In this study, we have elucidated the function of the Atg16L complex. This complex brings LC3 to the site of lipidation and catalyzes the final step of the reaction. On the basis of these results, we propose a dynamic localization model of LC3 lipidation (). On induction of autophagy, the Atg16L complex relocalizes from the cytosol to an undetermined membrane in an Atg16L-dependent manner. The high-energy Atg3-LC3 intermediate, which has been activated by Atg7, is recruited to the membrane via the interaction between Atg3 and Atg12 in the Atg16L complex. This brings LC3 into proximity with PE in the membrane, leading to subsequent lipidation.
Figure 7. The dynamic localization model of LC3 lipidation. On induction of autophagy, the Atg16L complex in the cytosol localizes on a yet undetermined membrane in an Atg16L-dependent manner. The high-energy Atg3-LC3 intermediate activated by Atg7 is recruited (more ...)
In vitro reconstitution experiments show that the minimum components necessary for Atg8/LC3 lipidation are Atg7, Atg3, Atg8/LC3, phospholipid-containing liposomes, and ATP (Ichimura et al., 2004
; Sou et al., 2006
). In the presence of Atg8/LC3 and PE at much higher concentrations than those seen in vivo, PE-containing micelles/vesicles and Atg8/LC3 will encounter each other at some frequency sufficient to support the lipidation reaction. The Atg12-Atg5 conjugate has been reported to facilitate the formation of Atg8-PE in vitro (Hanada et al., 2007
). Atg16 is necessary for efficient Atg8-PE formation in vivo; however, little additive effect of Atg16 on PE conjugation of Atg8 was observed in vitro (Hanada et al., 2007
). According to our model, the putative membrane factor that is recognized by Atg16 is probably not present on artificial liposomes. However, we show herein that Atg16L is directly involved in LC3 lipidation by specifying the site of the reaction. Based on both the in vitro and in vivo results, it is reasonable to propose that in vivo, the Atg16L complex functions as a scaffold on which LC3 is transferred from Atg3 to PE. In an analogy to the ubiquitination reaction, the complex may be regarded as an E3-like factor in the LC3 conjugation system in the sense that the E3 component plays a role in associating the substrate with the E2 enzyme (Suzuki et al., 2005
; Matsushita et al., 2007
). Generally, an E3 specifically recognizes its protein substrates; however, in this case, the recognition process may be attributed to the binding of the complex to the membrane. Thus, the Atg16L complex is a unique and new type of E3, which recognizes specific membranes as substrates. The mechanism by which LC3 is targeted to specific membranes has been mysterious, because PE is an abundant phospholipid that may be present in all membrane organelles, and unlipidated LC3 is a soluble protein. Our results show that membrane targeting of LC3 is defined by the Atg16L complex, and not some intrinsic targeting information within LC3. The in vivo target of LC3 is restricted to PE, but phosphatidylserine (PS) can be a target of LC3 in vitro (Sou et al., 2006
). The selectivity in vivo may be related to the membrane localization of the Atg16L complex.
Our results demonstrate that the site of Atg16L complex localization is the site of lipidation. This place is likely critical for autophagosome formation, because Atg16L complex forced to localize to the PM enables LC3 lipidation, but not autophagosome formation. The only known place where Atg5 (or Atg16L) localizes is the outer surface of the isolation membrane, the forming autophagosome in mammalian cells (Mizushima et al., 2001
). Therefore, the isolation membrane is the premier candidate for the location of LC3 lipidation. The riddle associated with this possibility is that recruitment of the Atg3-LC3 intermediate to forming autophagosomes does not explain how new lipid, including PE, is supplied. Recently, Nakatogawa et al. (2007)
showed that Atg8-PE is potent in causing the hemifusion of vesicles/micelles in vitro, and this property may be related to the membrane elongation step of autophagosome formation. An alternative possibility is that there may be an undetected, and probably transient, association of the Atg16L complex to a membrane structure that enables the lipidation of LC3. In this case, lipidated LC3 would be delivered to the autophagosome after lipidation, possibly together with the Atg16L complex. Such a membrane could be assigned as the source of autophagosomal membrane lipids. In the case of yeast, the PAS is such a candidate. Yeast genetic studies showing a reciprocal dependence of PAS localization of the Atg12 system and Atg8 can be explained by our model (Suzuki et al., 2007
). However, a membrane structure has not yet been detected at the PAS. Whether the PAS model is applicable to mammals is a further interesting question.
The PM-localized Atg16L complex bypasses two important factors that are normally critical for LC3 lipidation: starvation signaling and PI3K signaling (Kabeya et al., 2000
). This result indicates that the output of these signaling pathways is the targeting of the Atg16L complex to the source membrane. The simplest model is that the autophagy-specific PI3K complex, which includes Beclin-1, is activated by starvation, and the resulting phosphatidylinositol 3-phosphate recruits the Atg16L complex; however, another more complex mechanism may be involved. Most importantly, we showed that targeting of the Atg16L complex to the membrane is sufficient for LC3 lipidation. This indicates that targeting of the Atg16L complex is an important step in terms of the regulation of autophagosome formation.
We showed that overexpresssion of the Atg16L coiled-coil region affects only the localization of the Atg16L complex, but not its function as a lipidation catalyst. The mechanism of inhibition remains unknown, but we speculate that a factor associating with the Atg16L coiled-coil region is titrated out by the excess Atg16L. Such a factor would interact with the Atg16L complex and be necessary for its localization. We are now pursuing the identification of this factor, which must be key for autophagosome formation.
We have uncovered a direct functional linkage between the Atg12 and LC3 systems. The most important finding is that the Atg16L complex may specifically recognize the membrane origin of the autophagosome. Hence, the mechanism of membrane localization of the Atg16L complex is key to understanding membrane dynamics in autophagy. By pursuing the details, we are approaching the core of this longstanding fundamental question in autophagy.
Crohn's disease is a common form of chronic inflammatory bowel disease of unknown etiology (Travis et al., 2006
). Recently, an association between Crohn's disease and variant Atg16L has been reported, although the mechanisms by which the variant predisposes to intestinal inflammation is unknown (Consortium TWTCC, 2007
; Hampe et al., 2007
; Rioux et al., 2007
). Our findings may be an important step toward understanding this disease, by providing information about the precise function of Atg16L and the regulation of autophagy via Atg16L.