Intrinsic lipid binding activity of ATG 16L1 supports efficient membrane anchoring and autophagy
2019; Springer Nature; Volume: 38; Issue: 9 Linguagem: Inglês
10.15252/embj.2018100554
ISSN1460-2075
AutoresLeo J. Dudley, Ainara G. Cabodevilla, Agata N Makar, Martin Sztacho, Tim Michelberger, Joseph A. Marsh, Douglas R. Houston, Sascha Martens, Xuejun Jiang, Noor Gammoh,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle1 April 2019Open Access Transparent process Intrinsic lipid binding activity of ATG16L1 supports efficient membrane anchoring and autophagy Leo J Dudley Leo J Dudley Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ainara G Cabodevilla Ainara G Cabodevilla Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Agata N Makar Agata N Makar orcid.org/0000-0003-4661-1010 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Martin Sztacho Martin Sztacho Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Tim Michelberger Tim Michelberger Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Joseph A Marsh Joseph A Marsh Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Douglas R Houston Douglas R Houston Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Sascha Martens Sascha Martens orcid.org/0000-0003-3786-8199 Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Xuejun Jiang Xuejun Jiang Cell Biology Department, Memorial Sloan Kettering Cancer Centre, New York, NY, USA Search for more papers by this author Noor Gammoh Corresponding Author Noor Gammoh [email protected] orcid.org/0000-0001-9402-9581 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Leo J Dudley Leo J Dudley Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Ainara G Cabodevilla Ainara G Cabodevilla Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Agata N Makar Agata N Makar orcid.org/0000-0003-4661-1010 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Martin Sztacho Martin Sztacho Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Tim Michelberger Tim Michelberger Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Joseph A Marsh Joseph A Marsh Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Douglas R Houston Douglas R Houston Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, UK Search for more papers by this author Sascha Martens Sascha Martens orcid.org/0000-0003-3786-8199 Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria Search for more papers by this author Xuejun Jiang Xuejun Jiang Cell Biology Department, Memorial Sloan Kettering Cancer Centre, New York, NY, USA Search for more papers by this author Noor Gammoh Corresponding Author Noor Gammoh [email protected] orcid.org/0000-0001-9402-9581 Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK Search for more papers by this author Author Information Leo J Dudley1,‡, Ainara G Cabodevilla1,‡, Agata N Makar1, Martin Sztacho2, Tim Michelberger1, Joseph A Marsh3, Douglas R Houston4, Sascha Martens2, Xuejun Jiang5 and Noor Gammoh *,1 1Cancer Research UK Edinburgh Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 2Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, Vienna Biocenter, University of Vienna, Vienna, Austria 3Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, UK 4Institute of Quantitative Biology, Biochemistry and Biotechnology, University of Edinburgh, Edinburgh, UK 5Cell Biology Department, Memorial Sloan Kettering Cancer Centre, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +44 1316518526; E-mail: [email protected] The EMBO Journal (2019)38:e100554https://doi.org/10.15252/embj.2018100554 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 Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation. Synopsis Recruitment of core autophagy protein ATG16L1 to the phagophore, essential for autophagosome biogenesis, involves it intrinsic ability to bind phosphoinositides on pre-autophagosomal compartments, to promote LC3 lipidation and autophagosome maturation. The coiled-coil domain (CCD) of ATG16L1 interacts with phosphatidylinositol-3-phosphate and other phosphoinositides. CCD-lipid interaction is required for efficient recruitment of ATG16L1 to the phagophore. Disrupting the interaction between ATG16L1 and phosphoinositides inhibits LC3 lipidation. Introduction While some organelles, such as the endoplasmic reticulum (ER) or mitochondria, are generated by growing and budding from pre-existing organelles, autophagosome formation is initiated through the de novo nucleation of membranous structures (Joshi et al, 2017). This process requires the activity of distinct protein complexes that act to relay upstream signals in order to facilitate the growth of precursor membranes known as pre-autophagosomal structures (PAS; Lamb et al, 2013). Of these protein complexes, the ATG5 complex, comprised of the ATG12~ATG5 conjugate and ATG16L1, plays a pivotal role in both the nucleation of the PAS and the downstream conjugation of the ATG8 ubiquitin-like family of proteins (such as LC3) to phosphatidylethanolamine (PE; Sakoh-Nakatogawa et al, 2013). The conjugation of LC3 on the PAS facilitates the maturation of autophagosomes and the recruitment of cargo molecules for their subsequent lysosomal degradation. In order to better understand how autophagy-related complexes are activated and recruited to the growing PAS, recent studies have started to uncover the genetic and temporal hierarchy of these complexes in mammalian cells (Itakura & Mizushima, 2010; Koyama-Honda et al, 2013). Following inhibition of mTORC1 (e.g. by amino acid starvation or small molecule inhibitors), the ULK1 complex is relieved from its inhibitory phosphorylation by mTOR, resulting in its recruitment to the PAS independently of downstream autophagy complexes (Itakura & Mizushima, 2010). The ULK1 kinase can phosphorylate and activate members of the ATG14 complex, containing the class III PI3K kinase Vps34, which facilitates the recruitment of phosphatidylinositol 3-phosphate (PI3P) sensors (such as DFCP1, WIPI1 and WIPI2; Axe et al, 2008; Matsunaga et al, 2010). FIP200, a component of the ULK1 complex, and WIPI2b can both directly interact with ATG16L1, providing a mechanism for the localisation of the ATG5 complex to the PAS during mTORC1 inactivation (Gammoh et al, 2013; Nishimura et al, 2013; Dooley et al, 2014). Membrane recruitment of most autophagy complexes is pivotal for their role in autophagy. Ectopic recruitment to the plasma membrane of an ATG5-binding fragment of ATG16L1 results in aberrant and constitutive lipidation of LC3 (Fujita et al, 2008). Similar results were also obtained upon plasma membrane tethering of the ATG16L1 binding partner, WIPI2b (Dooley et al, 2014). These findings suggest that membrane localisation of ATG16L1 in cells is sufficient to drive the conjugation of LC3 to PE and that its regulation could provide means to fine-tune autophagy. Despite the above proposed hierarchy, it remains unknown how autophagy players are recruited during ULK1-independent autophagy (Gammoh et al, 2013). Furthermore, live-cell imaging analyses suggest that both the recruitment and displacement of autophagy proteins to the PAS occurs in an asynchronous manner, indicating that protein–protein interactions are not sufficient to stabilise autophagy complexes on membranes (Koyama-Honda et al, 2013). In addition, sequences that correspond to WIPI2b and FIP200 binding sites in mammalian ATG16L1 are absent in yeast Atg16 (Fujioka et al, 2010; Gammoh et al, 2013; Nishimura et al, 2013; Dooley et al, 2014). These observations suggest the existence of previously unknown mechanisms that can mediate the localisation of the ATG5 complex to membranes. Given the central role of the ATG5 complex during various forms of LC3 conjugation, including both canonical and non-canonical autophagy (Fletcher et al, 2018), we aimed to investigate how the ATG5 complex is recruited to autophagy-related membranes. In this study, we have identified highly conserved sequences within the coiled-coil domain (CCD) of ATG16L1 that mediate its direct interaction with lipids, thereby enhancing its PAS localisation and autophagic activity. Results ATG16L1 membrane targeting activity is retained in the absence of ATG5 or WIPI2 To investigate the membrane recruitment of the ATG5 complex, we first addressed the role of either ATG5 or ATG16L1 in PAS targeting. Since deletion of either protein can destabilise the other (Fig 1A, Nishimura et al, 2013), we generated knockout cell lines stably expressing GFP-tagged proteins to avoid any discrepancies resulting from reduction in protein levels or ectopic localisation (Li et al, 2017). As seen in Fig 1B, the recruitment of stably expressed ATG16L1 to punctate structures was not disrupted in the absence of ATG5. On the other hand, ATG5 showed a diffused pattern of staining in the absence of ATG16L1. Biochemical fractionation further confirmed the finding that ATG16L1 accumulates in membrane fractions in the absence of ATG5 (Fig 1C), as well as in the absence of ATG3 (Gammoh et al, 2013). These results suggest a role for ATG16L1 in the membrane targeting of the ATG5 complex. Previous studies show that the PAS localisation of an ATG16L1 mutant lacking both WIPI2b and FIP200 binding (ATG16L1∆FBD) was markedly reduced but not completely inhibited, suggesting that these interaction partners may act as signalling players that enhance the membrane recruitment of ATG16L1 (Gammoh et al, 2013; Nishimura et al, 2013). This was further confirmed in WIPI2−/− cells where ATG16L1 puncta formation was reduced but not fully inhibited (Fig 1D and E). Residual ATG16L1-positive puncta formed in WIPI2−/− cells were sensitive to Vps34 inhibition by 3-methyladenine (3′MA) treatment, in agreement with previous data showing the requirement of PI3P for the PAS recruitment of the ATG5 complex (Koyama-Honda et al, 2013). Overall, these findings suggest the existence of additional previously undescribed mechanisms that mediate the recruitment of ATG16L1 to the PAS. Figure 1. ATG16L1 membrane targeting activity is retained in the absence of ATG5 or WIPI2 Analyses of protein expression in lysates of various cell lines by Western blotting against the indicated antibodies. Fluorescence analyses of GFP-ATG5 or ATG16L1-GFP stably expressed in ATG5−/− or ATG16L1−/−. Cells were amino acid starved for 2 h prior to fixation and imaging of the GFP fluorescence. Scale bar: 10 μm. Assessment of ATG16L1 levels in the cytosolic (C) and membrane (M) fractions in lysates of the indicated cell lines using Western blot analyses and antibodies against ATG16L1. ATG16L1−/− and ATG5−/− stably expressed ATG16L1-GFP while ATG3−/− stably expressed Flag-S-ATG16L1. Antibodies against α-tubulin and integrin β1 were used as controls for fractionation. Exogenous (exo) ATG16L1 was detected using antibodies against ATG16L1. Lack of WIPI2 expression is confirmed by Western blot analyses in wild-type MEFs (WIPI2+/+) and WIPI2−/− cells. Immunofluorescence analyses of ATG16L1−/− and WIPI2−/− stably expressing Flag-S-ATG16L1. Cells were amino acid starved (AA starve) for 2 h in the presence or absence of 3′MA (and additional pretreatment for 30 min) followed by fixation and immunostaining using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm. Right panel represents quantification of three independent experiments and error bars depicting SEM values. *P ≤ 0.05, **P ≤ 0.01 (pairwise unpaired Student's t-test). Download figure Download PowerPoint PAS targeting activity of ATG16L1 lies within its CCD To identify the region within ATG16L1 required for its localisation to the PAS, we examined ATG16L1 puncta formation in U2OS cells expressing a series of ATG16L1 truncation mutants (depicted in Fig 2A). As seen in Fig 2B, the deletion of N-terminal sequences containing the ATG5 binding (fragment ∆1, residues 39–623) or the further downstream linker region (fragment ∆2, residues 120–623) did not affect puncta formation when compared to wild-type ATG16L1 (ATG16L1WT). On the other hand, subsequent fragments lacking the CCD of ATG16L1 (fragments ∆3 and ∆4, residues 206–623 and 336–623, respectively) were diffused in cells, suggesting that PAS targeting requires either the dimerisation of ATG16L1 or additional unknown activities within the CCD. To distinguish these two possibilities, we aimed to further delete sequences within the CCD that were predicted to be dispensable for dimerisation. Based on ATG16L1 structural predictions and comparisons to yeast Atg16 (Fujioka et al, 2010), we analysed conserved regions within the ATG16L1 CCD that were predicted to not contribute to the dimer-dimer interface. A combined deletion of amino acids 182–205 within the context of the ∆2 fragment (ATG16L1∆2∆182–205) resulted in a diffused pattern of staining, indicating the requirement of these residues of ATG16L1 for puncta formation (Fig 2C). We further confirmed that deleting residues 182–205 within the context of the full-length protein (ATG16L1∆182–205) did not interfere with the ability of ATG16L1 to interact with FIP200 and ATG5 or homodimerise (Fig 2D and E), whereas a truncation mutant lacking the CCD, but not the WD40 domain, was unable to homodimerise (Fig 2F). When expressed in ATG5−/− cells, the deletion mutant, ATG16L1∆182–205, exhibited a diffused pattern of staining (Fig 2G), whereas ATG16L1WT formed punctate structures. These findings confirm that sequences within the CCD are required for PAS targeting but dispensable for previously identified functions of ATG16L1, including dimerisation and binding to FIP200 and ATG5. Figure 2. PAS targeting activity of ATG16L1 lies within its CCD Schematic presentation of ATG16L1 fragments and mutants used in this study. The following fragments encompassed the indicated amino acids: wild type (WT): 1–623; ∆1: 39–632; ∆2: 120–623; ∆3: 206–623 and ∆4: 336–623. Mutant ∆2∆182–205 consists of the ∆2 fragment with an additional deletion in amino acids 182–205. All constructs contained a Flag-S tag at the N-terminal end. Fragments depicted in (A) were expressed in U2OS cells and amino acid starved for 5 h followed by fixation and immunostaining against S tag to detect S-ATG16L1. Scale bar: 10 μm. U2OS cells expressing Flag-ATG16L1∆2 or Flag-ATG16L1∆2∆182–205 were treated as in (B) and stained using antibodies against Flag tag (to detect ATG16L1, green) and FIP200 (red). Scale bar: 10 μm. Protein–protein interaction assay in 293T cells transiently transfected with the indicated Flag-S-tagged ATG16L1 constructs. S tag pull-down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Homodimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated Flag-S-tagged ATG16L1 constructs. S tag pull-down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Homodimerisation assay similar to (E). Flag-ATG16L1WT or Flag-ATG16L1∆182–205 were stably expressed in ATG5−/− cells and analysed by immunofluorescence using antibodies against Flag tag to detect ATG16L1. Scale bar: 9 μm. Download figure Download PowerPoint ATG16L1 binds liposomes through CCD sequences Having shown that the localisation of ATG16L1 to punctate structures requires sequences within its CCD of unknown function, we further investigated the relevance of these sequences in recruiting ATG16L1 to the PAS. Further analyses of the CCD domain region indicated the presence of a hydrophobic region and positively charged residues that could mediate direct lipid binding of ATG16L1 to membranes (Fig 3A). To address this possibility, we used a microscopy-based technique to test the recruitment of rhodamine-labelled small unilamellar vesicles (SUVs) to beads coated with ATG16L1-GFP (Fracchiolla et al, 2016), a sensitive approach to detect protein-lipid binding activities. As seen in Fig 3B and C, there was no significant recruitment of liposomes to ATG16L1-GFP-bound beads when using liposome preparations that contained phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidylserine (PS) suggesting an inability of ATG16L1 to bind these phospholipids. Consistent with the hypothesis that ATG16L1 can directly bind to autophagy-related membranes, liposome recruitment to ATG16L1-GFP beads was enhanced when incubated with PI3P-containing liposomes, an essential lipid for the biogenesis of autophagosomes (Axe et al, 2008). Moreover, liposome preparations containing phosphatidylinositol 4-phosphate (PI4P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) also resulted in enhanced recruitment to ATG16L1-GFP containing beads, indicating that these membrane phospholipids can also bind ATG16L1. The binding of ATG16L1 to liposomes was independent of ATG5 or its WD40 domain as a deletion mutant of ATG16L1 lacking its C-terminal half was able to bind liposomes when purified from ATG5−/− cells (Fig EV1A and B). We further confirmed the ability of wild-type ATG16L1 to bind PI3P in an independent assay using lipid-coated beads, where we also detected its binding to phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2) (Fig EV1C). In contrast, ATG16L1 did not significantly bind to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) when compared to control beads, suggesting a degree of specificity for phosphoinositides. Interestingly, this assay also revealed that ATG16L1 has low binding affinity for PE and PS, while also exhibiting an affinity for PA, although this was found to be non-significant compared to the control beads. Figure 3. ATG16L1 binds liposomes through CCD sequences Sequence alignment of ATG16L1 CCD segment from various species and ATG16L2 (Sc: Saccharomyces cerevisiae; Xt: Xenopus (Silurana) tropicalis; Hs: Homo sapiens; Mm: Mus musculus). Residues that mediate WIPI2b and FIP200 binding are highlighted in orange and magenta, respectively. Cyan-shaded residues (I171, K179 and R193) are exposed conserved residues predicted to not contribute to the dimer-dimer interface and are mutated in this study. Cherry-shaded residues are mutated in Figs 6 and 7. Microscopy-based protein-liposome binding assay. ATG16L1-GFP immobilised on beads incubated in the presence of rhodamine-labelled liposome preparations containing the indicated phosphoinositides. Scale bar: 50 μm. Quantification of relative liposome binding in (B). Structural modelling of ATG16L1 residues 160–205 (magenta helix) in the presence of lipid bilayer (green lines). Highlighted residues include I171, K179 and R193 as sticks, which are mutated in this study. A PI3P molecule is shown as a yellow stick embedded in the lipid bilayer and interacting with the highlighted positively charged residues of ATG16L1. Microscopy-based protein–liposome binding assay as in (B). ATG16L1WT- and ATG16L1LD-GFP immobilised on beads were incubated with rhodamine-labelled, PI3P-positive liposome preparations. Scale bar: 50 μm. Right panel shows quantification of liposome binding relative to ATG16L1WT from three independent experiments including SEM values. Protein–protein interaction assay in 293T cells transiently transfected with the indicated S-tagged ATG16L1 constructs. S tag pull-down was performed and protein complexes were analysed by immunoblotting using the indicated antibodies. Dimerisation assay in 293T cells transiently transfected with ATG16L1-GFP and the indicated S-tagged ATG16L1 constructs and analysed as in (F). Data information: Quantifications depict means and error bars (SEM) from at least three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (pairwise unpaired Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Further characterisation of ATG16L1 lipid binding activity Microscopy-based protein–liposome binding assay. ATG16L1∆WD40-GFP or GFP alone was purified from ATG5−/− cells and immobilised on beads followed by incubation in the presence of rhodamine-labelled liposome preparations containing PI3P. Scale bar: 50 μm. Quantification of relative liposome binding in (A) along with ATG16L1WT-GFP from Fig 3E. Quantifications depict means and error bars (SEM) from three independent experiments. ***P ≤ 0.001 (pairwise unpaired Student's t-test). Lipid binding experiment using the indicated lipid-coated beads incubated with recombinant wild-type ATG16L1 purified from insect cells. Bound proteins were analysed by Western blot using anti-ATG16L1 antibodies. Right panel shows quantifications depicting means and error bars (SEM) from at least three independent experiments. *P ≤ 0.05 (pairwise unpaired Student's t-test). Download figure Download PowerPoint The ability of ATG16L1 to bind PI3P suggests that positively charged residues, potentially located within or juxtaposed to amino acids 182–205 of its CCD, may mediate this interaction. Although previous studies have identified interactions between coiled-coil domains and phospholipids (Horikoshi et al, 2011; Zheng et al, 2014), the structural bases underlying these have not yet been directly elucidated. To explore the potential structural mechanism of ATG16L1-lipid interaction, we performed structural prediction analyses of a short region of mouse ATG16L1 CCD and modelled its interaction with PI3P embedded in a lipid bilayer. These analyses predict that ATG16L1 CCD could potentially interact with the negatively charged headgroup of PI3P by lying flat on the membrane surface (Fig 3D). Molecular dynamics simulation of the protein in association with PI3P in a model lipid bilayer indicated that this association was stable over the course of the trajectory. Interestingly, helices and coiled-coil domains have also been previously shown to interact with lipid bilayers by lying flat on the membrane surface, although adopting a different mechanism than that predicted for ATG16L1 (utilising primarily hydrophobic rather than electrostatic interactions; Pluhackova et al, 2015; Woo & Lee, 2016). Our homology modelling highlights three residues, including K179, R193 and the further upstream residue I171, which line the outer faces of the coiled-coil and are solvent-exposed, thereby free to interact with the phosphate groups of PI3P or PI(3,4)P2. These residues are conserved in yeast Atg16 but are missing from ATG16L2, a protein closely related to ATG16L1 that does not localise to the PAS despite its ability to bind ATG5 and homodimerise (Ishibashi et al, 2011). As predicted, mutation of these three residues in ATG16L1 to aspartic acid (I171D, K179D and R193D) strongly reduced the recruitment of liposomes to ATG16L1-coated beads (Fig 3E). Hence, we named this mutant ATG16L1LD for lipid binding-deficient ATG16L1 mutant. Given that these residues are adjacent to WIPI2b and FIP200 binding sites, we further confirmed that ATG16L1LD did not interfere with the ability of ATG16L1 to bind these two interactors nor interfere with ATG16L1 homodimer formation (Fig 3F and G). Overall, these data suggest that ATG16L1 contains residues within its CCD that mediate interactions to negatively charged lipids but are dispensable for its dimerisation and binding to WIPI2 and FIP200. Binding of ATG16L1 to lipids is required for PAS recruitment To dissect the functional relevance of ATG16L1 lipid binding in cells, we further analysed the localisation of ATG16L1WT and ATG16L1LD, stably expressed in ATG16L1−/− cells. When compared to ATG16L1WT, we observed a strong inhibition of ATG16L1LD recruitment to puncta positive for ATG5 and WIPI2 during amino acid starvation (Fig 4A and B), suggesting that ATG16L1 lipid binding is required for its efficient recruitment to the PAS. Additionally, the overall intensity of ATG16L1LD puncta was significantly reduced compared to that of ATG16L1WT, implying that ATG16L1 is recruited less efficiently to these sites (Fig 4C). Furthermore, enhanced formation of WIPI2-positive puncta, as observed by reconstituting ATG16L1−/− cells with ATG16L1WT, was impaired in ATG16L1LD-expressing cells, indicating that ATG16L1 lipid binding may influence early autophagic events (Fig 4B). Residual ATG16L1LD punctate structures could potentially be due to its ability to bind upstream autophagy players, including WIPI2b and FIP200, which have been proposed to facilitate its recruitment to the PAS (Gammoh et al, 2013; Nishimura et al, 2013; Dooley et al, 2014). To test this possibility, we stably expressed ATG16L1LD in WIPI2−/− cells and observed a further reduction in puncta formation of this mutant to levels comparable to background levels in mock infected cells (Fig 4D). Overall, these data suggest that the efficient recruitment of ATG16L1 to the PAS relies on its ability to interact with both PI3P through CCD sequences and protein binding partners, such as WIPI2b. Figure 4. Binding of ATG16L1 to lipids is required for PAS recruitment ATG16L1−/− stably expressing the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 h prior to immunofluorescence analyses using antibodies against Flag tag (green) and ATG5 (red). Scale bar: 9 μm. Right panels show quantifications of average number of Flag- and ATG5-positive dots per cell. Cells, as in (A), were immunostained using antibodies against S tag (to detect ATG16L1, red) and WIPI2 (green). Scale bar: 9 μm. Lower panel shows quantification of average number of WIPI2-positive dots per cell. Average intensities of individual Flag-ATG16L1 dots in (A). Underlying grey circles represent individual data points. Dots were quantified (n = 1,225 for ATG16L1WT; n = 334 for ATG16L1LD). WIPI2−/− cells reconstituted with the indicated Flag-S-ATG16L1 constructs were amino acid starved for 2 h prior to immunofluorescence analyses using antibodies against Flag tag (green) and FIP200 (red). Scale bar: 9 μm. Lower panel shows quantification of average number of Flag-positive dots per cell. Data information: Quantifications depict means and error bars (SEM) from at least three independent experiments. *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001 (pairwise unpaired Student's t-test). Download figure Download PowerPoint Binding of ATG16L1 to lipids is required for autophagy To examine the functional impact of disrupted PAS localisation in the lipid binding-deficient mutant, ATG16L1LD, we further assessed its ability to mediate LC3 lipidation in cells. When compared to ATG16L1−/− cells reconstituted with ATG16L1WT, ATG16L1LD-expressing cells exhibited a strong inhibition of LC3 lipidation during amino acid starvation (Fig 5A). To confirm that autophagy was inhibited, we measured the levels of p62, an adaptor protein that is degraded during autophagy, following amino acid starvation. Consistent with the LC3 lipidation results, p62 degradation was impaired in ATG16L1LD-expressing cells, suggesting that autophagic flux was al
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