ATG9 and ATG16 mulated mostly in the TGN if compared to control siRNA-treated are targeted to a different population of clathrin-coated vesicles, cells (Fig 4A and B, Supplementary Fig S7A). Moreover, lack of and whether additional components of endocytic machinery AP2 induced accumulation of p62 (Fig 4C) and significantly participate in their segregation or collision. impaired LC3 lipidation (Supplementary Fig S7B) consistent with In conclusion, we propose a dual role for TBC1D5: it regulates reported data [6]. Differently, the dominant-negative form of membrane trafficking, upon autophagy induction, and it contributes Dynamin 2 (K44A) dispersed ATG9 toward the plasma membrane to different routes of endosomal sorting including clathrin-AP2- (Fig 4D). mediated sorting and retromer-Rab7-dependent retrograde transport. Figure 3. TBC1D5 and ATG9 bind to AP2 complex. A HA-Flag-TBC1D5 expression was induced in T-REx HeLa cells for 20 h, lysates were subjected to GST pull-down, using GST and GST-AP2M1. Co-precipitated TBC1D5 was detected with anti-Flag antibody. B Control cells and TBC1D5 (shRNA#1)U2OS cell lysates subjected to SDS–PAGE, subsequently blotted with endogenous antibodies for AP2 subunits AP2A1 and AP2M1. C Immunofluorescent staining of AP2A1 in U2OS shRNA control, or shRNA#1 TBC1D5 cells treated with KU0063974 or DMSO (6 h). DU2OS cells stably expressing HA-Flag-ATG9A were fixed and stained for endogenous AP2A1 and TBC1D5. Regions of co-localization are indicated with arrows. EU2OS cells stably expressing HA-Flag-ATG9A were starved in EBSS (4 h) or treated with KU0063794 (6 h), subsequently fixed and stained with anti-AP2A1 antibody. FU2OS cells stably expressing mCherry-ATG9A were treated with KU0063794 (6 h), lysed in co-immunoprecipitation buffer. Lysates from DMSO- or KU0063794-treated cells were split and equal volumes were incubated with RFP-Trap beads or GFP-Trap beads, or agarose as a negative control, overnight at 4°C. Precipitated proteins were analyzed by SDS–PAGE. 396 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A BC E F ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 397 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic B C Figure 4.AP2 complex is required for ATG9 trafficking. AU2OS cells stably expressing HA-Flag-ATG9A transfected with siRNA targeting AP2A1 (40 nM), or siRNA AllStar control (40 nM) were treated with KU0063794 or DMSO (6 h) 4 days post-transfection, fixed, and stained with anti-HA antibody. B Co-localization of HA-Flag-ATG9A with TGN46. C Lysates of U2OS cells transfected with siRNA targeting AP2A1, or siRNA control AllStars analyzed by SDS–PAGE. DU2OS cells stably expressing mCherry-ATG9 were transiently transfected with GFP-Dynamin 2 or GFP-Dynamin 2 (K44A), 20 h post-transfection cells were fixed and immunostained with anti-TBC1D5 antibody. ATG9A and Dynamin 2 co-localization is indicated by arrows. Data information: Bar graphs (B), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. 398 EMBO reports Vol 15 |No 4 | 2014 ª 2014 The Authors Doris Popovic & Ivan Dikic TBC1D5 and AP2 complex regulate ATG9 in autophagy EMBO reports A B E F ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 399 EMBO reports TBC1D5 and AP2 complex regulate ATG9 in autophagy Doris Popovic & Ivan Dikic Figure 5. CME inhibition alters ATG9-TBC1D5 interaction. AU2OS cells stably expressing HA-Flag-ATG9A treated with DMSO or Dynasore (100 lM) 15 min, fixed and immunostained with anti-HA and anti-TBC1D5 antibodies. B Quantification of ATG9A and AP2 co-localization from experiment in (A), pictures from 3 independent experiments were analyzed and co-localization quantified, statistics is calculated as described in Supplementary Materials and Methods. C RGB-line profile across the vesicle indicated with arrow in (B); red—AP2; green—ATG9A; blue—TBC1D5. D Quantification of ATG9A and AP2 co-localization. Cells were starved in EBSS media (45 min) or pretreated with Dynasore (15 min) and subsequently starved for 45 min in combination with Dynasore. Control cells were treated with DMSO. E Quantification of LC3 puncta in cells stably expressing HA-Flag-ATG9A, treated and presented as in (D). 100 cells were quantified in 3 independent experiments. F T-REx HeLa cells were transfected with siRNA Control oligo or siRNA oligo#1 targeting AP2A1 (40 nM). 72 h post-transfection, expression of HA-Flag-TBC1D5 was induced with doxycycline (50 ng/ml), and 96 h post-transfection cells were treated with Dynasore for 15 min, or pretreated with Dynasore and subsequently starved in combination with Dynasore for additional 45 min. Cells were lysed in co-immunoprecipitation buffer, and lysates were incubated with Myc antibody or M2 antibody coupled with agarose overnight at 4°C. Beads were washed 3 times with incubation buffer and subjected to SDS–PAGE. G Proposed model for AP2 and TBC1D5 role in ATG9 trafficking toward autophagosomes. At steady state ATG9 traffics from Golgi to the endosomes and to the plasma membrane. Plasma membrane fraction gets internalized via AP2. During autophagy ATG9-AP2 vesicles redistribute toward autophagosomes via interaction of TBC1D5 with AP2 and LC3. Data information: Bar graphs (B, D, E), mean  s.d. n = 3, unpaired two-tailed t-test, P-values: ns P > 0.05,*P ≤ 0.05,**P ≤ 0.01, ***P ≤ 0.001. Materials and Methods and in vitro binding assays can be found in Supplementary Materials and Methods. Dynasore treatments Supplementary information for this article is available online: Dynasore (Sigma Aldrich Cat. Nr. D7693) was dissolved in DMSO http://embor.embopress.org according to the manufacturer, and used at final concentrations of 100 lM. Acknowledgements We thank Daniela Höller, David McEwan, Ivana Novak, Paolo Grumati, and Autophagy stimulation Aliaksandr Khaminets for critical reading and comments on the manuscript. The work was supported by the Cluster of Excellence “Macromolecular Autophagy was stimulated by starvation or chemical inhibition of Complexes” of the Goethe University Frankfurt (EXC115), LOEWE Oncology mTOR. First, cells were washed 2 times in starvation media (EBSS, Signaling Network, and the LineUB European Research Council Advanced Gibco Cell Culture) and subsequently incubated for 45 min or 4 h. Grant to ID. For mTOR inhibition, we used a KU0063794 inhibitor [29], dissolved in DMSO according to the manufacturer (Axon Medchem, Author contributions Cat. Nr. 1472), and used at a final concentration of 1 lM. Control D.P. conducted the experiments. D.P and I.D. designed the research, analyzed cells were treated with DMSO only. the data, and wrote the manuscript. Statistical analysis Conflict of interest The authors declare that they have no conflict of interest. Co-localization thresholded Pearson’s correlation coefficients were calculated in Volocity Demo software (Perkin Elmer). 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Garcia-Martinez JM, Moran J, Clarke RG, Gray A, Cosulich SC, Chresta TBC1D14 regulates autophagosome formation via Rab11- and ULK1- CM, Alessi DR (2009) Ku-0063794 is a specific inhibitor of the mamma- positive recycling endosomes. J Cell Biol 197: 659– 675 lian target of rapamycin (mTOR). Biochem J 421: 29– 42 ª 2014 The Authors EMBO reports Vol 15 |No 4 | 2014 401

Journal

EMBO Reports – Springer Journals

Published: Apr 1, 2014

Keywords: AP2; ATG9; autophagy; clathrin endocytosis; TBC1D5

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