The bulk degradative process of macroautophagy requires the dynamic growth of autophagosomes, which carry cellular contents to the lysosome for recycling. Atg9A, a multi-pass transmembrane protein, is an apical regulator of autophagosome growth, yet its regulatory mechanism remains unclear. Our work suggests that hypoxia (low glucose and oxygen) triggers a rearrangement of the small adapter protein 14-3-3ζ interactome. Our data suggest that the localization of mammalian Atg9A to autophagosomes requires phosphorylation on the C terminus of Atg9A at S761, which creates a 14-3-3z docking site. Under basal conditions, this phosphorylation is maintained at a low level and is dependent on both ULK1 and AMPK. However, upon induction of hypoxic stress, activated AMPK bypasses the requirement for ULK1 and mediates S761 phosphorylation directly, resulting in an increase in 14-3-3z interactions, recruitment of Atg9A to LC3-positive autophagosomes, and enhanced autophagosome production. These observations suggested to us that long unstructured C-terminus of Atg9A may be a site of protein docking and regulation. We used BioID, along with conventional interactomics, to map the C- and N-terminal proximity-based interactions of Atg9A. We identified a network of Atg9A C-terminal interactions that include members of the ULK1 complex. Using gel filtration, we find that Atg9A co-immunoprecipitates with the ULK1 complex in high molecular weight fractions. Moreover, phosphorylation of the Atg9A C-terminus at S761 occurs within the ULK1 complex under nutrient-replete conditions, while hypoxia triggers a redistribution of phosphorylated Atg9A to low molecular weight fractions. Probing these relationships further, we find that Atg13, a component of the ULK1 complex, directly interacts with Atg9A and is required for Atg9A C-terminal phosphorylation. Furthermore, a non-phosphorylatable mutant of Atg9A (S761A) accumulates with Atg13 in high molecular weight complexes. Together, these data suggest that Atg13 recruits Atg9A to the ULK1 complex at the phagophore assemble site (PAS) and that S761 phosphorylation triggers Atg9A retrieval from the PAS
College and Department
Physical and Mathematical Sciences; Chemistry and Biochemistry
BYU ScholarsArchive Citation
Weerasekara, Vajira Kaushalya, "Functional and Mechanistic Insight into the Role of ATG9A in Autophagy" (2017). Theses and Dissertations. 6644.
Autophagy, ATG9A, 14-3-3ζ, AMPK, ULK1, Cancer, Chemoresistance