Journal: bioRxiv

Article Title: The ULK1 effector BAG2 regulates autophagy initiation by modulating AMBRA1 localization

doi: 10.1101/2023.12.08.570815

Figure Lengend Snippet: (A) AMBRA1 binds stronger to SHA-BAG2 under starvation conditions. Shown is the relative enrichment of AMBRA1 in BAG2-AP as determined by MS of n=3 biological replicates. Box plots show spreads of log2 transformed MS ratios compared to neg. control experiments. Single values are highlighted as white dots. Error bars indicate 95% confidence intervals. (B-C) AMBRA1 binds stronger to SHA-BAG2 under starvation conditions as determined by AP-western blot analysis. (B) shows one representative western blot of n=3 biological replicates. Samples were normalized to protein amount prior analysis. Note: samples were run on one gel, black lines indicate cropping of unrelated lanes. (C) quantification of replicates shown in (B). *: p<0.05, t-test. Error bars indicate 95% confidence interval. (D) Endogenous AMBRA1 and BAG2 interact. Shown are relative abundances of annotated proteins in AMBRA1 IP-MS experiments. Starvation (1 h HBSS) leads to an increase in AMBRA1-BAG2 interaction as compared to growth conditions (DMEM). GAPDH is shown as negative control. Box plot shows log2-transformed relative fold changes of protein abundances (iBAQ values) compared to IgG control experiments normalized to AMBRA1 enrichment. Error bars indicate 95% confidence interval. Single values of n=3 biological replicates are highlighted as white dots. ***: p<0.001. (E) The interaction of AMBRA1 with BAG2 does not depend on HSC/HSP70 binding. Shown are relative abundances of annotated proteins in SHA-BAG2 AP-MS experiments. Compared to BAG2 WT (WT), BAG2 I160A (I160A) interacts less with CLCC1, HSPA4 and HSPA9. The interaction with AMBRA1 is not perturbed by the point mutation. Box plots show log2-transformed relative fold changes of protein abundances (iBAQ values) normalized to WT, error bars indicate 95% confidence interval. Single values of n=3 biological replicates are highlighted as white dots. **: p<0.01; ns: not significant. (F-G) Loss of BAG2 leads to perturbed recruitment of AMBRA1 to ER membrane. (F) Representative fluorescence images of ERGFP expressing HeLa WT and BAG2 KO/KO cells stained for AMBRA1 (Alexa 647) and subjected, or not, to nutrient deprivation, adding HBSS for 2 h. Scale bar: 5 μm. (G) Quantitative analysis of GFP and Alexa 467 overlayed area (μm 2 ) per cell reported as the mean ± SD, *P ≤ 0.05, **P ≤ 0.005 and ***P ≤ 0.0005 using ANOVA 2-way test for repeated samples. (H-I) Loss of BAG2 leads to increased number of WIPI2 dots. (H) WIPI2 puncta analysis using IF micrographs from untreated (DMEM) or starved (HBSS, 3 h) HeLa WT and BAG2 KO cells (left side), or BAG2 KO cells transfected with a lentiviral control vector (VC) or a vector encoding BAG2 WT (WT, right side). Representative pictures of n=3 biological replicates are shown. Scale bar = 10 μm. (I) Puncta quantification of (H). White dots represent the quantified average WIPI2 puncta per cell per image (5 to 23 cells per image, with a total of 12 images of n=3 biological replicates for BAG2 KO + VC samples, or 16 images of n=4 biological replicates for each other conditions). An unpaired student’s t-test was used to determine the significant differences. **: p<0.01; ***: p<0.001.

Article Snippet: For endogenous AMBRA1 immunoaffinity purifications (antibody ABC131, Millipore), 2×15 cm dishes of cells per replicate were lysed in AMBRA1 IP buffer (10 mM Tris, 150 mM NaCl, 10% glycerol, 0.5% NP-40, EDTA-free 1x protease inhibitors (Roche, 11-697-498-001), pH-8).

Techniques: Transformation Assay, Control, Western Blot, Protein-Protein interactions, Negative Control, Binding Assay, Mutagenesis, Membrane, Fluorescence, Expressing, Staining, Transfection, Plasmid Preparation