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

Abstract

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 vesicletrafficking and autophagy pathways, including a role for an ULK1independent ATG13 complex in regulating ATG9A.

Synaptic vesicle proteins and ATG9A self-organize in distinct vesicle phases within synapsin condensates

“…We next investigated the relation of ATG9A to SVs. As previously described 31, 32 , when expressed alone, ATG9A-EGFP was enriched in the Golgi complex, but also localized in scattered puncta throughout the cytoplasm (Fig. 3a).…”

“…However, substantial differences between the two biotinylated proteomes were also detected. Consistent with previous studies, ATG9A samples were enriched in subunits of AP4 (an heterotetrameric complex responsible for the sorting of ATG9A at the TGN) 31,32,34 , in AP4 interactors (tepsin and rusc2) 32,34,35 , in subunits of the PIKfyve complex (a phosphoinositide metabolizing complex that that function at late endosomes/lysosomes) 36 as well as in the autophagy factors ULK1 and ATG13 37 (Fig. 5c).…”

Structural basis for ATG9A recruitment to the ULK1 complex in mitophagy initiation

“…Removing the WF finger of ATG101 (WF to DD) had no effect on ATG9A binding. Our mutational analysis suggests that the hydrophobic groove drives ATG9A binding and explains the need for both ATG101 and ATG13 in binding ATG9A in vivo (Kannangara et al 2021).…”

“…The ATG9A, ATG13 and ATG101 form a multifunctional hub at an early stage of starvation-induced autophagy and mitophagy initiation (Dalle Pezze et al 2021; Karanasios et al 2016; Zachari et al 2019). A BioID mass spectroscopy approach revealed that ATG13:ATG101 can recruit ATG9A during p62-dependent autophagy, independent of FIP200 and ULK1 (Kannangara et al 2021). Deletion of ATG13 or ATG101 led to a mislocalization of ATG9A, leading in turn to an accumulation of p62 aggregates identical to the ATG9A KO phenotype (Kannangara et al 2021; Saitoh et al 2009).…”

“…A BioID mass spectroscopy approach revealed that ATG13:ATG101 can recruit ATG9A during p62-dependent autophagy, independent of FIP200 and ULK1 (Kannangara et al 2021). Deletion of ATG13 or ATG101 led to a mislocalization of ATG9A, leading in turn to an accumulation of p62 aggregates identical to the ATG9A KO phenotype (Kannangara et al 2021; Saitoh et al 2009). This study highlighted the importance of the ATG9A, ATG13 and ATG101 nexus and focused our attention on this subnode of the autophagy interaction network.…”