A study on the isolation and signaling of new autophagy regulators

Abstract

Autophagy is a cellular degradation system for maintaining cellular homeostasis under various stress signals such as nutrient deprivation, hypoxic condition, or endoplasmic reticulum stress. To overcome the stress conditions, cells utilize autophagy-specific proteins to generate double-membrane vesicle called autophagosome to sequester cytosolic components and subcellular organelles fusing with lysosome. However, the precise roles of ATGs and their regulatory mechanisms are still unknown. To identify novel regulators of autophagy, I employed cell-based functional screening assays using bimolecular fluorescence complementation (BiFC) method. I found an interaction between ULK1 and ATG9 in mammalian cells and utilized the interaction to identify novel regulators of autophagy upstream of ULK1, an initiation step. I established a cell-based screening assay employing N-terminal Venus-tagged ULK1 and C-terminal Venus-tagged ATG9 BiFC. By performing gain-of-function screening, I identified G6PT as an autophagy activator. G6PT enhanced the interaction between N-terminal Venus (VN)-tagged ULK1 and C-terminal Venus (VC)-tagged ATG9, and increased autophagic flux independent of its transport activity. G6PT negatively regulated mTORC1 activity, demonstrating that G6PT functions upstream of mTORC1 in stimulating autophagy Also, ATG7-VN/VCn-ATG12 BiFC assay was employed to isolate autophagy modulators which regulate ATG7, an E1-like activating enzyme for ATG12. Utilizing the assay, ZAK was isolated as a potent autophagy activator on elongation step. Expression of ZAK enhanced the fluorescence of ATG7-VN/VCn-ATG12 BiFC assay and increased the interaction between them. ZAK reduced the levels of p62 and ubiquitin conjugates increasing LC3 dot formation. Conversely, malfunction of ZAK expression inhibits autophagy flux. Interestingly, depletion of ZAK blocked amino-acid or serum starvation-induced autophagy to almost control level. Furthermore, ZAK interacted with ATG7 in mammalian cells. I hypothesize that ZAK is an essential regulator for autophagy activity functioning through its kinase activity and interaction with ATG7.