Sorting out “non‐canonical” autophagy

Abstract

Canonically, LC3 lipidation has been associated with autophagy pathways but it becomes increasingly clear that this modification can also occur during autophagy‐unrelated processes. In this issue, Florey and colleagues find that the WD40 domain of ATG16L1 is dispensable for LC3 lipidation during starvation‐induced autophagy but required for its lipidation during several other membrane‐based processes that are different from autophagy. This finding opens the door for the analysis of the functions of LC3 lipidation in these pathways.

Macroautophagy (hereafter autophagy) is a process for the delivery of intracellular material, referred to as cargo, into the lysosome for degradation. Autophagy can be triggered by starvation or the presence of intracellular cargoes and entails the de novo formation of a double‐membrane organelle termed autophagosome, within which the cargo is sequestered. During the canonical form of autophagy, a number of conserved AuTophaGy (ATG)‐related proteins act in a hierarchical manner to mediate the formation of autophagosomes. A hallmark of this process is the covalent attachment of ATG8 family proteins (here referred to as LC3) to the membrane lipid phosphatidylethanolamine (PE) on the nascent autophagosomal membrane. This lipidation reaction occurs analogous to the attachment of ubiquitin to target proteins and is mediated by the E1‐like enzyme ATG7 and the E2‐like enzyme ATG3. In addition, a protein complex composed of the ATG5, ATG12, and ATG16L1 proteins, the so‐called ATG12~ATG5‐ATG16L1 complex, acts in an E3‐like manner to promote the conjugation of LC3 to PE. The complex also specifies the site of LC3 lipidation (Fujita et al, 2008). LC3 lipidation, its subsequent membrane localization, and final degradation in the lysosome have long been used to assess autophagic activity (Kabeya et al, 2000). The interpretation of these results was based on the assumption that LC3 lipidation would only occur during autophagosome formation. However, it has become evident over the years that LC3 lipidation also occurs during processes that are unrelated to autophagy. For example, it was found that LC3 can be attached to the plasma membrane during several forms of phagocytosis, micropinocytosis, or viral infections. It was also reported that LC3 can be conjugated to endo‐lysosomal membranes (Florey et al, 2015). Many of these processes involving LC3 lipidation were often referred to as “non‐canonical” autophagy even though they are not autophagic pathways since they do not mediate the delivery of intracellular material into lysosomes (Galluzzi et al, 2017). Furthermore, in these pathways, LC3 conjugation occurs to single membranes and this membrane‐attached LC3 is not delivered into the lysosomal lumen. The function of the membrane‐attached LC3 in these “non‐canonical” pathways is in many cases not understood.

The upstream autophagy machinery including the ULK1 complex, the class III PI3KC1, and ATG9 is essential for canonical autophagy but not for most of the other processes entailing LC3 lipidation. In contrast, the current evidence suggests that LC3 lipidation universally requires the same conjugation machinery composed of ATG3, ATG7, and the ATG12~ATG5‐ATG16L1 complex. This has made it difficult to study the role of LC3 conjugation in the “non‐canonical” lipidation events since deletion of components of the conjugation machinery abolishes all forms of LC3 conjugation. In this issue of The EMBO Journal, Florey, Fletcher, Ulferts, and colleagues present a tool that may enable scientists to sort out the induction and role of LC3 lipidation during autophagy and autophagy‐unrelated processes (Fletcher et al, 2018). It has also the potential to allow the investigation of what the specific function and mechanism of action of LC3 lipidation during the autophagy‐unrelated pathways actually is.

In particular, Fletcher et al (2018) study the role of the ATG16L1 protein during LC3 conjugation. ATG16L1 contains multiple domains and protein interaction motifs. Its conserved N‐terminal domain binds to ATG5. This domain is followed by a coiled‐coil domain, a region binding FIP200 and WIPI2 called FBD (Gammoh et al, 2013; Dooley et al, 2014), and finally a C‐terminal WD40 domain. The latter two domains are, unlike the ATG5‐binding and the coiled‐coil domains, not present in Saccharomyces cerevisiae. The authors followed the localization of ATG16L1 upon the induction of multiple autophagy‐unrelated processes that are associated with LC3 lipidation. These included several forms of phagocytosis and treatment with the sodium/proton ionophore monensin, which promotes LC3 lipidation to endo‐lysosomal compartments (Jacquin et al, 2017). ATG16L1 localized to these LC3‐positive membranes in a manner that did not depend on PI3P and its effector WIPI2 indicating that it is autophagy independent. The authors then went on to dissect the domain requirements of ATG16L1 for this membrane recruitment. Employing stable cell lines, the authors could show that while the central FIP200 binding domain (FBD) is dispensable for its recruitment to the plasma membrane and endo‐lysosomal membranes, the C‐terminal WD40 domain is essential for the membrane localization of ATG16L1 during autophagy‐unrelated processes. In contrast, the recruitment of ATG16L1 during starvation‐induced autophagy required the FBD but not the WD40 domain. The loss of membrane recruitment of ATG16L1 correlated with a loss of LC3 lipidation. Individual residues in the WD40 domain were shown to be required for membrane localization of ATG16L1 and LC3 lipidation but the factors that mediate ATG16L1 recruitment remain to be identified, although TMEM59 and TMEM166/EVA1 could be involved (Boada‐Romero et al, 2013; Hu et al, 2016). Employing the truncated ATG16L1 lacking the WD40 domain, the authors went on to analyze the function of the ATG16L1 recruitment and LC3 lipidation in autophagy‐unrelated processes. They were able to demonstrate autophagy‐independent LC3 lipidation during influenza A virus infection. Finally, establishing a mouse model lacking the C‐terminal WD40 domain of ATG16L1, they detected a reduction of MHC class II antigen presentation in dendritic cells.

Figure 1. Model for the recruitment of ATG16L1 to membranes.

(Left) Induction of LC3 lipidation during autophagy depends on the ULK1 complex subunit FIP200 and WIPI2, which recruit ATG16L1 to the site of autophagosome formation via the FBD. LC3 becomes conjugated to the isolation membrane that will eventually give rise to a double‐membrane autophagosome. (Right) During autophagy‐unrelated, non‐canonical pathways involving LC3 lipidation, ATG16L1 is recruited via its C‐terminal WD40 domain and promotes the lipidation of LC3 to single membranes.

In summary, the study by Florey and colleagues opens the door for the dissection of the role of LC3 lipidation during autophagy‐unrelated pathways by using the C‐terminally truncated ATG16L1 as a tool, although some caution should be exercised since the WD40 may be required for certain types of selective autophagy (Fujita et al, 2013). It becomes increasingly clear that the LC3 lipidation machinery can be used as plugin by several cellular pathways, but also that autophagy pathways may not absolutely depend on LC3 lipidation (Tsuboyama et al, 2016). Thus, many earlier studies employing LC3 lipidation as measure for autophagic activity may need to be reassessed. It will also be interesting to see whether any of the ATG4 proteins, which remove LC3 from the membrane, is preferentially involved in non‐autophagic processes. Clearly, a lot of fascinating work lies ahead.

See also: K Fletcher et al (February 2018)