Figure 3

Elucidation of the roles of N- or C-terminal LIR motifs for autophagosome targeting of RavZ(ΔCA)-GFP. (A,B) Schematic diagram of GFP-fused RavZ mutant proteins and confocal images depicting the cellular localization of GFP-fused LIR motifs in RavZ proteins in MEF cells treated with 100 nM rapamycin (rapa) + 10 mM NH₄Cl. The bar graphs (B) illustrate the GFP fluorescence intensities of the autophagic membranes and the cytosol (the A/C ratio) (n = 75 for each group). ***P < 0.001 (n = 75 for each group) one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test). Scale bar: 10 μm. (C,D) mATG8 protein-binding properties of the GFP-fused LIR motifs of RavZ proteins using GST-pulldown assays and quantification analysis for the binding. Extended blot images including these data are presented in Supplementary Fig. 6B. The bar graphs (D) illustrate relative quantification of the level of bound GFP-constructs in GST pull-down assay. The levels of bound GFP-constructs intensity were normalized to the intensity of the expressed GFP-constructs (input). The data are presented as the mean ± SEM of three independent experiments. RavZ(ΔCA)_mLIR1/2-3-GFP, LIR1/2/3 mutant of RavZ(ΔCA)-GFP; RavZ(ΔCA)_mLIR1/2-GFP, LIR1/2 motif mutant of RavZ(ΔCA)-GFP; RavZ(ΔCA)_mLIR3-GFP, LIR3 motif mutant of RavZ(ΔCA)-GFP. RAP, GABARAP; RAP-L1, GABARAP-L1; RAP-L2, GABARAP-L2; N.S., not significant.