Macroautophagy (hereafter referred to as autophagy) is the bulk degradation and recycling of cytoplasmic materials by forming a double membrane vesicle called the autophagosome. Autophagosome formation is regulated by the coordinated action of a set of proteins. ATG9A is the only multispanning transmembrane protein that plays an essential role in autophagosome formation, yet its function is largely elusive. Previous studies have shown that the C-terminus of ATG9A plays an important role in regulating its trafficking and proper function in autophagy. In line with that idea, we previously identified an AMPK- and ULK1- mediated phosphorylation on the C terminus of ATG9A at S761, which is required for proper ATG9A trafficking and autophagic flux. In our current study, we employed a BioID-based proteomics approach and identified a network of ATG9A C terminal interactors that include members of the ULK1 complex, ATG13, and ATG101, as well as protein complexes within the ER, Golgi, and endosomal trafficking pathways, many of which provide new insight into ATG9A trafficking mechanisms. We discovered that ATG9A exists with ATG13 and ATG101 in a separate subcomplex outside the canonical ULK1 complex. We show that the ATG13-ATG101 subcomplex regulates ATG9A trafficking and basal P62 degradation.

L’autophagie est un mécanisme cellulaire complexe, nécessitant plus de 40 protéines ATGs (AuTophaGy related), impliqué dans le maintien de l’homéostasie cellulaire. Sa dérégulation a été décrite comme une cause possible de la tumorigénèse. Nos travaux ont montré dans une cohorte de 80 patientes atteintes de cancer du sein, que l’expression du gène codant la protéine ATG9A, jouant un rôle dans les étapes précoces de l’autophagie, est plus importante dans les tissus cancéreux des patientes de type triple négatif. Afin d’étudier le rôle d’ATG9A dans la lignée de cancer du sein triple négatif MDA-MB-436, nous avons développé deux modèles d’extinction du gène ATG9A à l’aide de sh-ARN ou de la technique CRISPR-Cas9. Ces modèles d’extinction présentent un blocage de l’autophagie via une diminution de la dégradation des autophagosomes. Nous avons également montré une inhibition des phénotypes cancéreux in vitro et in vivo des cellules sh-ATG9A comparé aux cellules contrôles. Cependant, nous n’avons observé aucune différence de phénotypes cancéreux entre le modèle CRISPR-Cas9, contrairement au modèle sh-RNA, nous avons émis l’hypothèse que l’ARNm d’ATG9A pourrait jouer un rôle dans la maintenance des phénotypes cancéreux via l’expression d’une isoforme de la protéine ATG9A, exprimée après mutation de la séquence d’ATG9A par le système CRISPR-Cas9 ou via son interaction avec des ARN non codants régulateurs. Si cette hypothèse est confirmée, cet ARNm pourrait devenir une cible thérapeutique dans les cancers du sein triple négatif pour lesquels aucune thérapie ciblée n’existe actuellement. / Autophagy is an intracellular process which contributes to the maintenance of cell homeostasis. The deregulation of this complex process, which requires more than 40 ATG proteins, has been shown to be involved in tumor development. In our laboratory, we analyzed a cohort of 80 breast cancers and demonstrated that ATG9A gene expression is increased in triple negative breast cancer samples compared to adjacent healthy tissues. We then studied the role of ATG9A in the triple negative breast cancer cell line MDA-MB-436 using two extinction models created with the sh-RNA or the CRISPR-Cas9 technology. Our two extinction models presented a blockade of autophagy, due to a decrease of autophagosome degradation. We also observed a decrease of in vitro and in vivo cancer phenotypes, such as proliferation, invasion or in vivo tumor growth, of sh-ATG9A cells compared to control cells. However, we did not observe any difference of cancer phenotypes between the CRISPR-CAS9 cells and the control ones. Since we still detected the presence of the ATG9A mRNA in the CRISPR models but not in the sh-RNA models, we hypothesized that this mRNA might play a role in the maintenance of breast cancer phenotypes in these cells, either by the expression of a truncated isoform of the ATG9A protein from the mutated ATG9A mRNA obtained after the action of the CRISPR-Cas9 system, or its interaction with non-coding mRNAs. If proven, this could establish ATG9A mRNA as a potential therapeutic target in triple negative breast cancers for which no targeted therapy is currently available.

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

Autophagy, as the name suggests, is a cellular process of self-eating in which cytoplasmic debris is engulfed by a double membrane vesicle dubbed the autophagosome and is ultimately degraded and recycled by proteases in the lysosome. The process is initiated by a group of core ATG proteins, including a multi-pass transmembrane protein called ATG9A. Although ATG9A has been shown to be essential for both stress induced and basal autophagy, its mechanism and interaction network remain largely illusive. Our current study employs BioID proteomics to identify a network of interactors, including regulators of membrane fusion and vesicle trafficking, such as TRAPP, EARP, GARP, exocyst, AP-1 and AP-4 complexes, as well as members of the ULK1 autophagy kinase complex. Further investigations confirm that two components of the ULK1 complex, ATG13 and ATG101, directly interact with ATG9A. Using CRISPR, we show that deletion of ATG13 or ATG101 disrupts ATG9A trafficking and causes an accumulation of ATG9A at p62/SQSTM1-positive ubiquitin clusters. Lentivirus reconstitution and split-mVenus approaches using an ULK1 binding deficient mutant of ATG13 reveal that ATG9A interacts with ATG13 and ATG101 in an ULK1-independent manner. Together, these data reveal ATG9A interactions in vesicle trafficking and autophagy pathways, including a role for an ULK1- independent ATG13 complex in regulating ATG9A.

14-3-3 proteins are among a family of phospho-binding proteins that are known to regulate many essential cellular mechanisms. By binding to sites of phosphorylation, 14-3-3s are integrated into multiple signaling pathways that govern critical processes, such as apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. These processes are crucial for tumorigenesis and 14-3-3 proteins are known to play a central role in facilitating cancer progression. In this study, my colleagues and I discover two novel 14-3-3 interacting proteins, ATG9A and PTOV1, that are both vital to essential cellular functions and describe various mechanisms that these two proteins regulate. ATG9A is a multi-pass transmembrane lipid scramblase that is found primarily as a homotrimer in the ER or small ATG9A vesicles. It is essential in the cellular recycling process called autophagy and is believed to act at the earliest stages of autophagy by providing the seed for the growth of the double membrane vesicle called an autophagosome. Previous work in our lab demonstrated that upon hypoxic stress, AMPK, the master nutrient-sensing kinase, phosphorylates S761 on the C-terminus of ATG9A. This triggers the binding of 14-3-3ζ to contribute to ATG9A function in hypoxia induced autophagy. Despite this revelation, the exact function of ATG9A is still poorly understood, especially in unstimulated conditions where autophagy functions at a basal level and AMPK is inactive. In this study, we sought to understand ATG9A function more broadly by identifying novel interactors of ATG9A and the role ATG9A plays in basal autophagy. To do this, we employed BioID mass spectrometry and various biochemical approaches to identify LRBA as a bona fide ATG9A interactor and autophagy regulator. Furthermore, using deuterium labeling and quantitative whole proteome mass spectrometry, and various other biochemical techniques, we show that ATG9A regulates the basal degradation of p62 and is recruited to sites of basal autophagy by active poly-ubiquitination to initiate basal autophagy. PTOV1 is an oncogenic protein that is poorly understood. Our current understanding of PTOV1 is limited to a few studies, which demonstrate that PTOV1 is highly expressed in primary prostate tumor samples and is correlated with metastasis, drug resistance, and poor clinical outcomes. In this study, we identify a mechanism by which SGK2, a poorly understood kinase, phosphorylates PTOV1 at S36 to trigger 14-3-3 binding at that site to increase PTOV1 stability in the cytosol and increase c-Jun expression. Upon SGK2 inhibition, 14-3-3 releases PTOV1 and PTOV1 is shuttled into the nucleus where HUWE1, an E3 ubiquitin ligase, ubiquitinates PTOV1 and initiates PTOV1 degradation by the proteasome. This is the first detailed mechanism of regulation identified for the poorly understood oncogene, PTOV1, and sheds light on potential therapeutic targets for cancer treatments.