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Deciphering the role of a membrane-targeting domain in assisting endosomal and autophagic membrane localization of a RavZ protein catalytic domain
Jui-Hee Park1 (Graduate student), Seung-Hwan Lee1 (Graduate student), Sang-Won Park1 (Graduate student), Yong-Woo Jun1 (Graduate student), Kun-Hyung Kim2 (Researcher), Myungjin Kim2 (Senior Researcher), Jin-A Lee2 (Professor), Deok-Jin Jang1,* (Professor)
1Department of Ecological Science, Kyungpook national University,
2Brain Research Core Facilities Center, Korea Brain Research Institute,
3Department of Biological Science and Biotechnology, Hannam University
Abstract
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, but to autophagic membranes in autophagy-induced cells. In autophagic membranes, GFP-CA-MT delipidated overexpressed or endogenous mATG8s-PEs. Furthermore, GFP-CAツメ3-MT, an メ3 helix deletion within the CA domain, failed to localize to the endosomal or autophagic membranes and could not delipidate overexpressed mATG8s-PEs. 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.
Abstract, Accepted Manuscript(in press) [Submitted on September 7, 2020, Accepted on November 26, 2020]
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