Golgi-associated LC3 lipidation requires V-ATPase in noncanonical autophagy

Abstract

Autophagy is an evolutionarily conserved catabolic process by which cells degrade intracellular proteins and organelles in the lysosomes. Canonical autophagy requires all autophagy proteins (ATGs), whereas noncanonical autophagy is activated by diverse agents in which some of the essential autophagy proteins are dispensable. How noncanonical autophagy is induced and/or inhibited is still largely unclear. In this study, we demonstrated that AMDE-1, a recently identified chemical that can induce canonical autophagy, was able to elicit noncanonical autophagy that is independent of the ULK1 (unc-51-like kinase 1) complex and the Beclin1 complex. AMDE-1-induced noncanonical autophagy could be specifically suppressed by various V-ATPase (vacuolar-type H+-ATPase) inhibitors, but not by disturbance of the lysosome function or the intracellular ion redistribution. Similar findings were applicable to a diverse group of stimuli that can induce noncanonical autophagy in a FIP200-independent manner. AMDE-1-induced LC3 lipidation was colocalized with the Golgi complex, and was inhibited by the disturbance of Golgi complex. The integrity of the Golgi complex was also required for multiple other agents to stimulate noncanonical LC3 lipidation. These results suggest that the Golgi complex may serve as a membrane platform for noncanonical autophagy where V-ATPase is a key player. V-ATPase inhibitors could be useful tools for studying noncanonical autophagy.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells

“…As seen from the representative blots, and consistent with the known ability of ConA and other V-ATPase inhibitors to stall autophagic flux [47], ConA treatment increased total LC3B, mainly as LC3B-II in Panc-1 cells and LC3B-I in BxPC-3 cells, and increased p62 in both cell lines ( Figure 5A-C). A requirement for V-ATPase activity for LC3 lipidation, as seen in BxPC-3 cells, is a hallmark of unconventional autophagy [48], and this finding seems consistent with a previous report of differential regulation of LC3 lipidation in BxPC-3 and Panc-1 cells [49]. a3 KD modestly increased LC3B-II/LC3B-I in both cell types ( Figure 5D,E), with little if any change in p62 ( Figure 5D,F).…”

Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer

“…However, it should be noted that the relationship between V‐ATPase and autophagy is complex particularly as the V‐ATPase has a well‐established role in activation of lysosomal acid hydrolases that mediate proteolysis during autophagy 80, 81, 82. Moreover, although V‐ATPase inhibition can result in increased autophagic markers in some settings,74 V‐ATPase is required for activation of noncanonical autophagy 83. In a very recent study, the autophagy‐related protein ATG5 was demonstrated to displace ATP6V1E1 from V‐ATPase, causing it to accumulate in exosomes 84.…”

Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes

“…During amino acid or glucose starvation, membrane recruitment of ATG16L1 involves its binding to WIPI2b 11 , an effector protein of phosphatidyl 3-phosphate (PI(3)P) or PI(5)P 12,13 . However, ATG16L1 targeting to membranes may be independent of WIPI2b during endomembrane LC3 lipidation [14][15][16] .…”