Monitoring LC3- or GABARAP-positive autophagic membranes using modified RavZ-based probes.

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.