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Mammalian ATG8 proteins (mATG8s) are essential for selective autophagy because they recruit various proteins with LC3- interacting region (LIR) motifs to autophagic membranes. The RavZ protein, secreted by Legionella pneumophila, and mammalian ATG4B possess functional LIR motifs that participate in lipidated mATG8 deconjugation on autophagic membranes. RavZ comprises three functional LIR motifs at the N- and Cterminal sides of its catalytic domain (CAD). This study demonstrated that LIR motifs at the N-terminal side of the CAD of RavZ are involved in autophagic membrane targeting and substrate recognition, while LIR motif at the C-terminal side facilitate autophagic membrane targeting. Our results also revealed that the C-terminal LIR motif in human ATG4B is pivotal in delipidating LC3B-phosphatidylethanolamine (PE), but it plays a minor role in pro-LC3B priming in the cytosol. Therefore, introducing a functional LIR motif to the N-terminal of ATG4B does not affect LC3B-PE delipidation. This study clearly described the position-dependent roles of LIR motifs in RavZ and ATG4B in cellular contexts.

The bacterial effector protein RavZ from a pathogen can impair autophagy in the host by delipidating the mammalian autophagy- related gene 8 (mATG8)-phosphatidylethanolamine (PE) on autophagic membranes. In RavZ, the membrane-targeting (MT) domain is an essential function. However, the molecular mechanism of this domain in regulating the intracellular localization of RavZ in cells is unclear. In this study, we found that the fusion of the green fluorescent protein (GFP) to the MT domain of RavZ (GFP-MT) resulted in localization primarily to the cytosol and nucleus, whereas the GFP-fused duplicated-MT domain (GFP-2xMT) localized to Rab5- or Rab7-positive endosomes. Similarly, GFP fusion to the catalytic domain (CA) of RavZ (GFP-CA) resulted in localization primarily to the cytosol and nucleus, even in autophagy-induced cells. However, by adding the MT domain to GFP-CA (GFP-CA-MT), the cooperation of MT and CA led to localization on the Rab5-positive endosomal membranes in a wortmannin-sensitive manner under nutrient-rich conditions, and to autophagic membranes in autophagy-induced cells. In autophagic membranes, GFP-CA-MT delipidated overexpressed or endogenous mATG8-PE. Furthermore, GFP-CAΔα3-MT, an α3 helix deletion within the CA domain, failed to localize to the endosomal or autophagic membranes and could not delipidate overexpressed mATG8-PE. Thus, the CA or MT domain alone is insufficient for stable membrane localization in cells, but the cooperation of MT and CA leads to localization to the endosomal and autophagic membranes. In autophagic membranes, the CA domain can delipidate mATG8-PE without requiring substrate recognition mediated by LC3-interacting region (LIR) motifs. [BMB Reports 2021; 54(2): 118-123].