The remarkable conservation of autophagy-related proteins and processes between yeast and higher eukaryotes has contributed to a rapid advance in our understanding of this process in multicellular animals. The data presented here extend this conservation to the mechanisms of autophagy regulation by nutrient-dependent TOR signaling. As in yeast, Drosophila Atg13 forms a complex with Atg1, stimulates its activity and is highly phosphorylated in a nutrient and TOR-dependent manner. Although the relevant substrates of Atg1 remain to be identified, these results suggest that this fundamental pathway from nutrient signal to autophagic induction has been largely retained between yeast and metazoans. This regulatory link between TOR and Atg1/Atg13 thus represents one of the most highly conserved TOR outputs described to date.
Despite this conservation, we also identify critical differences in the behaviors of these proteins, which likely stem in part from differences in the physiology of lower versus higher eukaryotes. While this manuscript was under review, Chan et al. (2008)
reported an essential role in autophagy for human Atg13 using RNAi-mediated knockdown in cultured cell lines and described interactions between Atg13 and Ulk1 and Ulk2 similar to those reported here. In this issue of Molecular Biology of the Cell
, Hosokawa et al. (2009)
and Jung et al. (2009)
also describe related interactions between mammalian Atg13 and Ulk kinases. Together, these studies point to a model whereby Atg13 is phosphorylated and interacts with Atg1 under both fed and starved conditions in metazoans, in contrast to the growth-dependent phosphorylation and dissociation of Atg13-Atg1 observed in yeast. Although the greater nutrient buffering capacity of multi- versus unicellular animals probably contributes to these differences, the requirement for significant basal rates of autophagy in the maintenance of large, long-lived metazoan cells may dictate that the Atg1-Atg13 complex remain partially active even under fed conditions. Furthermore, although Atg1 can clearly associate with phosphorylated Atg13, dephosphorylation of TOR-dependent sites on Atg13 may be masked by increased phosphorylation by Atg1, but may nonetheless contribute to regulation of Atg1-Atg13 interaction or activity. The observation that Atg1 kinase activity plays a role under both fed and starved conditions in supporting hyperphosphorylation of Atg13 (Supplemental Figure S3C) suggests that rather than switching Atg1 kinase between off and on states, nutrient signals may instead affect its substrate specificity or accessibility.
Metazoan Atg1-Atg13 complexes have also accrued additional regulatory mechanisms that have not been described in yeast, including negative feedback from Atg1 to TOR, phosphorylation of Atg13 by Atg1, and nutrient-dependent effects on the stability of these proteins. These additional layers of regulation are in keeping with the elaboration of intracellular signaling pathways in metazoans. The observation that Atg13 phosphorylation is both TOR- and Atg1-dependent under fed conditions suggests a model whereby phosphorylation by one of these kinases may serve as a priming event for the other. In addition, the role of Atg1-dependent phosphorylation of Atg13 under starvation conditions remains an important question. Although the Atg1-independent localization of Atg13 to autophagosomes and its activation of Atg1 indicate that Atg13 functions upstream of Atg1, the finding that Atg13 acts as a substrate for Atg1-dependent phosphorylation raises the possibility that Atg13 may also act to transduce signals downstream of Atg1. Identification of additional Atg1 substrates and Atg13 interacting proteins may help clarify this issue.
Given its positive role in autophagy induction, the ability of Atg13 to inhibit autophagy when overexpressed was unexpected. This may in part reflect a dominant negative effect of Atg13 overexpression causing titration of Atg1 complexes or competition with other Atg1 substrates. However, the observation that Atg13 stimulates TOR-dependent phosphorylation of Atg1 and that Atg1 levels increase in Atg13
mutant cells suggests that Atg13 has both positive and negative roles in autophagy induction. These opposing activities of Atg13 are reminiscent of the mTOR complex I component Raptor, which plays an essential role in TOR signaling yet also inhibits TOR activity under starvation conditions (Kim et al., 2002
). Our results suggest that Atg13 may play an analogous role, switching between states of promoting or inhibiting autophagy, thereby sharpening the response to changes in nutrient conditions. These findings suggest that the relative ratio of Atg13 to Atg1 or to other components of the complex may play an important role in dictating its activity. In this regard, the differential regulation of Atg1 and Atg13 levels by TOR could provide an additional mechanism whereby TOR signaling can influence autophagic activity. Although the autophagy-defective phenotypes of Atg1
mutants reveals the dominant positive roles of these genes, disruption of other putative components of this complex leads to autophagy induction (unpublished data), suggesting some components may play primarily negative roles.
In conclusion, our results demonstrate that Atg1-Atg13 complexes play an essential, conserved role in promoting autophagy in response to TOR inactivation, with additional regulatory functions unique to metazoans. Further insight into the regulation of this complex and identification of its targets may lead to additional means of manipulating autophagy rates for therapeutic purposes.