V-ATPase is a universal regulator of LC3 associated phagocytosis and non-canonical autophagy

Abstract

Non-canonical autophagy is a key cellular pathway in immunity, cancer and neurodegeneration, characterised by Conjugation of ATG8 to endolysosomal Single-Membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1) and infection (influenza), dependent on the ATG16L1 WD40-domain, and specifically K490. However, the factor(s) associated with non-canonical ATG16L1 recruitment, and CASM induction, remain unknown. Here, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase directly recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis, and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM, and indicate that SopF evolved in part to evade non-canonical autophagy.

The ATG5 interactome links clathrin-mediated vesicular trafficking with the autophagosome assembly machinery

“…Indeed, ATG16 has been found to bind directly to ATG5 [ 44 ] to reinforce its lipid binding capabilities [ 45 ], although yeast Atg16 is dispensable for the E3-like activity of the ATG12–ATG5 conjugate in vitro [ 2 ]. We also detected ATP6V0D1 (ATPase, H+ transporting, lysosomal V0 subunit D1)—a component of the V 0 domain of the V-ATPase in both WT and GFP-ATG5 K13 ° R interactomes ( Table S2 ), which is consistent with recent descriptions of ATG16L1 and ATG5 interacting with the ATP6V1A (ATPase, H+ transporting, lysosomal V1 subunit A) component during non-canonical LC3 lipidation pathways [ 46 ] and during bacterial infection [ 47 ]. Of further note, GFP-ATG5 K13 ° R also interacted strongly with ATG16L2—an isoform of mammalian ATG16 that binds to ATG12–ATG5 but cannot support autophagosome assembly due to variations in the coiled coil central section [ 48 ].…”

When the Phagosome Gets Leaky: Pore-Forming Toxin-Induced Non-Canonical Autophagy (PINCA)

“…It is very likely that LC3 recruitment to perforated phagosomes during PINCA is also activated by the V-ATPase-ATG16L1-axis, which might represent a general pathway to recruit LC3 to damaged, yet not ruptured compartments. Furthermore, it is reasonable that also during LAP, V-ATPase is responsible for ATG16L1 recruitment and subsequent LC3 conjugation, since ROS production by Nox2 is not sufficient to induce LAP, when V-ATPase is inhibited ( Hooper et al., 2021 ). It is tempting to speculate that LC3 conjugation during LAP is not triggered by Nox2-generated ROS but by V-ATPase-induced ATG16L1 recruitment.…”

“…Due to this, it is plausible that bacterial toxin-induced pore formation during PINCA can also induce osmotic imbalances within phagosomes in macrophages. Wether these osmotic imbalances can trigger V-ATPase-coupled ATG16L recruitment and LC3 lipidation during PINCA, as observed during other non-canonical autophagy pathways ( Florey et al., 2015 ; Fletcher et al., 2018 ; Xu et al., 2019 ; Hooper et al., 2021 ), are interesting topics for future studies.…”

“…A recent preprint study by Hooper et al. indicates that also during LAP, V-ATPase is responsible for ATG16L1 recruitment and subsequent conjugation of LC3 onto the phagosomal membrane ( Hooper et al., 2021 ) ( Figure 1 ). LC3 lipidation resembles the conjugation of ubiquitin to proteins ( Slobodkin and Elazar, 2013 ), therefore similar terms were used for the enzymes of the two ubiquitin like conjugation systems, which carry out the process ( Ichimura et al., 2000 ).…”

“…To the PI3P platforms formed by the PI3KC3 complex, a so far unknown factor binds to which ATG16L1 is recruited via its WD40 domain together with the LC3 conjugation machinery consisting of ATG3, ATG7, ATG5 and ATG12. A preprint study ( Hooper et al., 2021 ) marks the V-ATPase as possible candidate, which recruits ATG16L1 and the LC3 conjugation machinery to the phagosome during LAP. ROS production by Nox2 leads to oxidative inactivation of the protease ATG4, which prevents ATG4-mediated deconjugation of LC3 and stabilization of the LAPosome.…”