Autophagosome formation is initiated at phosphatidylinositol synthase‐enriched ER subdomains
2017; Springer Nature; Volume: 36; Issue: 12 Linguagem: Inglês
10.15252/embj.201695189
ISSN1460-2075
AutoresTaki Nishimura, Norito Tamura, Nozomu Kono, Yuta Shimanaka, Hiroyuki Arai, Hayashi Yamamoto, Noboru Mizushima,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoArticle11 May 2017free access Transparent process Autophagosome formation is initiated at phosphatidylinositol synthase-enriched ER subdomains Taki Nishimura Corresponding Author [email protected] orcid.org/0000-0003-4019-5984 Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Norito Tamura Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Search for more papers by this author Nozomu Kono Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yuta Shimanaka Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroyuki Arai Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan AMED-CREST, Japan Agency for Medical Research and Development, Tokyo, Japan Search for more papers by this author Hayashi Yamamoto Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Noboru Mizushima Corresponding Author [email protected] orcid.org/0000-0002-6258-6444 Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Taki Nishimura Corresponding Author [email protected] orcid.org/0000-0003-4019-5984 Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Norito Tamura Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Search for more papers by this author Nozomu Kono Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yuta Shimanaka Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan Search for more papers by this author Hiroyuki Arai Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan AMED-CREST, Japan Agency for Medical Research and Development, Tokyo, Japan Search for more papers by this author Hayashi Yamamoto Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Noboru Mizushima Corresponding Author [email protected] orcid.org/0000-0002-6258-6444 Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Author Information Taki Nishimura *,1,†, Norito Tamura1,2, Nozomu Kono3, Yuta Shimanaka3, Hiroyuki Arai3,4, Hayashi Yamamoto1 and Noboru Mizushima *,1 1Department of Biochemistry and Molecular Biology, Graduate School and Faculty of Medicine, The University of Tokyo, Tokyo, Japan 2Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan 3Department of Health Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan 4AMED-CREST, Japan Agency for Medical Research and Development, Tokyo, Japan †Present address: Medical Research Council Laboratory for Molecular Cell Biology, University College London, London, UK *Corresponding author. Tel: +44 020 7679 7208; E-mail: [email protected] *Corresponding author. Tel: +81 3 5841 3440; Fax: +81 3 3815 1490; E-mail: [email protected] EMBO J (2017)36:1719-1735https://doi.org/10.15252/embj.201695189 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The autophagosome, a double-membrane structure mediating degradation of cytoplasmic materials by macroautophagy, is formed in close proximity to the endoplasmic reticulum (ER). However, how the ER membrane is involved in autophagy initiation and to which membrane structures the autophagy-initiation complex is localized have not been fully characterized. Here, we were able to biochemically analyze autophagic intermediate membranes and show that the autophagy-initiation complex containing ULK and FIP200 first associates with the ER membrane. To further characterize the ER subdomain, we screened phospholipid biosynthetic enzymes and found that the autophagy-initiation complex localizes to phosphatidylinositol synthase (PIS)-enriched ER subdomains. Then, the initiation complex translocates to the ATG9A-positive autophagosome precursors in a PI3P-dependent manner. Depletion of phosphatidylinositol (PI) by targeting bacterial PI-specific phospholipase C to the PIS domain impairs recruitment of downstream autophagy factors and autophagosome formation. These findings suggest that the autophagy-initiation complex, the PIS-enriched ER subdomain, and ATG9A vesicles together initiate autophagosome formation. Synopsis The autophagy initiating ULK complex is recruited to an endoplasmic reticulum subdomain enriched in phosphatidylinositol synthase and subsequently translocates to ATG9A-positive autophagosome precursors in a phosphatidylinositol-dependent manner. ULK complex components associate with two distinct membranes: the ER membrane and ATG9A-enriched membrane. Upon autophagy initiation, the ULK complex is recruited to an ER subdomain enriched in phosphatidylinositol synthase (PIS). Phosphatidylinositol in the PIS-enriched membrane is required for autophagosome formation. The ULK complex translocates to ATG9A-positive autophagosome precursors in a PI3P-dependent manner. Introduction Macroautophagy (hereafter referred to as autophagy) is a catabolic process that is highly conserved among eukaryotes. When autophagy is induced, a part of the cytoplasm is surrounded by a membrane sac, termed the isolation membrane (also known as the phagophore). The isolation membrane expands to form a double-membrane autophagosome, which subsequently fuses with lysosomes for degradation of engulfed materials. Autophagosome formation involves a number of autophagy-related (ATG) proteins (Xie & Klionsky, 2007; Mizushima et al, 2011). Systematic hierarchical analysis revealed that the serine/threonine kinase Atg1 (UNC-51-like kinases (ULKs) in mammals) complex is the most upstream unit among the ATG proteins (Suzuki et al, 2007; Itakura & Mizushima, 2010). This complex is composed of Atg1, Atg13, Atg17, Atg29, and Atg31 in yeast and ULK1 (or ULK2), ATG13, FIP200 (also known as RB1CC1 and partially homologous to yeast Atg11 and Atg17), and ATG101 in mammals (Mizushima et al, 2011). Atg17 and FIP200 are proposed to act as a scaffold for recruitment of downstream Atg proteins (Ragusa et al, 2012; Suzuki et al, 2013). Upon nutrient deprivation, Atg1/ULK is activated and phosphorylates downstream effectors, such as BECLIN1 (Russell et al, 2013), ATG14 (Park et al, 2016), and Atg9/ATG9A (Papinski et al, 2014; Zhou et al, 2017), to induce autophagosome formation. Thus, it is critical to understand the membrane structures/domains on which the activated Atg1/ULK complex acts. It is well known that early autophagic structures are formed in close apposition to the endoplasmic reticulum (ER)-related subdomains such as the omegasome (Axe et al, 2008), ER-mitochondrial contact site (Hamasaki et al, 2013), ER exit site (Graef et al, 2013; Suzuki et al, 2013), ER-Golgi intermediate compartment (ERGIC; Ge et al, 2013), and isolation membrane-associated tubular/vesicular structures (Uemura et al, 2014). In mammalian cells, the ULK complex is translocated to punctate structures in tight association with the ER by both phosphatidylinositol (PI)3P-dependent and -independent mechanisms (Itakura & Mizushima, 2010; Karanasios et al, 2013). It was also reported that the ULK complex localizes to transferrin receptor-positive recycling endosomes (Longatti et al, 2012). However, the precise target membrane of the Atg1/ULK complex remains unknown. Here, we performed systematic biochemical analysis using various ATG knockout cells and found that the ULK complex first localizes to PI synthase (PIS)-enriched ER subdomains and then translocates to the ATG9A-positive autophagosome precursors in a PI3P-dependent manner. PI in the PIS-enriched membrane is required for autophagosome formation. Results The autophagy-initiation complex associates with two distinct membrane structures To analyze the autophagic membranes, cell homogenates of mouse embryonic fibroblasts (MEFs) were subjected to flotation analysis. As for wild-type (WT) MEF homogenates, ATG proteins, except for the membrane protein ATG9A and membrane-bound microtubule-associated protein light chain 3 (LC3)-II, mostly remained in the bottom cytosolic fractions (Fig 1A). Consistent with a previous report (Chan et al, 2009), small proportions of the ULK complex components were found in floated membrane fractions under growing conditions. Flotation of FIP200, ATG13, ATG101, and WIPI2 into lighter density fractions became clearer under starvation condition (Fig EV1A), indicating that autophagic membranes were floated. In order to enrich these autophagic membranes, we used several ATG-deficient cell lines, in which autophagosome formation is blocked at various steps leading to accumulation of autophagic intermediate structures at specific stages under growing conditions (Fig EV1B; Itakura & Mizushima, 2010; Kageyama et al, 2011; Kishi-Itakura et al, 2014). Figure 1. Flotation of ATG proteins derived from ATG14 KO and ATG3 KO cell homogenates A. OptiPrep membrane flotation analysis of WT MEFs cultured in regular DMEM (Growing). After fractionation, each fraction was centrifuged at 100,000 × g for 40 min to enrich the membranes. Arrow indicates the position of ATG14. B. Autophagosome formation. ATG14 and ATG3 are required for the nucleation and elongation or closure steps during autophagosome formation, respectively. C, D. ATG14 KO (C) and ATG3 KO MEFs (D) were cultured in regular DMEM and subjected to OptiPrep flotation analysis as described above. Asterisks indicate the flotation of ATG proteins from ATG14 KO and ATG3 KO homogenates. Arrow in (D) indicates the position of ATG14. E, F. ATG3 KO MEFs stably expressing FLAG-ATG9A were cultured in regular DMEM (E) or starvation medium in the presence of 200 nM wortmannin (Starv. + WM) (F) for 2 h. Note that wortmannin treatment significantly reduced the amounts of the ULK complex components, WIPI2, and ATG5 in the top fraction (compare † in E with †† in F). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. FIP200 is recruited to membrane structures independently of the other ULK complex components and ATG9A A. WT MEFs stably expressing FLAG-ATG9A were cultured in starvation medium for 2 h and subjected to OptiPrep membrane flotation analysis as in Fig 1A. Asterisk indicates the fractions of floated Atg proteins. B. Validation of ATG KO cell lines. WT and various ATG KO MEFs were analyzed by immunoblotting using the indicated antibodies. C–F. FIP200 KO (C), ULK1/2 DKO (D), ATG13 KO (E), and ATG101 KO MEFs (F) were cultured in regular DMEM and subjected to OptiPrep membrane flotation analysis. Asterisks indicate the flotation of FIP200 into middle-density fractions. G. The indicated ATG KO MEFs were cultured in starvation medium for 1 h and analyzed by immunofluorescence microscopy using anti-FIP200 antibody. Scale bar, 10 μm. H. WT MEFs stably expressing GFP-ATG9A were cultured in starvation medium in the presence or absence of 200 nM wortmannin for 1 h. Cells were fixed and analyzed by immunofluorescence microscopy using anti-FIP200 and WIPI2 antibodies. Scale bar, 10 μm. Download figure Download PowerPoint We first analyzed homogenates obtained from ATG14 knockout (KO) and ATG3 KO MEFs. ATG14, a component of the PI 3-kinase complex, and ATG3 are required for the nucleation step and the elongation or closure step during autophagosome formation, respectively (Fig 1B; Mizushima et al, 2011; Kishi-Itakura et al, 2014). Thus, early autophagosome precursors and elongating isolation membranes accumulate in ATG14 KO and ATG3 KO cells, respectively. As for the ATG14 KO homogenate, in addition to ATG9A and LC3-II, the ULK complex components (FIP200, ATG13, ATG101, and ULK1) were floated into middle-density fractions (fractions 5–7) even under growing conditions (Fig 1C, *). On the other hand, the isolation membrane-resident proteins WIPI2 and ATG5 were not clearly floated. These results suggest that these middle-density fractions contain autophagic precursor membranes, to which the ULK complex is recruited in a PI 3-kinase-independent manner. Similarly, when ATG3 KO homogenates were used, the ULK complex components and autophagy-specific PI 3-kinase complex components (ATG14, VPS34, and BECLIN1) were floated into the middle-density fractions (fractions 4–7; Fig 1D, **). In contrast to ATG14 KO homogenates, the ULK complex components, WIPI2, and ATG5 were also enriched in the top (lightest) fraction (fraction 1) in ATG3 KO homogenates (Fig 1D, ***), suggesting that the top fraction contains elongating isolation membranes. To confirm whether the flotation of these ATG proteins was dependent on formation of the isolation membrane, the effect of acute inhibition of the PI 3-kinase, which is required for isolation membrane elongation (Kishi-Itakura et al, 2014), was examined using wortmannin. The amounts of the ULK complex components, WIPI2, and ATG5 in the top fraction were markedly reduced in ATG3 KO cells after wortmannin treatment (Fig 1E and F), suggesting that isolation membranes are indeed enriched in the top fraction. On the other hand, the flotation of the ULK complex components into the middle-density fractions was unaffected by wortmannin treatment (Fig 1F), which is consistent with the results in ATG14 KO cells (Fig 1C). While the puncta of WIPI2, a PI3P-binding protein, were sensitive to wortmannin, a significant proportion of the FIP200 puncta was not (Fig EV1H). These results suggest that the ULK complex is sequentially recruited to the two distinct membrane structures with middle and light densities in PI3P-independent and -dependent manners, respectively. These structures can be separated using ATG14 KO and ATG3 KO cells. FIP200 tethers the ULK complex subunits to membranes Next, we examined which component of the ULK complex determines its membrane targeting. In FIP200 KO cells, ATG13, ATG101, and ULK1 were not clearly floated into the middle-density fractions (Fig EV1C). By contrast, FIP200 was floated in all of the ULK1/2 double KO (DKO), ATG13 KO, and ATG101 KO cells (Fig EV1D–F). ATG101 and ULK1 were not clearly floated in ATG13 KO cells (Fig EV1E). Consistently, FIP200 localized to punctate structures in ATG13 KO, ATG101 KO, and ULK1/2 DKO MEFs (Fig EV1G). These results suggest that FIP200 has the ability to target membranes independently of the other components of the ULK complex. FIP200 is co-purified with the ER-related membranes and ATG9A-positive isolation membranes Our flotation analysis demonstrated that a significant population of the ATG9A-positive membranes was also collected in the middle- and light-density fractions, in which the ULK complex components accumulated when autophagosome formation was blocked (Fig 1C and D, asterisks). In mammals, it has been reported that ATG9A dynamically associates with the isolation membrane, but is not contained in the isolation membrane or autophagosomal membranes (Orsi et al, 2012; Lamb et al, 2016). On the other hand, in yeast, Atg9-containing vesicles are thought to be a seed membrane for autophagosome biogenesis and Atg9 is indeed incorporated into autophagic membranes (Yamamoto et al, 2012). These findings prompted us to investigate the relationship between the ULK complex and ATG9A vesicles in these fractions. When ATG9A vesicles were purified from the middle-density fractions derived from non-starved WT (Fig EV2A) and ATG14 KO MEF homogenates (Fig EV2B), RAB1A (a mammalian Ypt1 homolog) was co-precipitated (Fig 2A–C). This is consistent with previous reports that Ypt1 is recruited to Atg9 vesicles in yeast (Kakuta et al, 2012). However, FIP200 was not detected in these FLAG-ATG9A immunoprecipitates from the middle-density fractions of WT or ATG14 KO MEFs (Fig 2B and C), although both ATG9A and FIP200 were present in these fractions in ATG14 KO MEFs (Fig EV2B). These data suggest that FIP200 is mainly recruited to membrane structures other than ATG9A vesicles in ATG14 KO MEFs. In line with this, FIP200 was clearly floated to the middle-density fractions in the absence of ATG9A (Fig EV2C). Furthermore, FIP200 formed punctate structures in ATG9A KO MEFs (Fig EV1G), as did ULK1 (Itakura et al, 2012; Orsi et al, 2012). These results suggest that the ULK complex can target some membrane structures other than ATG9A vesicles at an early step during autophagosome formation. We further found that the middle-density fractions contained the ER membranes, and in these fractions of ATG14 KO homogenates, FIP200 was co-precipitated with ER membranes purified with FLAG-tagged SEC61B (Fig 2D). These results suggest that the ULK complex localizes to the ER-related membrane in the absence of autophagy-specific PI 3-kinase. Click here to expand this figure. Figure EV2. Separation of autophagy-related membranes in ATG9A KO and ATG14 KO cells A–C. The indicated MEF cells were cultured in regular DMEM and subjected to OptiPrep floatation analysis as shown in Fig 1E. Asterisks indicate the flotation of FIP200 into middle-density fractions (B, C). Download figure Download PowerPoint Figure 2. FIP200 is co-purified with the ER membranes from ATG14 KO MEF homogenates A. Experimental scheme of autophagic membrane purification. Cells stably expressing FLAG-ATG9A were cultured in regular DMEM and subjected to OptiPrep flotation analysis. FLAG-ATG9A-enriched membranes were precipitated from the middle-density fractions (fractions 4–7 mixture) or the top fraction (fraction 1) under detergent-free conditions using anti-FLAG antibody-coated magnetic beads. B, C. Purification of ATG9A-positive membranes. WT (B) and ATG14 KO MEFs (C) expressing FLAG-ATG9A were cultured in regular DMEM and subjected to OptiPrep flotation analysis as described in the legend to Fig 1. FLAG-ATG9A-enriched membranes were purified from middle-density fractions derived from WT (B) (Fig EV2A) and ATG14 KO MEF homogenates (C) (Fig EV2B). The resulting precipitates were examined by immunoblot analysis with the indicated antibodies. D. The ER membranes were purified from middle-density fractions derived from ATG14 KO MEF homogenates by immunoprecipitation with FLAG-GFP-SEC61B. An asterisk indicates non-specific signals. The intensities of the bands in the input and IP fractions were measured using ImageJ software. The ratio of IP to input was calculated. Each value was normalized to the ratio of IP to input of FLAG-tagged protein (co-IP efficiency). Download figure Download PowerPoint Although FIP200 and ATG9A were not co-purified in the middle-density fractions of ATG14 KO homogenate, FIP200 and WIPI2, another isolation membrane protein, were co-purified with FLAG-ATG9A from the top fraction of ATG3 KO homogenates (Fig 3A). These results suggest that FIP200 resides on isolation membranes together with ATG9A in ATG3 KO MEFs. In contrast to ATG14 KO homogenates (Fig 2C), small amounts of FIP200 and WIPI2 were co-precipitated with FLAG-ATG9A from the middle-density fractions of ATG3 KO homogenates (Fig 3B). Similarly, SEC61B and GFP-ATG14 were co-precipitated with FLAG-ATG9A in these middle fractions (Fig 3B and C). SEC61B was co-precipitated with FLAG-ATG9A from the middle-density fractions of ATG3 KO homogenates more efficiently than from those of WT or ATG14 KO homogenates (Fig 2B and C). These data suggest that ATG9A-containing autophagosome precursor membranes are associated with the ER in ATG3 KO cells. Overall, we conclude that FIP200 can be recruited to two different membranes: the ER-related membrane (ATG14 KO, fractions 4–7) and the isolation membrane (ATG3 KO, fraction 1; Fig 3D). The co-purification of ATG9A, FIP200, WIPI2, ATG14 and SEC61B from the middle-density fractions of ATG3 KO homogenates would reflect the ER-isolation membrane contact (ATG3 KO, fractions 4–7; Fig 3D). Figure 3. FIP200 and the ER membrane are co-purified with ATG9A-positive membranes from ATG3 KO MEF homogenates A–C. Purification of ATG9A-positive membranes. ATG3 KO MEFs expressing indicated constructs were cultured in regular DMEM and subjected to OptiPrep flotation analysis as shown in Fig 1E. FLAG-ATG9A-enriched membranes were purified from the top fraction (fraction 1) (A) and middle-density fractions (fractions 4–7) (B, C). The immunoprecipitation efficiency was calculated as in Fig 2. D. Summary of autophagic membrane purification. FIP200 was co-precipitated with the ER membranes purified from middle-density fractions of ATG14 KO homogenates (ATG14 KO, fractions 4–7; Fig 2D). The FLAG-ATG9A immunoprecipitates from the middle-density fractions of ATG3 KO homogenates contained the ER marker SEC61B and GFP-ATG14 as well as small amounts of FIP200 and WIPI2, which might reflect the ER-isolation membrane contact (ATG3 KO, fractions 4–7; Fig 3B and C). FIP200 and the isolation membrane protein WIPI2 were co-precipitated with ATG9A-enriched membrane from the top fraction of ATG3 KO homogenates (ATG3 KO, fraction 1; Fig 3A). Download figure Download PowerPoint FIP200 is recruited to the PIS-enriched ER subdomain at the initiation stage In spite of the fact that FIP200 associates with the ER membrane at an early stage of autophagy, it does not show an ER-like reticular pattern but a punctate distribution. This led us to hypothesize that FIP200 might be recruited to an ER subdomain. The ER is known to be the major site of cellular phospholipid synthesis, which would be required for autophagosome biogenesis. Recent studies have demonstrated that several phospholipid-metabolizing enzymes are segregated on the ER membrane (English & Voeltz, 2013; Pol et al, 2014). To characterize the ER subdomain that associates with FIP200, we screened phospholipid biosynthetic enzymes involved in the de novo synthesis and remodeling pathways that colocalized with FIP200 (Fig 4A; Shindou & Shimizu, 2009; Pol et al, 2014). To facilitate the accumulation of FIP200 at the ER subdomain, cells were treated with wortmannin. We found that phosphatidylinositol synthase (PIS), cholinephosphotransferase 1 (CPT1), choline/ethanolaminephosphotransferase 1 (CEPT1), phosphatidylserine synthase 1 (PSS1), and phospholipase D1A (PLD1A) were colocalized with FIP200 in these wortmannin-treated cells under starvation conditions (Fig 4B). Among these enzymes identified, we focused on PIS because this enzyme catalyzes the formation of PI, which is a precursor of PI3P required for autophagosome formation (Kihara et al, 2001). It was reported that PIS localizes to unique compartments: PIS is located in a highly dynamic ER-related compartment (Kim et al, 2011) and leading edges of ER tubules (English & Voeltz, 2013). When autophagosome formation was blocked at an early stage by wortmannin treatment, more than 70% of the FIP200 puncta were colocalized with PIS-GFP (Fig 5A and B), suggesting that FIP200 is recruited to PIS puncta at an early stage. PIS-GFP was not degraded in response to starvation unlike p62, a typical autophagy substrate (Fig EV3), ruling out the possibility that PIS accumulates as an autophagic substrate. In these wortmannin-treated starved cells, PIS and FIP200 double-positive puncta did not colocalize with the ER exit site marker SEC31A or mitochondria (Fig 5C and D). Furthermore, clearly separated PIS structures did not colocalize with ERGIC53 under wortmannin-treated conditions (Fig EV4). These results suggest that the PIS puncta are distinct from the ERGIC, ER exit site, and ER-mitochondrial contact site. The colocalization of PIS and FIP200 was also observed in ATG14 KO MEFs, in which early autophagic structures accumulated (Fig 5A). Also, we observed biochemically that FIP200 associated with PIS-enriched membranes purified from ATG14 KO MEFs (Fig 5E). This association disappeared in the presence of detergent, suggesting that the FIP200 co-precipitation was not simply caused by non-physiological aggregation but in a membrane-dependent manner. Moreover, in line with our model (Fig 3D; ATG14 KO, fractions 4–7), co-precipitation of ATG9A with PIS-enriched membranes was not observed (Fig 5E). Overall, we conclude that FIP200 is recruited to the PIS-enriched ER subdomain at an early stage of autophagy, upstream of the action of PI 3-kinase. Figure 4. Several phospholipid biosynthetic enzymes colocalize with FIP200 in wortmannin-treated cells under starvation conditions The phospholipid synthesis pathways in mammalian cells. De novo synthesis (top) and remodeling (bottom) pathways are shown. Enzymes that colocalize with endogenous FIP200 in WT MEFs cultures in starvation medium in the presence of 200 nM wortmannin for 1 h are shown in red, and those that do not colocalize are shown in blue. WT MEFs stably expressing the indicated GFP- or FLAG-tagged enzymes were cultured in starvation medium in the presence or absence of 200 nM wortmannin for 1 h. The cells were fixed and stained with anti-FIP200 antibody. Subcellular distributions of the indicated enzymes and FIP200 were examined by immunofluorescence microscopy. The cells were classified into three categories based on the colocalization of the indicated enzyme and FIP200: clear colocalization (red), partial colocalization (green), and no colocalization (blue). More than 30 cells were analyzed for each protein. Download figure Download PowerPoint Figure 5. FIP200 is recruited to PIS-enriched ER subdomains at the initiation stage of autophagy WT or ATG14 KO MEFs stably expressing PIS-GFP were cultured in regular DMEM or starvation medium in the presence of wortmannin (WM) for 1 h. Scale bar, 10 μm. Quantification of the number of FIP200+PIS− and FIP200+PIS+ puncta in growing cells and wortmannin-treated starved cells. The number of puncta was quantified from more than 60 randomly selected cells from three independent samples as described in the Materials and Methods. Data represent mean ± SEM. *P < 0.0001 vs. growing condition. Differences were statistically analyzed by two-tailed Mann-Whitney U-test. HeLa cells stably expressing PIS-GFP were cultured in starvation medium in the presence of wortmannin for 1 h. Cells were analyzed by immunofluorescence microscopy using anti-FIP200 and anti-SEC31A antibodies. Scale bar, 10 μm. WT MEFs stably expressing PIS-GFP were cultured in starvation medium in the presence of wortmannin for 1 h. For mitochondrial staining, cells were treated with 50 nM Mitotracker Red CMXRos for 15 min before fixation. Scale bar, 10 μm. PIS-GFP-FLAG was precipitated from the middle-density fractions of ATG14 KO MEF homogenates in the absence or presence of 1% Triton X-100. The resulting precipitates were examined by immunoblot analysis with the indicated antibodies. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. The effect of starvation on PIS-GFP expressionWT MEFs stably expressing PIS-GFP were cultured in growing or starvation conditions in the presence of the indicated inhibitor for 4 h. Then, 200 nM wortmannin (WM), 100 nM bafilomycin A1 (BafA1), 10 μM MG132, and 5 μg/ml brefeldin A (BFA) were used. Note that p62 but not PIS-GFP was degraded in an autophagy-dependent manner under starvation conditions. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. No colocalization of PIS puncta with ERGIC53 in wortmannin-treated cells under starvation conditionsWT MEFs stably expressing PIS-GFP were cultured in starvation medium in the presence of 200 nM wortmannin for 1 h. Cells were analyzed by immunofluorescence microscopy using anti-ERGIC53 antibody. Scale bar, 10 μm. Download figure Download PowerPoint The ULK complex forms a punctate structure on PIS-positive membranes To further characterize the localization of FIP200, we used a super-resolution structured illumination microscope (SR-SIM), which has ~120-nm lateral resolution. In starved cells, approximately 20% of FIP200 puncta colocalized with PIS-GFP almost completely (Fig 6A right top panels, and C), whereas more than 50% of FIP200 puncta only partially associated with PIS-GFP-positive structures (Fig 6A right bottom panels, and C). These results indicate that FIP200 at least partially colocalizes with PIS-GFP even under wortmannin-untreated condition. By contrast, most FIP200 puncta colocalized with PIS-GFP puncta in wortmannin-treated
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