ATG9A‐mediated turnover of p62 condensates requires ubiquitin and occurs independently of the LC3‐lipidation machinery

Abstract

Autophagy is a tightly controlled cellular recycling process that requires a host of autophagy machinery to form a double membraned vesicle called the autophagosome. This process is most understood in the context of stress‐induced autophagy, with little known about autophagosome biogenesis in basal (nutrient replete) conditions. To understand the regulation of basal autophagy, our work has focused on the poorly understood protein ATG9A, a multi‐pass transmembrane lipid scramblase that is essential for basal autophagy. To broadly understand the role ATG9A plays in basal autophagy, we utilized a quantitative proteome‐level MS/MS approach to measure how ATG9A affects protein flux. We show that loss of ATG9A in basal conditions impairs the degradation of autophagy adaptors, particularly p62/SQSTM1. Using a panel of ATG knock‐out cells, we demonstrate that the lipid transferase proteins ATG2A, ATG2B, and ATG9A promote the basal autophagic turnover of p62 and TAX1BP1 over other autophagy adaptors and do so independently of the LC3‐lipidation machinery. Furthermore, we demonstrate that ATG2A and ATG9A lipid transferase activity regulates the rate of p62 condensate degradation. Finally, we show in CRISPR knock‐in cell lines that ubiquitin is required for recruiting ATG9A to p62 condensates. Taken together, our data suggest that the lipid transferase activity of ATG9A and ATG2A is vital to basal autophagic regulation of protein homeostasis, and that ubiquitination is an apical signal that initiates recruitment of ATG9A to p62 condensates.