Journal
SCIENTIFIC REPORTS
Volume 9, Issue -, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41598-019-53372-2
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Funding
- National Research Foundation [NRF-2017R1D1A3B03030972, NRF-2018R1D1A1B07048822]
- National Scientist Honor Program [HI18C0158]
- Korea Health Technology RD project [HI18C0158]
- Bio & Medical Technology Development Program of the National Research Foundation (NRF) - Ministry of Science ICT [2017M3A9G7073521]
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Xenophagy is a selective lysosomal degradation pathway for invading pathogens in host cells. However, invading bacteria also develop survival mechanisms to inhibit host autophagy. RavZ is a protein secreted by Legionella that irreversibly delipidates mammalian autophagy-related protein 8 (mATG8) on autophagic membranes in host cells via efficient autophagic membrane targeting. In this study, we leveraged the autophagic membrane-targeting mechanism of RavZ and generated a new autophagosome probe by replacing the catalytic domain of RavZ with GFP. This probe is efficiently localized to mATG8-positive autophagic membranes via a synergistic combination between mATG8 protein-binding mediated by the LC3-interacting region (LIR) motifs and phosphoinositide-3-phosphate (PI3P) binding mediated by the membrane-targeting (MT) domain. Furthermore, the membrane association activity of this new probe with an MT domain was more efficient than that of probes with a hydrophobic domain that were previously used in LIR-based autophagosome sensors. Finally, by substituting the LIR motifs of RavZ with selective LIR motifs from Fycol or ULK2, we developed new probes for detecting LC3A/B- or GABARAP subfamily-positive autophagic membranes, respectively. We propose that these new RavZ-based sensors will be useful for monitoring and studying the function of mATG8-positive autophagic membranes in different cellular contexts for autophagy research.
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