Elucidation of different roles of LIR motifs of the modified RavZ protein using LIR(Fy). (A) Schematic of the generation of eGFP-tagged deconjugase-Fy and its LIR motif-deficient mutants. (B) Confocal images showing the autophagosome formation of mRFP-LC3B (upper) or mRFP-GABARAPL1 (lower) protein co-expressed with RavZ proteins (RavZ, wild type) or deconjugase-Fy mutants in HKO cells upon autophagy induction (100 nM rapamycin [Rapa], 4 h). Scale bar: 10 μm. (C) Bar graphs illustrating the autophagosome spot number for each cell. Values are presented as means ＋ SEM. *** P < 0.001, one-way ANOVA with the Newman–Keuls multiple comparison test. N.S., not significant. (D) Schematic of the generation of eGFP-tagged enzyme-deficient mutant (C258S) of deconjugase-Fy and its LIR motif-deficient mutants. (E) Confocal images showing the cellular localization of RavZC258S or enzyme-deficient mutants containing one or more LIR(Fy) co-expressed with mRFP-LC3B (upper) or mRFP-GABARAPL1 (lower) protein in HKO cells upon autophagy induction (100 nM bafilomycin A1 (BafA1) in Earle’s balanced salt solution (EBSS) for 2 h, ＋EBSS, BafA1). Scale bar: 10 μm. (F) Bar graphs illustrating the quantification of the autophagosome and cytosol (A:C) ratio of GFP fluorescence in each cell. Values are presented as means ＋ SEM . ***P < 0.001, one-way ANOVA with the Newman–Keuls multiple comparison test. N.S., not significant. The numbers above the bar graph represent the number of replicates for each experiment.
