Abstract
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 Fyco1 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.
Highlights
Xenophagy is selective autophagy by which host cells degrade invading pathogens in lysosomes[1,2]
RavZ(ΔCA)-GFP was efficiently localized to autophagic membranes through mammalian autophagy-related protein 8 (mATG8) binding mediated by light chain 3 (LC3)-interacting region (LIR) motifs and phosphatidylinositol 3-phosphate (PI3P) binding mediated by an MT domain within the RavZ protein
It has been reported that RavZ protein secreted from Legionella is targeted to autophagic membranes and delipidates mATG8-PE on autophagic membranes in cells[19,20]
Summary
Xenophagy is selective autophagy by which host cells degrade invading pathogens in lysosomes[1,2]. RavZ(ΔCA)-GFP was efficiently localized to autophagic membranes through mATG8 binding mediated by LIR motifs and PI3P binding mediated by an MT domain within the RavZ protein. Results and Discussion Generation and cellular localization of RavZ(ΔCA)-GFP into LC3 or GABARAP-positive autophagic membranes in an MT domain, LIR1/2 motif, or LIR3 motif-dependent manner.
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