EMBO/SourceData huggingface dataset:
Figure 2A Representative kymographs of LysoTracker-labeled lysosomes in proximal dendrites of hippocampal neurons infected with scrambled shRNA (shSc), LAMTOR2 shRNA (shLAMTOR2), or Raptor shRNA (shRaptor). Scale bar, 5 µm. B Quantitative analysis of lysosomal movement from kymographs (N = 44, 24, and 19 neurons for shSc, shLAMTOR2, and shRaptor, respectively, from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Sidak's post-test ***P < 0.001 as compared to shSc; n.s., not significant.C, D FRAP analysis of dendritic lysosomal movement in Raptor KD neurons. C Representative images of a neuron at 0 and 4 min after photobleaching. D Quantification of the lysosomes in C undergoing retrograde (red) or anterograde (cyan) transport (N = 7 and 8 neurons for shSc and shRaptor, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.E LAMTOR1 KD did not affect trafficking of vesicles labeled with Alexa 594-conjugated transferrin (red, Tf 594) in dendrites. Shown are dendritic segments (upper) and kymographs (lower). Scale bar, 5 µm. F Quantitative analysis of vesicular movement from kymographs (N = 10 neurons from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Mann-Whitney U test ***P < 0.001 as compared to shSc; n.s., not significant.
Figure 3A Quantification of the percent of lysosomes moving in the anterograde or retrograde direction in neurons infected with AAV expressing either LAMTOR1 shRNA (shLAMTOR1) or scrambled shRNA (shSc) and Accell lyspersin siRNA or control siRNA. Neurons were imaged with LysoTracker to visualize lysosomal trafficking in dendrites. N = 17-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.C-F Quantification of the percent of lysosomes moving in the anterograde or retrograde direction. C, E, F Cultured hippocampal neurons were infected with AAV expressing either shLAMTOR1 or shSc; they were treated with vehicle control or ML-SI1 (20 µM, C, n = 16-49 neurons from 3-10 independent experiments), or ML-SA1 (20 µM, E, n = 13-49 neurons from 3-10 independent experiments), or CilioD (20 µM, F, n = 12-49 neurons from 3-10 independent experiments), and imaged with LysoTracker to visualize lysosomal trafficking in dendrites. D Neurons were infected with shLAMTOR1 or shSc AAV and Accell TRPML1 siRNA or control siRNA; they were imaged as described above. N = 7-31 neurons from 3-6 independent experiments. Data information: Data with error bars are represented as means ± SEM. ***P < 0.001 compared with shSc or shSc/Accell siControl; ##P < 0.01, ###P < 0.001 compared with shLAMTOR1 or shLAMTOR1/Accell siControl; &&P < 0.01, &&&P < 0.001 compared with shSc/Accell siLyspersin; n.s., not significant; two-way ANOVA with Tukey's post-test.
Figure 4F Quantification of co-IP results. N = 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test (F). *P < 0.05, ***P < 0.001.
Figure 5A Interactions between LAMTOR1 and TRPML1 in mouse hippocampus. Binding of LAMTOR1 to TRPML1 in vivo was disrupted by systemic administration of the TAT-2031 peptide. Wes protein analysis with anti-LAMTOR1 and -TRPML1 antibodies of immunoprecipitation performed with anti-LAMTOR1 antibodies or negative control anti-HA antibodies using whole hippocampal homogenates from naïve, TAT or TAT-2031-treated mice. B Quantification of the relative abundance of TPRML1 pulled down by LAMTOR1 in naïve, TAT or TAT-2031-treated mice. N = 3 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.C Representative images from proximity ligation assay (PLA) performed on brain slices from naïve, TAT or TAT-2031-treated mice. Evidence of proximity between LAMTOR1 and TRPML1 is indicated by the appearance of red puncta. Nuclei are counterstained with DAPI (blue). Scale bar, 10 μm. D Quantification of the number of PLA signals in CA1 from naïve, TAT or TAT-2031-treated mice. N = 6 mice for each group. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by one-way ANOVA with Dunnett's post-test **P < 0.01, ***P < 0.001.
Figure 6D TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1 Ca2+ release in both control and LAMTOR1 KD cells. Data information: traces represent the mean values of each group.G Treatment with GPN (200 µM), BAPTA-AM (20 µM), or ML-SI1 (20 µM) blocked ML-SA1-induced TRPML1-GCaMP6m responses in neurons. Data information: traces represent the mean values of each group.I Both LAMTOR1 KD and TAT-2031 treatment (10 µM) increased ML-SA1-induced TRPML1-GCaMP6m responses in neurons. J Quantification of peak responses as shown in I. N = 6-13 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.K LAMTOR1 KD increased PI(3,5)P2 (0.5 µM)-induced TRPML1-GCaMP6m responses and the blocking effect of ML-SI1 treatment (20 µM). L Quantification of peak TRPML1-GCaMP6m responses as shown in K. N = 6-16 cells from 3 independent experiments. Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post hoc analysis *P < 0.05, **P < 0.01, ***P < 0.001, ###P < 0.001, n.s., not significant. traces represent the mean values of each group.
Figure 7A-D Effects of LAMTOR1 KD and ML-SI1 treatment on TBS-induced LTP (A) or LFS-induced LTD (C) in CA1. B, D Means ± SEM of fEPSPs measured 40 min after TBS (B, n = 4-5 slices from 4-5 mice) or LFS (D, n = 4-8 slices from 4-8 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms.G-J Effects of TAT-2031 treatment on TBS-induced LTP (G) or LFS-induced LTD (I) in CA1. H, J Means ± SEM of fEPSPs measured 40 min after TBS (H, n = 6 slices from 6 mice) or LFS (J, n = 3-4 slices from 3-4 mice) in different groups. Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by Student's t-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT Insets show representative traces of evoked fEPSPs before and 40 min after TBS/LFS. Scale bar 0.5 mV/10 ms.K Effects of LAMTOR1 KD and treatment with a calcineurin inhibitor, FK506, on LFS-induced LTD in CA1. L Means ± SEM of fEPSPs measured 45 min after LFS in different groups (n = 3-4 slices from 3-4 mice). Data information: Slopes of fEPSPs were normalized to the average values recorded during the first 10 min baseline Statistical significance was assessed by two-way ANOVA with Tukey's post-test *P < 0.05, **P < 0.01, ***P < 0.001 compared with shSc, Vehicle, or TAT, #P < 0.05, ##P < 0.01, ###P < 0.001 compared with shLAMTOR1. Insets show representative traces of evoked fEPSPs before and 40 min after LFS. Scale bar 0.5 mV/10 ms.
Figure 8LAMTOR1 KD significantly reduced levels of GluA1 phosphorylation and total GluA1 as well as the ratio of p-GluA1 to GluA1 in hippocampal neurons, and this effect was reversed by ML-SI1 treatment. A Representative images of CA1 pyramidal neurons stained with anti-p-GluA1 S845 (magenta), anti-GluA1 (red), and anti-GFP (green) antibodies. Scale bar = 20 µm.E Representative images of proximal dendrites of hippocampal neurons stained with anti-GluA1 (green), anti-LAMP2 (red), and anti-GFP (gray) antibodies. Note that GluA1 labeled with Alexa Fluor 633 secondary antibodies was false-colored green and GFP false-colored gray to better show colocalization of GluA1 with LAMP2. Arrowheads indicate clearly co-localized puncta. Scale bar, 5 µm.
Figure 9A Mice received 10 min of training in an environment with two identical objects and received a retention test 24 h later in which one object was replaced with a novel one. LAMTOR1 shRNA-injected mice exhibited a significant deficit 24 h after training, which was reversed by ML-SI1 treatment (N = 7-18 mice). B % freezing for different experimental groups in context memory (N = 7-19 mice). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's post-test. *P < 0.05, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1.
Figure 1Peptide array dot blot analysis to identify LIR motifs in the indicated SNARE proteins. Amino acids for the identified LIRs from positive signals are marked on each SNARE. Amino acids are denoted as amino acid single letter codes in the blot.Co-immunoprecipitation (Co-IP) analysis of interactions between FLAG-tagged Stx16/Vti1a/Stx6 and EGFP-tagged LC3B or GABARAP overexpressed in HEK293T cells. * indicates mouse IgG heavy chain of precipitated mouse-anti-FLAG antibody.GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and GST-tagged mAtg8 proteins.GST pull-down analysis of interactions between wild type (WT) or LIR-mutant (L219A/V222A) Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.GST pull-down analysis of interactions between WT or different types of LIR-mutant Stx16 and GST-tagged LC3C or GABARAP. Lower panel shows percentages of WT or LIR-mutant Stx16 bound to GST-LC3C or GST-GABARAP.GST pull-down analysis of interactions between radiolabeled Myc-Stx16 and WT or LDS-mutant GABARAP (GABARAP-Y49A and -Y49A/F104A). Lower panel shows percentages of Stx16 bound to WT or LDS-mutant GABARAP. Data shown as means ± SEM of precipitated Stx16, n = 3.
Figure 3WT or STX16/STX17DKO HeLa-YFP-Parkin cells were treated with CCCP (10 µM) or oligomycin A (5 µM) and antimycin A (10 µM) (OA) for 16 hours, and subjected to high-content microscopy (HCM) analysis of mitochondria clearance. Masks: blue, nuclei; red, mitochondria stained with mitochondrial DNA (mtDNA) antibody. Scale bar: 20 µm.   Quantifications of mtDNA by object count or total area per cell in WT or STX16/STX17DKO HeLa-YFP-Parkin cells treated as in (A); Data shown as means ± SEM of mtDNA object count (upper panel) or object total area (lower panel) per cell; minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).   WT or STX16/STX17DKO Huh7 cells were treated with H2O2 (0.4 mM) for indicated time points, and autophagic clearance of peroxisomes was measured by the protein levels of PMP70, PEX14 and p62.   Quantifications of peroxisomal proteins PMP70 and PEX14 for WT or STX16/STX17DKO Huh7 cells treated as in (C); Data shown as means ± SEM of PEX14 or PMP70 and β-actin ratios, n = 3; †, not significant; *, p < 0.05; **, p < 0.01 (two-way ANOVA).   WT, STX16KO, STX17KO or STX16/STX17DKO THP-1 cells were infected with Mycobacterium tuberculosis (Mtb), followed by starvation to induce autophagy. Autophagic clearance of infected Mtb was measured by Mtb colonies grown on Middlebrook 7H11 agar plates; Data shown as means ± SEM of Mtb colonies, n = 4; *, p < 0.05; **, p < 0.01 (two-way ANOVA).
Figure 4Validation of STX16/STX17 DKO by Western blot analysis in HEK293 cells stably expressing RPS3-Keima (left panel) and HCT116 cells stably expressing RPL28-Keima (right panel).WT or STX16/STX17DKO HEK293 RPS3-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16 /STX17DKO HEK293 RPS3-Keima cells treated as in (C). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).  WT or STX16/STX17DKO HCT116 RPL28-Keima cells were cultured in full medium or starved for 8 hours, and subjected to HCM analysis of Keima puncta accumulated in autolysosomes with the excitation wavelength at 560 nm. Masks: white, cells identified based on nuclei; red, Keima puncta detected with the excitation and emission wavelengths of 560 nm and 620 nm, respectively; lower panel shows Keima puncta without masks. Scale bar: 20 µm.   Quantifications of autolysosomal Keima puncta in WT or STX16/STX17DKO HCT116 RPL28-Keima cells treated as in (E). Data shown as means ± SEM of Keima puncta per cell, minimum 1000 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; **, p < 0.01 (two-way ANOVA).
Figure 5(A) WT or STX16KO HeLa cells were starved in EBSS for 2 h and subjected to HCM analysis of LAMP2 and LC3 puncta. Masks: white, cells identified based on nuclei; red, LC3 puncta; green, LAMP2 puncta; yellow, overlap of LC3 and LAMP2. Scale bar: 20 µm.(B-D) Quantifications of LC3 puncta per cell (B), LC3 and LAMP2 overlap area per cell (C), and LAMP2 puncta per cell (D) in WT or STX16KO cells treated Data shown as means ± SEM of puncta or overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (two-way ANOVA).(E) Western blot analysis of LAMP1 and LAMP2 protein levels in WT and separate clones of STX16KO HeLa, Huh7, and U2OS cells.(F) Western blot analysis of LAMP2 protein levels in WT and separate clones of STX6KO or VTI1AKO HeLa cells.(G) Time course of mTOR inactivation during EBSS starvation in WT vs. STX16KO HeLa cells analyzed by Western blotting for mTOR substrates ULK1 and 4E-BP1.(H) WT or STX16KO HeLa cells were starved in EBSS for 1 h and subjected to HCM analysis of mTOR puncta. Masks: white, cells identified based on nuclei; red, mTOR puncta. Scale bar: 20 µm. (I) Quantifications of mTOR puncta in WT or STX16KO cells treated as in (H). Data shown as means ± SEM of mTOR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; **, p < 0.01 (two-way ANOVA).
Figure 7WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 90 min and subjected to HCM analysis of overlaps between LAMP2 and Stx16. Masks: white, cells identified based on nuclei; green, LAMP2 puncta; red, Stx16 puncta; yellow, overlap of LAMP2 and Stx16. Scale bar: 20 µm.Quantifications of overlaps between LAMP2 and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LAMP2 and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).Quantifications of overlaps between LBPA and Stx16 in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells grown in full medium or treated Data shown as means ± SEM of LBPA and Stx16 overlap area per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; *, p < 0.05; **, p < 0.01 (one-way ANOVA).WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells were starved in EBSS for 1 hour, followed by starvation with the presence of LTR (100 nM) for additional 30 min, and subjected to HCM analysis of LTR. Scale bar: 20 µm.Quantifications of LTR puncta in WT, LC3TKO, GABARAPTKO or HexaKO HeLa cells treated Data shown as means ± SEM of LTR puncta per cell, minimum 500 cells were counted each well from at least 12 wells, 3 independent experiments; †, not significant; *, p < 0.05; **, p < 0.01 (one-way ANOVA).
Figure 1a, Cells (wt, ∆atg18 or ∆atg18/∆atg21/∆hsv2 ()), with and without a plasmid expressing wt or a mutant form of Atg18) were grown in YPD, vacuoles were stained with FM4-64 and imaged by confocal microscopy. At least 10 confocal sections spaced at 300 nm were assembled into maximum projections. Images show cells before (0 min) and 15, 30 and 60 min after induction of vacuolar fragmentation by addition of 0.4 M NaCl. Red: FM4-64. Gray: DIC. Scale bar 5 µm.b, The number of vacuoles per cell was quantified for the experiment in a. 150 cells from three independent experiments were counted for each condition. Values represent the mean, error bars the SEM.
Figure 2atg18 cells expressing plasmids carrying Atg18-GFP or Atg18FGGG-GFP, a mutant in the lipid binding sites, were stained with FM4-64. a, Confocal z-stacks were taken before and 2, 6 and 10 min after addition of 0.4 M NaCl. Stacks were processed into maximum projections using ImageJ. Scale bar: 5 µm.b, Same experiment as in a, but in a fab1 or vps34 background.d, Localisation of Atg18-GFP 5 min after salt shock. Experiment as in a. Scale bar: 1 µm.
Figure 4b,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for b, the wildtype peptideb,c Their secondary structure was analyzed by circular dichroism spectroscopy in the presence of increasing concentrations of hexafluoro-2-propanol (HFIP), in the presence of 6 mM small unilamellar vesicles (SUVs, 70% egg PC and 30 % PS, total phospholipid concentration approx. 6 mM), or of or control buffer only (blue). Spectra are shown for c, the SLoop peptide.d, Vacuole morphology of cells expressing Atg18Sloop-GFP, imaged before and after osmotic shock as in Fig 1a. Scale bar: 5 µm.e, Number of vacuoles per cell in d, assayed as in Figure 1b. n=3.f, Vacuole morphology of cells expressing Atg18DLoop-GFP, imaged as in d.g, Number of vacuoles per cell in e, assayed as in Fig. 1b. n=3.
Figure 5Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for a, Atg18wtBinding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for b, Atg18FGGG;Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for c, Atg18DLoop;Binding of recombinant Atg18 variants to liposomes. SUVs were made of EPC with 5% of the indicated phosphoinositides, or of EPC and the indicated fraction of PS. Cholesterol had been added to 20 mol% of the total phospholipid amount. Liposomes were incubated with the purified proteins for 30 min at room temperature (25°C), centrifuged and the supernatants (S) and pellets (P) analyzed by SDS-PAGE and Coomassie staining. Binding is shown for d, Atg18SLoop.e, The bands from a-d were quantified from 3 independent experiments. The mean with SEM is shown.
Figure 6a. Autophagy measured with the pho8∆60 assay in atg18 cells complemented with wt, FGGG, SLoop or DLoop alleles of ATG18.b. Same assay as in a, but in atg18 atg21 cells complemented with wt or SLoop versions of ATG18 and ATG21. Atg21SLoop contains K426F and F429K substitutions. Helical wheel projections for both Atg21 helices are given. The arrows indicate the magnitude of the hydrophobic moment.c. Vacuole fragmentation in atg2 cells, assayed 15 min after a moderate hypertonic shock as in Fig. 1.d. Number of vacuoles per cell in c, assayed as in Figure 1b. n=3.
Figure 7a, Time lapse analysis of Atg18 added to liposomes. 10 µL of 10 µM purified recombinant Atg18wt was added to 100 µL buffer containing GUVs with 5% PI3P and 1% PI(3,5)P2. The sample was imaged on a spinning disc confocal microscope. Most of the tubules underwent scission into small vesicles (See movie EV6). Scale bar 5 µm. Areas in boxes 1 and 2 of c are shown at higher magnification in b and c. Scale bars: 2.5 µm.d, Quantification of fission activity. For each movie, the whole frame was divided into squares of 10 per 10 µm and the number of small vesicles per 100 µm2 counted. The mean of three independent experiments is shown with SEM.
Figure 8a, 1µM Atg18 was added to wells containing sedimented GUVs with 10% PI3P or PI(3,5)P2 while imaging a single plane by spinning disc microscopy. Membrane tension exerted by Atg18 binding suffices to induce liposome collapse, which allows to better judge the mobility of the tubular or clustered membranes that remain. Images are extracted from movies EV2 to EV5. Scale bar 5 µm.b, Atg18 was covalently labeled with the green fluorescent dye Alexa488 TFP (ThermoFisher) and used in experiments as in a, with rhodamine-phosphatidylethanolamine-labeled (Rh-PE) GUVs containing the indicated phosphoinositides. Scale bar 5 µmc, Effect of 1µM of purified recombinant Atg18 variants on GUVs containing 10mol% of the indicated phosphoinositides. Vesicles are shown after 1 to 3 min of incubation.
Figure 9a, Cross-linking of Atg18. Purified recombinant Atg18wt or Atg18FGGG (1.5 µM) was incubated with liposomes containing 5% PI3P, PI(3,5)P2, PI(4,5)P2 or 15% PS as a control for negative charge. After addition of a cleavable cross-linker or buffer only, liposomes were pelleted, dissolved in sample buffer, incubated in the presence or absence of 100 mM dithiothreitol (DTT) for 5 min at room temperature and analysed by SDS-PAGE and Coomassie staining. Cross-link products migrate at the upper limit of the separating gel.b, Cross-linking experiment with recombinant Atg18wt as in a, performed in the presence of increasing concentrations of liposomes containing 5% PI(3,5)P2.