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A substrate localization model for the selective regulation of TORC1 downstream pathways

ABSTRACT Target of rapamycin complex 1 (TORC1) is a protein kinase complex conserved in eukaryotes that coordinates diverse cellular processes critical for cell growth to environmental conditions. Previous studies have shown that TORC1 is localized mainly in the lysosome/vacuoles, and its localization is important for signaling to downstream pathways. We recently demonstrated that signaling to Sch9, an S6K-related substrate of TORC1 in budding yeast, was selectively suppressed upon oxidative stress, which was mediated by the delocalization of phosphatidylinositol 3, 5-bisphosphate (PI[3,5]P2) from vacuolar membranes following stress. We propose that TORC1 downstream pathways can be regulated separately via the modulation of organelle localization of a specific target protein.

Target of rapamycin complex 1 (TORC1) is an evolutionarily conserved protein kinase complex in eukaryotes which, acting as a signaling hub, contributes to the coordination of cellular processes critical for cell growth, such as protein and lipid synthesis, or uptake and recycling of amino acids [1]. A variety of environmental cues affect TORC1 activity and thereby its downstream processes, including nutrient levels, growth factors, and cellular stresses [2]. Recent lines of evidence have shown the importance of TORC1 subcellular localization on endomembrane structures, such as lysosomes or vacuoles, in the regulation of its activity. In mammals, TORC1 is activated by localization on the cytoplasmic surfaces of lysosomes via the Ragulator-Rag complex, which senses amino acid availability [3].
The budding yeast Saccharomyces cerevisiae is a model organism suitable for the molecular dissection of TORC1 regulation. Rapamycin is a drug that selectively inhibits TORC1 activity, and screening for mutants showing different sensitivities to rapamycin enriched those affecting TORC1-dependent processes. Indeed, TOR1/2, the catalytic components of TORC1, were identified genetically in this organism [4]. In yeast, components of the Ego-Gtr (Ragulator-Rag) complex are largely conserved and localized to vacuoles, but TORC1 remains associated with the vacuolar membranes upon inactivation [5][6][7]. The close relationship between the regulation of TORC1 activity and its lysosomal/vacuolar localization suggests that intracellular amino acid levels may be sensed somewhere near the lysosome/vacuole, because these organelles serve as a storage site for amino acids.
Genetic studies in yeast have identified roles for TORC1 in the regulation of multiple physiological functions through downstream signaling branches, each of which is involved in proteosynthesis, the stress response, nitrogen metabolism, uptake of nitrogen sources, and autophagy, among others. When yeast cells were subjected to stresses except for nutrient starvation, such as hyperosmolarity, oxidative stress, or heat shock, the downstream branches of yeast TORC1 do not always respond uniformly to changes in the environment [8,9]. These observations indicate that TORC1 downstream pathways can be regulated separately.
One possible mechanism for the selectivity of downstream signaling is the difference in inherent quality of each phosphorylation site as a TORC1 substrate [10]. In this 'substrate quality' model, the downstream effectors of TORC1 respond differentially by temporal and intensity alterations in TORC1 kinase activity according to changes in the levels of nutrients and growth factors. Such differential responses would serve to prioritize and/ or coordinate the diverse functions of TORC1 in each situation. Although the difference in substrate quality to a certain kinase is useful for the hierarchical regulation of downstream functions, it may be difficult to explain the mechanism underlying reciprocal regulation. For example, the Ego complex is strictly required for normal activity of a branch involved in protein phosphatase 2A (PP2A) but is less critical for the Sch9 branch of TORC1 downstream signaling, whereas deletion of the HOPS complex severely reduces the activity of the Sch9 branch without affecting that of PP2A [9].
In a recent study [9], we demonstrated that the change in substrate localization also contributes to the specific regulation of TORC1 downstream pathways (Fig. 1). Sch9 is a direct target of TORC1 [11], and signal transduction between TORC1 and Sch9 requires vacuolar localization of Sch9, which is mediated by the PI(3,5) P 2 -binding C2 domain of Sch9 [12]. Following oxidative stress, the PI(3,5)P 2 content in vacuolar membranes is reduced, suppressing the Sch9 branch, despite the fact that other TORC1 downstream pathways are scarcely affected. Furthermore, the artificial tethering of Sch9 to vacuoles by other lipid-binding domains recovered TORC1-Sch9 signaling. Although incomplete rescue of Sch9 phosphorylation by tethering implies that TORC1 activity itself may be repressed to some extent, we concluded that TORC1 signaling to the Sch9 branch is regulated by the change in Sch9 localization upon oxidative stress (Fig. 2). Because inhibition of the Sch9 branch causes the activation of Rim15 and Sod2 [13,14], the physiological importance of the selective suppression of Sch9 under oxidative stress would be to confer resistance to this type of stress. Moreover, the change in distribution, but not the amount, of PI(3,5)P 2 underlies the regulation, suggesting that spatiotemporal alterations in the localization of inositol phospholipids contribute to the selective suppression of TORC1 downstream pathways.
Our results revealed that the phosphorylation of Sch9 by TORC1 exclusively requires the vacuolar localization of Sch9, which may be because Sch9 has less affinity to TORC1 than the other TORC1 substrates (Fig. 2). Indeed, Atg13 is a direct target of TORC1 [15] but is not enriched in vacuoles [9], and its phosphorylation by TORC1 is less affected by vacuolar integrity [9]. The relocation of TORC1 and its associated proteins has been described upon other types of stresses; TORC1 is accumulated in stress granules after heat stress [16] or is sequestered to inhibited domains near vacuoles after glucose withdrawal [17]. In addition to these observations, our study demonstrated that TORC1 signaling is regulated by the relocation of a specific target protein, Sch9. To date, studies on TORC1 have not addressed substrate dynamics; future studies should focus on differences in the substrates to elucidate the regulation of TORC1 signaling in vivo.
The reversible phosphorylation of phosphatidylinositol is critical for the determination of organelle identity and regulates many biological processes [18,19]. Among the phosphatidylinositol phospholipids, PI(3,5)P 2 is less abundant in yeast, and the level of this phospholipid increases transiently in response to hyperosmotic stress [20]. We demonstrated that the delocalization of PI(3,5) P 2 from vacuoles following oxidative stress is primarily due to the loss of Fab1, the sole PI3P 5-kinase in yeast, from the vacuolar membrane [9]. Fab1 associates with vacuoles through its PI3P-binding FYVE domain, indicating that phosphatidylinositol dynamics within cells is altered or perturbed during the stress response. The mechanism remains unclear at present: the synthesis and/or transfer of specific lipid molecules may be regulated, actively or passively, by cellular stresses; or a physiological change in cells may induce the delocalization of a specific lipid according to its physicochemical nature [21].
Lastly, our results highlight the strategy used by cells to cope with a variety of stressors. Our finding that the signal from TORC1 to the Sch9 branch is notably more sensitive to stress signals due to the localization of Sch9  suggests that localization in concert with TORC1 activity effect a more nuanced mode of regulation. In practice, this means that Sch9 can be blocked more easily than alternative pathways. In mammalian cells, the signal from TORC1 to the translational regulator S6K1, the functional homolog of Sch9, decreases during even mild amino acid starvation, but the TORC1 signal to ULK1, which modulates autophagy, declines only under conditions of severe starvation [10]. Therefore, it appears that cells employ a systematic approach in TORC1 signaling, dynamically prioritizing the regulation of TORC1dependent branches in response to stress, with the inhibition of translation being the immediate top priority. The localization-dependent control of a TORC1 substrate in budding yeast may form one element of such a regulatory system.

Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.