We identified a mammalian ortholog of yeast Atg13, and found that mammalian Atg13 is incorporated into the ~3-MDa protein complex together with ULK1 and FIP200. Furthermore, we showed that mTORC1 also interacts with this huge complex in a nutrient dependent manner.
This and previous studies (Young et al., 2006
; Chan et al., 2007
; Hara et al., 2008
) demonstrated that each component of the ULK1–Atg13–FIP200 complex is essential for autophagy, likely for autophagosome formation. In the present study, we observed accumulation of LC3-II in Atg13 siRNA-treated cells (A). We cannot rule out the possibility that the effect of Atg13 siRNA was not sufficient to completely inhibit LC3 conversion. However, we have observed similar phenomenon in FIP200−/−
cells (Hara et al., 2008
). The amount of LC3-II was increased during starvation in these cells, which was not further increased by lysosome inhibitor treatment. Accumulation of LC3-II was also observed in Atg14 and Vps34 siRNA-treated cells (Itakura et al., 2008
) and Beclin 1 siRNA-treated cells (Zeng et al., 2006
). Even in yeast, it is known that neither the Atg1 complex nor the Atg14-PI3K complex is essential for Atg8-PE formation (Suzuki et al., 2001
; Obara et al., 2008
). We speculate that inhibition of these Atg proteins may cause ectopic accumulation of LC3-II on unknown aberrant membranes, not on autophagosome.
Although (m)TOR is known to be a main regulator of autophagy, how (m)TOR regulates the autophagy factors has not yet been determined. We showed that mTORC1 directly interacts with ULK1 and phosphorylates ULK1 and Atg13. These results were also independently found by another group (Jung et al., 2009
). Furthermore, Tor-dependent regulation/phosphorylation of Atg1 and Atg13 was also demonstrated in Drosophila
(Chang and Neufeld, 2009
). We could not find in the ULK1 and Atg13 sequences a classic TOR signaling (TOS) motif, which is thought to be a potential recognition sequence by raptor. However, raptor recognition sequences may not be as strictly conserved as previously predicted (Oshiro et al., 2007
). Importantly, mTORC1-ULK1 binding and ULK1 phosphorylation were rapidly accelerated by nutrient replenishment. We propose that mTORC1 suppresses autophagy through direct interaction with the ~3-MDa ULK1–Atg13–FIP200 complex. We do not think that this is the only regulatory mechanism of autophagy because it has also been reported that there is an mTOR-independent pathway for autophagy induction (Sarkar et al., 2005
), and the effect of starvation is larger than rapamycin treatment (Supplemental Figure S3). Determining whether this ~3-MDa autophagy complex can also be regulated by factors other than mTORC1 is therefore an important future issue.
The mammalian ULK1–Atg13–FIP200 complex shares common features with the yeast Atg1–Atg13–Atg17 complex. Association with each component appears to be interdependent. In yeast, Atg1–Atg17 interaction largely depends on Atg13 (Cheong et al., 2005
; Kabeya et al., 2005
). In the present article, we showed similar interdependency among the mammalian factors; FIP200 and Atg13 are important for ULK1-Atg13 and ULK1-FIP200 interactions, respectively (, B and E). In addition, the C-terminal region of ULK1 is important for binding to Atg13 (E and Supplemental Figure S4; Chan et al., 2009
) and FIP200 (Hara et al., 2008
). In yeast, the C-terminal region of Atg1 is also known to be required for interaction with Atg13 (Cheong et al., 2008
). Because of these similarities, we again propose that FIP200 could be a true mammalian counterpart of yeast Atg17, although it is possible that FIP200 has additional functions similar to those of other Atg17-interacting proteins such as Atg11, Atg29, and Atg31 because counterparts of these factors have not yet been identified in mammals.
However, there are several apparent differences between the mammalian and yeast complexes. In yeast, the Atg1–Atg13 interaction is enhanced during starvation, whereas it is stable in mammals. Yeast Atg13 is dramatically dephosphorylated during starvation, whereas this event is only modest in mammalian cells. More importantly, Atg13 is thought to be an upstream factor of Atg1 in yeast for the following reasons: 1) overexpression of Atg1 partially restores the autophagy-defective phenotype of the atg13
mutant (Funakoshi et al., 1997
), 2) Atg13 is important for Atg1 kinase activity (Kamada et al., 2000
), and 3) PAS targeting of Atg1 is mildly affected in Δatg13
, whereas in the Δatg1
mutant PAS targeting of Atg13 is enhanced (Suzuki et al., 2007
). However, we demonstrated in this study that mTORC1 directly binds and phosphorylates ULK1 and that raptor-ULK1 interaction is not mediated by Atg13. We therefore postulate that Atg13 might function as a regulator in the complex, rather than a signal mediator between (m)TOR and Atg1/ULK1, although we do not rule out the possibility that the signaling mechanisms of yeast and mammalian cells are different. Further analysis of the function of the ULK1–Atg13–FIP200 complex will provide more insight into the link between autophagy regulation and autophagosome formation.