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Fig. 3. Role of the LIR motifs of RavZ(ΔMT), an MT domain-deletion mutant, on delipidation of LC3B or GARARAP protein in rapamycin-induced autophagy. (A) Confocal images showing cellular localization of GFP-LC3B, GFP-GABARAP, or GFP-LC3C co-expressed with 3xFLAG-RavZ(ΔMT) protein or the indicated mutants in MEFs upon autophagy induction (100 nM rapamycin). (B) The bar graphs illustrate the GFP-LC3B-, GFP-GABARAP-, or GFP-LC3C-positive autophagosome spot numbers per cell (n = 20 for each group). The expression of 3xFLAG-RavZ(ΔMT), but not 3xFLAG-RavZ(ΔMT)mLIR1/2–3, significantly reduces the number of GFP-LC3B− or GFP-GABARAP-positive autophagic membranes, whereas only the expression of 3xFLAG-RavZ, but not 3xFLAG-RavZ(ΔMT) or the indicated mutants, affects the number of GFP-LC3C-positive autophagic membranes in rapamycin or rapamycin + CQ-treated cells compared to either mRFP or the 3xFLAG-RavZC258S catalytic mutant. Data are presented as mean ± SEM. N.S. (not significant). *P < 0.05, ***P < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test). WT, 3xFLAG-RavZ wild-type; C258S, 3xFLAG-RavZC258S, a catalytically inactive mutant; ΔMT, 3xFLAG-RavZ(ΔMT), a MT-domain deletion mutant; ΔMTmLIR1/2, 3xFLAG-RavZ(ΔMT)mLIR1/2, a point mutation in the LIR1 and 2 motifs in 3xFLAG-RavZ(ΔMT); ΔMTmLIR3, 3xFLAG-RavZ(ΔMT)mLIR3, a point mutation in the LIR3 motif in 3xFLAG-RavZ(ΔMT); ΔMTmLIR1/2–3, 3xFLAG-RavZ(ΔMT)LIRm1/2–3, a point mutation in all LIR motifs.
BMB Reports 2019;52:700~705
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