BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters

ATG9A, the only multi-pass transmembrane protein among core ATG proteins, is an essential regulator of autophagy, yet its regulatory mechanisms and network of interactions are poorly understood. Through quantitative BioID proteomics, we identify a network of ATG9A interactions that includes members of the ULK1 complex and regulators of membrane fusion and vesicle trafficking, including the TRAPP, EARP, GARP, exocyst, AP-1, and AP-4 complexes. These interactions mark pathways of ATG9A trafficking through ER, Golgi, and endosomal systems. In exploring these data, we find that ATG9A interacts with components of the ULK1 complex, particularly ATG13 and ATG101. Using knockout/reconstitution and split-mVenus approaches to capture the ATG13-ATG101 dimer, we find that ATG9A interacts with ATG13-ATG101 independently of ULK1. Deletion of ATG13 or ATG101 causes a shift in ATG9A distribution, resulting in an aberrant accumulation of ATG9A at stalled clusters of p62/SQSTM1 and ubiquitin, which can be rescued by an ULK1 binding-deficient mutant of ATG13. Together, these data reveal ATG9A interactions in vesicle-trafficking and autophagy pathways, including a role for an ULK1-independent ATG13 complex in regulating ATG9A. Synopsis This study presents the ATG9A proximity interactome, revealing interactions with vesicle trafficking complexes, core autophagy regulators, and an ULK1-independent ATG13-ATG101 complex that promotes the basal turnover of ubiquitin-rich p62/SQSTM1 clusters. BioID reveals ATG9A interactions with trafficking regulators, including an ULK1-independent ATG13-ATG101 complex. Loss of ATG13 causes a build-up of ATG9A at stalled ubiquitin-rich p62/SQSTM1 clusters. Reconstitution of ATG13-deficient cells with an ULK1-binding defective ATG13 rescues ATG9A trafficking. Graphical Abstract This study presents the ATG9A proximity interactome, revealing interactions with vesicle trafficking complexes, core autophagy regulators, and an ULK1-independent ATG13-ATG101 complex that promotes the basal turnover of ubiquitin-rich p62/SQSTM1 clusters.