Endomembrane damage sensing by V-ATPase recruits ATG16L1 for LC3 lipidation in situ

Area of Science:

• Cellular Biology
• Immunology
• Microbiology

Background:

• Selective autophagy, mediated by LC3 lipidation, is crucial for cellular defense against pathogens and internal threats.
• The V-adenosine triphosphatase (V-ATPase)-ATG16L1 pathway is essential for recruiting LC3 to pathogen-containing vacuoles.
• The Salmonella effector SopF interferes with this pathway by targeting V-ATPase, inhibiting ATG16L1 interaction.

Purpose of the Study:

• To elucidate the precise mechanism by which SopF disrupts the V-ATPase-ATG16L1 interaction and inhibits LC3 lipidation.
• To identify the specific modification and site on V-ATPase targeted by SopF.
• To determine if this mechanism is conserved across different cellular compartments and stimuli.

Main Methods:

• Investigated the interaction between SopF, V-ATPase, and ATG16L1 using biochemical assays.
• Utilized site-directed mutagenesis to identify the critical residue in V-ATPase targeted by SopF.
• Assessed LC3 lipidation and autophagic flux in response to SopF expression and V-ATPase mutations under various conditions, including pH perturbation.

Main Results:

• Demonstrated that host ARF GTPase binding facilitates SopF-dependent ADP-ribosylation of the Gln124 residue on the ATP6V0C/V0C subunit of V-ATPase.
• Showed that SopF or a ATP6V0C^Q124A mutation prevents LC3 activation triggered by pH changes in endolysosomes and the Golgi apparatus.
• Confirmed that disruption of the proton gradient in acidic compartments is a universal signal for V-ATPase-ATG16L1-induced LC3 lipidation.

Conclusions:

• SopF disrupts selective autophagy by ADP-ribosylating V-ATPase at Gln124, thereby inhibiting the V-ATPase-ATG16L1 axis.
• Proton gradient disruption in acidic cellular compartments serves as a key signal for V-ATPase-ATG16L1-mediated LC3 lipidation.
• This study reveals a conserved mechanism by which pathogens can subvert host autophagy for their survival.