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, 52 (12), 700-705

LIR Motifs and the Membrane-Targeting Domain Are Complementary in the Function of RavZ

Affiliations

LIR Motifs and the Membrane-Targeting Domain Are Complementary in the Function of RavZ

Sang-Won Park et al. BMB Rep.

Abstract

The bacterial effector protein RavZ is secreted by the intracellular pathogen Legionella pneumophila and inhibits host autophagy through an irreversible deconjugation of mammalian ATG8 (mATG8) proteins from autophagosome membranes. However, the roles of the LC3 interacting region (LIR) motifs in RavZ function remain unclear. In this study, we show that a membrane-targeting (MT) domain or the LIR motifs of RavZ play major or minor roles in RavZ function. A RavZ mutant that does not bind to mATG8 delipidated all forms of mATG8-phosphatidylethanolamine (PE) as efficiently as did wild-type RavZ. However, a RavZ mutant with a deletion of the MT domain selectively delipidated mATG8-PE less efficiently than did wild-type RavZ. Taken together, our results suggest that the effects of LIR motifs and the MT domain on RavZ activity are complementary and work through independent pathways. [BMB Reports 2019; 52(12): 700-705].

Conflict of interest statement

CONFLICTS OF INTEREST

The authors have no conflicting interests.

Figures

Fig. 1
Fig. 1
Binding efficiency of the RavZ protein and RavZ mutants to mATG8 proteins in vitro. (A) Schematic diagram of 3xFLAG-RavZ protein and its mutants. LIR, LC3 interacting region; CAT, Catalytic domain; MT, Membrane targeting domain. WT, 3xFLAG-RavZ wild-type; C258S, 3xFLAG-RavZC258S, a catalytically inactive mutant; mLIR1/2–3, 3xFLAG-RavZmLIR1/2–3, a point mutation in the consensus sequences (W/F/Y-X-X-I/L/V → A-X-X-A) within all LIR motifs from 3xFLAG-RavZ. (B) mATG8 protein-binding property of 3xFLAG-RavZ using GST pulldown assays. 3xFLAG-RavZ binds to GST-LC3A/B and GST-GABARAP/-L1/L2 proteins, whereas 3xFLAG-RavZmLIR1/2–3 did not bind to any of the mATG8 proteins. Upper panel: anti-FLAG, Lower panel: Coomassie blue staining.
Fig. 2
Fig. 2
Role of the RavZ LIR motifs on the delipidation of LC3B or GARARAP protein in rapamycin-induced autophagy. (A, B) Confocal images (A) showing cellular localization of GFP-LC3B or GFP-GABARAP, co-expressed with 3xFLAG-RavZ protein or the indicated RavZ mutants in MEFs upon autophagy induction (100 nM rapamycin (Rapa) + 50 μM CQ, 4 h). Arrows are representative of autophagic membranes. Scale bar: 10 μm. The bar graphs (B) illustrate the autophagosome spot numbers per cell (n = 20 for each group). 3xFLAG-RavZ and 3xFLAG-RavZmLIR1/2–3 reduce the number of GFP-LC3B or GFP-GABARAP-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 < 0.001 (one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test). (C) Depletion of endogenous LC3B-II by RavZ protein and the indicated mutants using Western blot analysis in HEK293T cells. Representative Western blots of three independent experiments of endogenous LC3B in cells expressing 3xFLAG-RavZ protein or the indicated mutants in MEFs upon autophagy induction (100 nM rapamycin). 3xFLAG-RavZ or 3xFLAG-RavZmLIR1/2–3 reduces the LC3B-II levels more than does either GFP or 3xFLAG-RavZC258S.
Fig. 3
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.
Fig. 4
Fig. 4
Schematic model of the molecular mechanism of autophagosome targeting of RavZ proteins and RavZ mutants. (A) RavZ proteins can target autophagic membranes via two complementary pathways. One major pathway is mediated by the α3 helix in the catalytic domain and the MT domain via weak membrane association and PI3P binding. Another pathway is mediated by the α3 helix in the catalytic domain and the LIR motifs, via weak membrane association and mATG8 binding. The catalytic domain of RavZ hydrolyzes the amide bond between the C-terminal glycine residue and an adjacent aromatic residue of all forms of mATG8 proteins on autophagic membranes. Therefore, RavZ proteins delipidate mATG8-PE irreversibly. However, it is not clear whether the LIR motifs ahead of the catalytic domain may be involved in substrate recognition on autophagic membranes. (B) The mATG8 binding-deficient RavZ mutant (RavZmLIR1/2–3) can target autophagic membranes by the α3 helix in the catalytic domain and the MT domain via weak membrane association and PI3P binding. The catalytic domain of RavZmLIR1/2–3 can delipidate all forms of mATG8-PE on autophagic membranes. (C) RavZ(ΔMT), the MT domain deletion mutant of RavZ, can target autophagic membranes by the α3 helix in the catalytic domain and the LIR motifs, via weak membrane association and mATG8 binding. The catalytic domain of RavZ(ΔMT) can delipidate LC3B-PE or GABARAP-PE, but not LC3C-PE, on autophagic membranes in an LIR motif-dependent manner.

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