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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

BMB Reports 2021년 54권 2호 p.118 ~ 123
박주희, 이승환, 박상원, 전용우, 김건형, Jeon Pu-Reum, 김명진, 이진아, 장덕진,
소속 상세정보
박주희 ( Park Jui-Hee ) - Kyungpook National University College of Ecology and Environment Department of Ecological Science
이승환 ( Lee Seung-Hwan ) - Kyungpook National University College of Ecology and Environment Department of Ecological Science
박상원 ( Park Sang-Won ) - Kyungpook National University College of Ecology and Environment Department of Ecological Science
전용우 ( Jun Yong-Woo ) - Kyungpook National University College of Ecology and Environment Department of Ecological Science
김건형 ( Kim Kun-Hyung ) - Korea Brain Research Institute
 ( Jeon Pu-Reum ) - Hannam University College of Life Science and Nanotechnology Department of Biological Sciences and Biotechnology
김명진 ( Kim Myung-Jin ) - Korea Brain Research Institute
이진아 ( Lee Jin-A ) - Hannam University College of Life Science and Nanotechnology Department of Biological Sciences and Biotechnology
장덕진 ( Jang Deok-Jin ) - Kyungpook National University College of Ecology and Environment Department of Ecological Science

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, 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.

키워드

Autophagy; Delipidation; mATG8; Membrane-targeting domain; RavZ

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