We have discovered a previously unrecognized autophagy-inducing condition, which is referred to as NNS-induced autophagy. The observation of NNS-induced autophagy implies that, besides severe nitrogen or carbon starvation, autophagy can also be regulated by relatively more subtle changes in medium composition and cellular metabolic state. The nature of these metabolic changes and how they are sensed by the autophagy machinery remain unknown and will be an area for future investigation.
Importantly, NNS-induced autophagy plays a critical role in maintaining cellular homeostasis upon changes in medium composition and metabolic state, because blocking NNS-induced autophagy by disruption of the autophagy core machinery leads to a severe growth phenotype upon switch to SL medium. Thus NNS-induced autophagy might be important for enabling cells to rapidly adapt to changing growth environments.
Another interesting feature of NNS-induced autophagy is that such autophagy is carbon source-dependent, unlike starvation-induced autophagy. Autophagy is not induced upon switch to SD medium, in which glucose is the carbon source (). Consistently, glucose addition to SL medium completely blocked the induction of autophagy upon switch to SL (Supplemental Figure S9). These results imply that NNS-induced autophagy is strictly controlled by cellular metabolic state and may be subject to glucose repression. For example, the up-regulation of mitochondrial respiratory function triggered by lactate, a nonfermentable carbon source, might be essential for NNS-induced autophagy. Alternatively, the abundance of a rich, fermentable carbon source such as glucose may repress the pathways that regulate NNS-induced autophagy. On the basis of these observations, we predict that studies of NNS-induced autophagy will improve our understanding of how the induction of autophagy is intimately coordinated with metabolism.
In addition, we propose that prototrophic strains of S. cerevisiae
, such as CEN.PK, will be valuable for the study of the metabolic regulation of autophagy. Such prototrophic strains do not have genomic lesions that disrupt the biosynthetic pathways for certain essential metabolites, such as amino acids and nucleotides, which could impact the regulation of autophagy and other metabolic processes (Boer et al., 2008
; Tu, 2010
; Tu et al., 2005
). Therefore the metabolic behavior of prototrophic cells is presumably more realistic and representative of wild yeast cells.
By conducting a visual screen designed based on these considerations, we have identified a protein complex, termed “the Iml1p complex,” which is selectively required for NNS-induced autophagy and regulates autophagosome formation. Thus our study has revealed the existence of proteins that specifically regulate NNS-induced autophagy as opposed to nitrogen-starvation-induced autophagy. We propose that the Iml1p complex might be evolutionarily conserved for three reasons. First, all three components of the complex—Iml1p, Npr2p, and Npr3p—are predicted to have orthologues in higher eukaryotes. Second, the Npr2p-Npr3p interaction has previously been shown to be conserved in human cells (Neklesa and Davis, 2009
). Finally, we have demonstrated that Iml1p contains an evolutionarily conserved domain (RANS domain), which is not only important for the formation of the Iml1p complex but also critical for NNS-induced autophagy.
Interestingly, the dephosphorylation of Atg13p indicates that TORC1 might be inactivated upon switch from the rich YPL medium to minimal SL medium (). Our identification of additional factors that are selectively involved in the regulation of NNS-induced autophagy suggests, however, that the molecular mechanisms underlying NNS-induced autophagy might be distinct from those regulating nitrogen-starvation-induced autophagy. Moreover, our findings suggest the possibility that the upstream signals that feed into TORC1 might be distinct under these two autophagy-inducing conditions, assuming that NNS-induced autophagy functions through TORC1. Because disruption of the Iml1p complex has no effect on rapamycin-induced autophagy (Supplemental Figure S5), we speculate that the Iml1p complex does not function at steps downstream of TORC1. Moreover, the failure of the Iml1p complex to regulate Atg13p phosphorylation implies that the Iml1p complex does not regulate TORC1 activity (). Therefore our study suggests that additional mechanisms could differentially regulate autophagy depending on the inducing stimulus. Future studies will be required to identify the upstream signals that trigger NNS-induced autophagy and to elucidate how the Iml1p complex regulates NNS-induced autophagy.