ATG16L2 binds the complex ATG5:ATG12, as originally shown in a mouse model system (Ishibashi et al. 2011) and subsequently confirmed with human proteins (Wible et al. 2019). Formation of the ATG16L2:ATG5:ATG12 complex interferes with the formation of the ATG16L1:ATG5:ATG12 complex and the subsequent formation of autophagosomes (Ishibashi et al. 2011, Wible et al. 2019, reviewed in Don Wai Luu et al. 2022). Knockdown of ATG16L2 in human neuroblastoma cell line H4 increases autophagy flux, corroborating the role of ATG16L2 as a negative regulator of autophagy (Lipinski et a. 2010).

The role of ATG16L2 in the regulation of autophagy was reported to be tissue-specific (Khor et al. 2019) and may differ under resting and stress conditions (Wang et al. 2022). Atg16l2 knockout mice are viable (Khor et al. 2019). In conditionally Atg16l1-knockout mouse T lymphocytes, the knockout of Atg16l2 does not exacerbate the Atg16l1-knockout phenotype (Khor et al. 2019). In the intestinal epithelium, the conditional knockout phenotype of Atg16l1 is reversed by the knockout of Atg16l2 (Khor et al. 2019). Knockout of Atg16l2 alone alters the cellular architecture of mouse intestinal epithelium, causing a significant decrease in the number of Paneth cells, which are known to secrete antimicrobials and function in intestinal barrier maintenance (Khor et al. 2019).

Besides its role in autophagy, ATG16L2 is also implicated in the innate immune response to intracellular pathogens where it was shown to regulate the activation of NLRP3 inflammasomes (Wang et al. 2022) and NLRC4 inflammasomes (Wen et al. 2024).