• BMB Reports
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• v.52 no.12
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• pp.700-705
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• 2019

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
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• v.54 no.2
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• pp.118-123
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• 2021

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-CA△α3-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.

• Molecules and Cells
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• v.41 no.1
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• pp.27-34
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• 2018

The cytoplasm in mammalian cells is a battlefield between the host and invading microbes. Both the living organisms have evolved unique strategies for their survival. The host utilizes a specialized autophagy system, xenophagy, for the clearance of invading pathogens, whereas bacteria secrete proteins to defend and escape from the host xenophagy. Several molecules have been identified and their structural investigation has enabled the comprehension of these mechanisms at the molecular level. In this review, we focus on one example of host autophagy and the other of bacterial defense: the autophagy receptor, NDP52, in conjunction with the sugar receptor, galectin-8, plays a critical role in targeting the autophagy machinery against Salmonella; and the cysteine protease, RavZ secreted by Legionella pneumophila cleaves the LC3-PE on the phagophore membrane. The structure-function relationships of these two examples and the directions of future research will be discussed.