Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

Ubiquitination is required to recruit Atg proteins

To determine the order in which Ub as well as LC3 and other Atg proteins are recruited to the transfected beads, we performed live-cell imaging of bead-transfected NIH3T3 cells stably expressing mStr-tagged Ub and the GFP-tagged Atg proteins LC3, Atg5, WIPI1, Atg14L, and ULK1 (Fig. 2, A and B; and Videos 3–7). The trafficking of each Atg protein greatly differed (Videos 3–7). The time lag between the localization of each Atg protein and Ub was quantified (Fig. 2 B). All of the Atg proteins were recruited to the transfected beads after Ub recruitment. Remarkably, LC3 and WIPI1 localized to the beads after the other Atg proteins, suggesting that the autophagic machinery recognizes the ubiquitinated substrate independently of an LC3-mediated mechanism.

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Figure 2.

Ubiquitination and recruitment of Atg proteins. (A and B) NIH3T3 cells stably expressing mStr-Ub and GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, or ULK1 were transfected with Effectene-coated latex beads for 30 min. Then, live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after Ub localization was measured for at least 30 cases for each combination. Each value represents the mean ± SD. Statistical analysis was performed by Student’s unpaired t test. *, P < 0.05; NS, not significant. (C) NIH3T3 cells stably expressing GFP-tagged Ub, LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated beads for 3 h in the presence or absence (mock) of 30 µM UBEI-41 (a ubiquitin E1–specific inhibitor) and subjected to immunocytochemistry for galectin3. The percentages of GFP-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD. Statistical analysis was performed by Student’s unpaired t test. *, P < 0.05. (D and E) Parent NIH3T3 cells, Atg4B mutant overexpressing NIH3T3 cells (D), wild-type MEFs, and Atg5-KO MEFs (E) stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.

Next, to directly demonstrate the necessity of ubiquitination, we used a Ub-activating enzyme (E1)–specific inhibitor (UBEI-41) because it enables blockade of ubiquitination instantly in cultured cells (Yang et al., 2007). As expected, the number of Ub-positive beads among galectin3-positive beads was drastically reduced upon UBEI-41 treatment (Fig. 2 C, Ub). Galectin3 was recruited to transfected beads even in UBEI-treated cells, indicating that internalization of beads and endosomal rupture occurred in our experimental condition. We also found that administering UBEI-41 strongly inhibited LC3 recruitment (Fig. 2 C, LC3). Moreover, the localization of all of the other representative Atg proteins was severely affected by UBEI-41 treatment. These results indicate that ubiquitination plays an important role in recruiting not only LC3 but also other Atg proteins to invading pathogens.

Likewise, we systematically quantified the recruitment of Atg proteins to galectin3-positive structures in autophagy-deficient cells (Atg5-KO, Atg14L-KO, Atg9L1-KO, and FIP200-KO cells as well as cells expressing an Atg4B mutant in which the LC3 system is blocked) stably expressing a series of GFP-tagged Atg proteins (Fig. 2, D and E; and Fig. S1; Kuma et al., 2004; Fujita et al., 2008a; Hara and Mizushima, 2009; Matsunaga et al., 2009; Saitoh et al., 2009). The ULK1 complex, Atg9L1, and the Atg16L1 complex were recruited to galectin3-positive structures independently of other Atg proteins (Fig. 2, D and E; and Fig. S1), which is consistent with autophagy against Salmonella and Parkin-mediated mitophagy (Itakura et al., 2012; Kageyama et al., 2011), and our live-cell imaging data (Fig. 2, A and B). Altogether, it is clear that before LC3 recruitment, the three core autophagy machineries, notably the Atg16L1 complex, the ULK1 complex, and Atg9L1, independently recognize the ubiquitinated endosomes containing bacteria or beads.

Ub moieties vary from mono-Ub to extended chains, linked through seven lysine residues in Ub or via the N-terminal methionine residue (linear; Iwai and Tokunaga, 2009). It has been recently reported that K63- and linear-linked Ub chains clearly colocalize with invading Salmonella (van Wijk et al., 2012). To examine the presence of K48- and K63-linked Ub chains to the invading Salmonella or transfected beads, we used K48- or K63-specific antibodies (Newton et al., 2008). Both K48- and K63-linked Ub signals were well colocalized with poly-Ub signals (Fig. S2 A). This result and the previous report show that different Ub linkages, including K48, K63, and linear, could be part of the Ub coat on the invading Salmonella and transfected beads.

It remains enigmatic which linkage of Ub is required for selective autophagy. To test loss of function of K63-linked or linear-linked Ub chains, Ubc13^fl/fl, HOIL-1L KO, and their control mouse embryonic fibroblasts (MEFs) were challenged with Salmonella (Yamamoto et al., 2006; Tokunaga et al., 2009). Ubc13 is an E2 enzyme crucial for generating K63-linked chains (Hofmann and Pickart, 1999), and HOIL-1L is an accessory protein of the LUBAC E3 ligase complex that is very important for efficient formation of linear-linked Ub chains (Tokunaga et al., 2009). We found that neither Ubc13^fl/fl nor HOIL-1L KO blocked LC3 recruitment to the Ub-positive Salmonella (Fig. S2, B and C). Collectively, these results indicate that autophagic machinery targeting did not depend solely on K63- or linear-Ub linkages.

The WD β-propellers of Atg16L1 directly interact with Ub

How do the three core autophagy machineries recognize the Ub-positive substrate? We focused on Atg16L1 because it contains a C-terminal WD β-propeller domain, whose function is unknown, but the same domain in several other proteins reportedly functions as an Ub-binding domain (Pashkova et al., 2010). Therefore, we tested the possibility that Ub directly interacts with the WD β-propellers of Atg16L1. First, we examined whether endogenous Atg16L1 coimmunoprecipitated with polyubiquitinated proteins. As shown in Fig. 3 A, Atg16L1 was detected in FK2 (a monoclonal antibody against poly Ub)-immunoprecipitated samples, but not in a control sample immunoprecipitated with IgG. To examine the interaction more directly, a GST pull-down experiment was performed. Purified GST or GST-Ub was immobilized to glutathione Sepharose beads (Fig. S3 A) and incubated with lysates of HEK293T cells transiently expressing a series of FLAG-tagged Atg16L1 constructs, including full-length Atg16L1, the WD β-propellers alone, or Atg16L1 lacking the complete WD region (ΔWD; Fig. 3 B). Full-length and the WD β-propellers bound to GST-Ub more efficiently than to GST alone. In contrast, the ΔWD mutant did not bind to GST-Ub (Fig. 3 C). Next, we prepared recombinant trigger factor–FLAG-tagged WD β-propellers of Atg16L1 using bacteria (Fig. S3 B). A GST pull-down experiment was performed with these recombinant proteins. The WD β-propellers bound to GST-Ub more efficiently than to GST alone (Fig. 3 D). From these results, we concluded that there is a direct interaction between the WD β-propellers of Atg16L1 and Ub.

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Figure 3.

The WD β-propellers of Atg16L1 directly interact with Ub. (A) HeLa cells were lysed with 1% Triton X-100 lysis buffer and subjected to immunoprecipitation with FK2- or control IgG-immobilized beads. The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies. (B) Schematic diagram of Atg16L1 (Full) and its deletion constructs. (C) Purified recombinant GST or GST-Ub–immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG-tagged Atg16L1 constructs for 1 h at 25°C with gentle agitation. The beads were washed three times with ice-cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG. (D) Purified GST or GST-Ub–immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor (TF) or TF-FLAG-WD β-propellers for 1 h at 25°C with gentle agitation. Washing and elution were performed as described in C. The eluted samples were subjected to SDS-PAGE and Western blotting for FLAG.

Atg16L1 is recruited by binding to Ub and FIP200

Although Atg16L1 could bind to Ub, deleting the WD β-propellers only minimally affected Atg16L1 recruitment to invading Salmonella (Fujita et al., 2009), suggesting that binding to Ub is not sufficient for Atg16L1 recruitment and additional interacting partner(s) functions in the recruitment. Thus, we searched for the Atg16L1 complex–interacting proteins using mass spectrometry. We found that ULK1 and FIP200 coimmunoprecipitated with the Atg16L1 complex, which consists of Atg16L1, Atg12, and Atg5 (Table S1). Because ULK1 forms a large protein complex including ULK1/2, Atg13, FIP200, and Atg101 (Mizushima, 2010), we performed a direct yeast two-hybrid assay with these proteins and found that Atg16L1 directly interacts with FIP200 (Fig. 4 A). The interaction was also confirmed by immunoprecipitation experiments (Fig. 4, B and C). Thus, we found that Atg16L1 binds to FIP200 in addition to Ub.

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Figure 4.

The WD β-propellers of Atg16L1 recruit LC3 in the absence of FIP200. (A) Yeast two-hybrid interactions of the ULK1 complexes with other human Atg proteins. ULK1, ULK2, Atg13, FIP200, and Atg101-AD fusions (rows 2–6) or control AD constructs (row 1, Empty) were coexpressed with control DBD constructs (column 1, Empty) or Atg protein–DBD fusions (columns 2–35) and tested for positive yeast two-hybrid interactions (top) or cotransformation (control, bottom). White lines divide images derived from the same plate, and red lines divide images from different plates. Atg16L1 positively interacted with FIP200 in this assay. (B) FIP200 binds to exogenously expressed Atg16L1. Myc-Atg16L1 coprecipitations with an empty vector control (lane 1) or One-STrEP-FLAG (OSF)-FIP200 (lane 2). (C) FIP200 binds endogenous Atg16L1. Atg16L1 coprecipitations with an empty vector control (lane 1) or OSF-FIP200 (lane 2). (D) Wild-type or FIP200-KO MEFs stably expressing the empty vector, full-length Atg16L1, or ΔWD mutant were incubated in growth medium (−) or Earle’s balanced salt solution (EBSS) (+) for 1 h and examined by Western blotting using the indicated antibodies. We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein (Fujita et al., 2009) because free Atg16L1 molecules not complexed with the Atg12–Atg5 conjugate are preferentially degraded by the ubiquitin–proteasome system. In control cells (Empty), two Atg16L1 splicing variants, the α- and β-forms, were detected. In full-length β-form–replaced cells (Full), the α-form was not detected, whereas in ΔWD-replaced cells (ΔWD) both the α- and β-forms were minimally detected. Thus, we successfully obtained cells that express only endogenous levels of the full-length or ΔWD mutant. (E–L) Atg16L1-replaced wild-type or FIP200-KO MEFs were infected with Salmonella (MOI = 10) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub (E and F) or LC3 and Ub (I and J). The percentages of Atg16L1-positive (G and H) or LC3-positive per Ub-positive bacteria were enumerated (K and L). At least 50 Salmonella were counted. The average ± SD is shown for three independent experiments. Statistical analysis was performed by Student’s t test. *, P < 0.05; NS, not significant. Bar, 5 µm.

Next, to test the roles of these interactions in autophagy, we generated Atg16L1-replaced wild-type and FIP200-KO cells (Fig. 4 D). We previously reported that stably expressing an exogenous Atg16L1 construct can replace the endogenous Atg16L1 protein with the exogenous one (Fujita et al., 2009; see legend of Fig. 4 for more details). In sharp contrast to wild-type cells, in FIP200-KO cells the recruitment of Atg16L1 to Ub-positive Salmonella and beads depended on the WD β-propellers (Fig. 4, E and H; and unpublished data). Next, we examined the effect of deleting the WD β-propellers on LC3 recruitment to Ub-positive Salmonella or beads because the Atg16L1 complex functions as an E3-like factor in the LC3 system (Fujita et al., 2008b). In wild-type cells, LC3 recruitment was not remarkably affected by deleting the WD β-propellers as was previously reported (Fig. 4, I and K; Fujita et al., 2009). On the contrary, deleting the WD β-propellers significantly decreased the localization of LC3 to Ub-positive Salmonella and beads in FIP200-KO cells (Fig. 4, J and L; and unpublished data). We also confirmed that LC3 lipidation was totally dependent on the WD β-propellers of Atg16L1 (Fig. 4 D). These results show that the localization of the Atg16L1 complex to Ub-positive Salmonella or beads involves interactions with both Ub via the WD β-propellers and FIP200, resulting in PE conjugation of LC3. Because the ULK1–FIP200 complex recruitment also depends on ubiquitination (Fig. 2 C), Atg16L1 must recognize Ub on the target through two mechanisms, one of which is direct recognition and the other is indirect.

To address the impact of the WDR-mediated mechanisms on the order of the Atg16L1 and ULK1–FIP200 complex recruitment, we quantified the localization of ULK1 and Atg16L1 in Ub-positive Salmonella in Atg16L1-reconstituted cells. The deletion of WDR did not affect the percentage of Atg16L1-positive in ULK1-GFP–positive population (Fig. S4 B). In sharp contrast to this, the deletion of WDR significantly decreased the percentage of Atg16L1-positive in ULK1-GFP–negative population (Fig. S4 C). These results suggest that the FIP200 is recruited to the Ub-positive Salmonella before Atg16L1 in the absence of WDR-mediated mechanisms.

DNA engineering and recombinant retroviruses

The pMRX-IRES-puro and pMRX-IRES-blast vectors were gifts from S. Yamaoka (Tokyo Medical and Dental University, Tokyo, Japan; Saitoh et al., 2003). To generate recombinant retroviruses, cDNAs corresponding to mStrawberry (mStr)-tagged galectin3, mStr-Ub, GFP-Ub, and GFP-p62 were subcloned into the pMRX-IRES-puro vector. Various human Atg16L1β (isoform-1) mutants were cloned into the pMRX vector, including full-length (1–607), ΔWD repeat domain (1–249), and the Crohn’s disease–associated mutant (T300A; Fujita et al., 2009). In addition, pMRX constructs were generated to encode various GFP-tagged Atg proteins, including LC3 (N terminus), Atg5 (N terminus), WIPI1 (N terminus), Atg14L (N terminus), Atg9L1 (C terminus), and ULK1 (C terminus; Kageyama et al., 2011), as previously reported. Recombinant retroviruses were prepared as previously described (Saitoh et al., 2003). To prepare recombinant proteins, cDNA corresponding to Ub was subcloned into the pGEX6P-1 vector, and cDNA corresponding to FLAG-tagged wild-type or the Crohn’s disease–associated mutant of the WD β-propellers of Atg16L1 was subcloned into the pCold-TF vector (Takara Bio Inc.).

Cell culture and retroviral infections

Plat-E cells were provided by T. Kitamura (The University of Tokyo, Tokyo, Japan; Morita et al., 2000). HeLa cells, HEK293T cells, NIH3T3 cells, wild-type MEFs, and autophagy-deficient MEFs (Atg5-KO, Atg14L-KO, Atg9L1-KO, Atg16L1-Δ/Δ, and FIP200-KO) were grown in DMEM supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 5 U/ml penicillin, and 50 U/ml streptomycin in a 5% CO₂ incubator at 37°C (Kuma et al., 2004; Fujita et al., 2008a; Saitoh et al., 2008, 2009; Hara and Mizushima, 2009; Matsunaga et al., 2009; Nishimura et al., 2013). Stable transformants were selected in growth medium with 1 µg/ml puromycin or 5 µg/ml blasticidin (Invivogen).

Western blotting

Samples were subjected to SDS-PAGE, and transferred to polyvinylidene fluoride membranes. The membranes were blocked with TBST (TBS and 0.1% Tween 20) containing 1% skim milk and were then incubated overnight at 4°C with primary antibodies 1,000–3,000× diluted in the blocking solution. Membranes were washed three times with TBST, incubated for 1 h at room temperature with 10,000× dilutions of HRP-conjugated secondary antibodies (GE Healthcare) in the blocking solution, and washed five times with TBST. Immunoreactive bands were then detected using ECL plus (GE Healthcare) and a chemiluminescence detector (LAS-3000, Fujifilm; Kimura et al., 2009).

Bacterial infections and bead transfections

Salmonella enterica serovar Typhimurium (SR-11 x3181) was provided by the Research Institute of Microbial Disease, Osaka University (Osaka, Japan). Bacteria were grown overnight at 37°C, and then sub-cultured at 1:33 for 3 h in LB without antibiotics. The bacterial inocula were prepared by pelleting at 10,000 g for 2 min, and were then added to host cells at a multiplicity of infection (MOI) of 10–100 at 37°C with 5% CO₂ (Kageyama et al., 2011). Bead transfections were performed as previously reported (Kobayashi et al., 2010). Transfection reagent-coated beads were prepared by mixing the beads (NH₂-, 17145–5; PolySciences, Inc.) with Effectene transfection reagent (301425; QIAGEN), according to the manufacturer’s instructions except that bead suspension was used instead of DNA solution. The resulting bead mixture (∼100 µl) was further mixed with 1 ml of growth medium, and added to cells by replacing the medium. After incubation with the bead mixture for 1 h at 37°C in a CO₂ incubator, the cells were washed twice with fresh growth medium to remove unattached beads, and further incubated for the time indicated in each experiment.

Immunofluorescence and microscopy

Cells were cultured on coverslips, fixed with 3% PFA in PBS for 10 min, and permeabilized with 50 µg/ ml digitonin in PBS for 5 min. Cells were then treated with 50 mM NH₄Cl⁻/PBS for 10 min at room temperature and blocked with PBS containing 3% BSA for 15 min. Primary antibodies were diluted 1:500 or 1:1,000, and Alexa Fluor–conjugated secondary antibodies (Invitrogen) were diluted 1:1,000 in PBS containing 3% BSA. Coverslips were incubated with primary antibodies for 60 min, washed six times with PBS, and incubated with secondary antibodies for 60 min. Samples were mounted using Slow Fade Gold and observed with an laser confocal microscope (FV1000; Olympus). The microscope images were taken using the FV1000 confocal laser-scanning microscope system equipped with a 100×/NA 1.40 oil immersion objective lens. Fluorochromes associated with the secondary antibodies were Alexa Fluor 405, 488, 568, or 594. Image acquisition software used was Fluoview (Olympus). The images were adjusted using Photoshop CS4 software (Adobe). For live-cell imaging, cells were grown in DMEM D6434 (Sigma-Aldrich) supplemented with 10% FBS with antibiotics on a glass-bottom dish (D310300; Matsunami Glass) and transfected with Effectene-coated latex beads for 30 min as previously described (Kobayashi et al., 2010). After beads transfection, the glass-bottom dish was mounted onto the microscope stage, which was equipped with a humidified environment chamber (MI-IBC; Olympus) that maintained the dish at 37°C with 5% CO₂. Images were acquired using an inverted microscope (model IX81; Olympus) equipped with a 60×/1.40 NA oil immersion objective (Olympus), a xenon lamp, a cooled charge-coupled device camera (CoolSNAP HQ; Roper Scientific), and a ZDC system under the control of MetaMorph v7.6.5.0 (Molecular Devices, MDS Analytical Technologies).

GST pull-down assay

GST and GST-Ub were purified from Escherichia coli lysates over glutathione Sepharose 4B (GE Healthcare) and dialyzed in PBS. 30 µg of each GST protein immobilized on 30 µl of glutathione Sepharose 4B was used per binding reaction. Mammalian cell lysates or bacterial cell lysates were used as the input proteins. HEK293T cells were transiently transfected with plasmids encoding the indicated Atg16L1 constructs, homogenized in PBS containing protease inhibitors (complete protease inhibitor cocktail; Roche), and then cleared by centrifugation and filtration (0.22 µm). Alternatively, E. coli BL-21 (DE3) cells expressing trigger factor–fused FLAG-WD β-propellers of Atg16L1 were sonicated in PBS containing 0.01% Triton X-100 and protease inhibitors and cleared by centrifugation and filtration (0.22 µm). Immobilized beads and cell lysates were incubated in a total volume of 500 µl PBS containing 0.1 mg/ml BSA and 0.01% Triton X-100 with gentle agitation for 1 h at 25°C. The beads were washed three times with ice-cold PBS and the bound complexes were eluted with 50 mM reduced glutathione in PBS and then subjected to SDS-PAGE and Western blot analyses.

Fractionation of bead–autophagosomes

Bead–autophagosomes were isolated using a modification of the method described by Desjardins et al. (1994). HeLa cells were transfected with Effectene-coated beads and incubated for 3 h. The cells were harvested with a silicone rubber scraper and homogenized in buffer (20 mM Hepes-KOH, pH 7.4, 80 mM sucrose, 220 mM mannitol, 10 mM N-ethylmaleimide, 1 mM PMSF, and a protease inhibitor cocktail [Roche]) by repeatedly passing (∼15 times) through a 1-ml syringe with a 25-gauge needle. The homogenate was centrifuged at 1,000 g for 10 min to remove the cytosol and microsomes. The bead–autophagosomes were isolated by flotation on a discontinuous gradient composed of 1 ml each of increasing concentrations of sucrose solutions (60, 40, 35, 30, 25, 20, 15, 10, and 5%; all sucrose solutions were wt/wt in 3 mM imidazole, pH 7.4). The resulting pellet was resuspended in a 25% sucrose solution and loaded on top of a 30% sucrose solution. The samples were centrifuged in a swinging bucket rotor (SW41Ti; Beckman Coulter) for 1 h at 100,000 g at 4°C. The bead-autophagosome–containing fraction was collected from the interface of the 10 and 15% sucrose solution. The bead–autophagosomes were resuspended in 2 ml of ice-cold PBS and pelleted by centrifuging for 10 min at 20,000 g at 4°C.

Yeast two-hybrid assays

Yeast two-hybrid assays were performed using the Matchmaker GAL4 Yeast Two Hybrid 3 system (Takara Bio Inc.) as previously reported (Langelier et al., 2006). Saccharomyces cerevisiae AH-109 was cotransformed with the cloning vectors, pGADT7 or pGBKT7, containing the inserts of interest. The transformed yeast cells were incubated on yeast nitrogen base and glucose with minus Leu, minus Trp selection for 3 d at 30°C. 10–100 colonies were resuspended in a liquid culture of Sabourand dextrose broth (minus Leu, minus Trp), and incubated on Sabourand dextrose broth (minus Leu, minus Trp, minus Ade, minus His) plates for 3 d.

Identification of co-purifying proteins by mass spectrometry

HEK293T cells were seeded (3 × 10⁶ cells/55-cm² dish) and cotransfected with 3 µg each of plasmids encoding members of the Atg16L1 complex (Atg12, Atg5, and Atg16L1) using polyethylenimine (25,000 kD; Polysciences, Inc.) as previously described (Durocher et al., 2002). The cells were harvested 48 h after transfection by incubating in 300 µl lysis buffer (50 mM Tris, pH 7.4, and 150 mM NaCl) supplemented with proteinase inhibitor cocktail (Roche) and 1% Triton X-100. Lysates were clarified by centrifugation (18,000 g, 10 min, 4°C) and incubated with Strep-Tactin Sepharose (30 µl slurry, 2 h, 4°C; IBA GmbH). The matrix was washed four times in wash buffer (20 mM Tris, pH 7.4, and 150 mM NaCl) supplemented with 0.1% Triton X-100, and the purified Atg16L1 complexes were eluted with 2.5 mM desthiobiotin. The co-purified proteins were identified by mass spectrometry. Briefly, the co-purified proteins were identified after SDS-PAGE and band excision. The proteins were digested with trypsin and identified by separating the peptide mixtures using nano-flow liquid chromatography with online tandem mass spectrometry (LC-MS/MS). Tandem mass spectra were acquired automatically and then searched against a nonredundant human database from the NCBI database with the Mascot Server (Matrix Science).

Statistics

All values in the figures are shown with standard deviation. Statistical analyses were performed using a two-tailed unpaired t test. P values <0.05 were considered statistically significant.

The authors thank R.Y. Tsien for the gift of mStrawberry cDNA; S. Yamaoka for providing pMRX-IRES-puro and pMRX-IRES-bsr; T. Kitamura for providing the Plat-E cells; N. Mizushima for the anti-Atg16L1 antibody; K. Okamoto for helpful discussions; S. Cox for English editing of the manuscript; and R. Tsukahara and H. Akai for technical assistance. LC-MS/MS analysis was performed in the DNA-chip Development Center for Infectious Diseases (RIMD, Osaka University).

This work was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT; to N. Fujita, S. Kobayashi, T. Haraguchi, T. Noda, and T. Yoshimori); and the Takeda Science Foundation (to T. Yoshimori and T. Noda).