TORC1 organized in inhibited domains (TOROIDs) regulate TORC1 activity

Abstract

The Target of Rapamycin (TOR) is a eukaryotic serine/threonine protein kinase that functions in two distinct complexes, TORC1 and TORC2, to regulate growth and metabolism1,2. GTPases, responding to signals generated by abiotic stressors, nutrients, and, in metazoans, growth factors, play an important3, but poorly understood role in TORC1 regulation. Here, we report that, in budding yeast, glucose withdrawal, which leads to an acute loss of TORC1 kinase activity4, triggers a similarly rapid Rag GTPase-dependent redistribution of TORC1 from being semi-uniform around the vacuolar membrane to a single, vacuole-associated cylindrical structure visible by super-resolution optical microscopy. 3D reconstructions of cryo-electron micrograph images of these purified cylinders demonstrate that TORC1 oligomerizes into a higher-level hollow helical assembly which we name a TOROID (TORC1 Organised in Inhibited Domain). Fitting of the recently described mammalian TORC1 structure into our helical map revealed that oligomerisation leads to steric occlusion of the active site. Guided by the implications from our reconstruction, we present a TOR1 allele that prevents both TOROID formation and TORC1 inactivation in response to glucose withdrawal demonstrating that oligomerisation is necessary for TORC1 inactivation. Our results reveal a novel mechanism by which Rag-GTPases regulate TORC1 activity and suggest that the reversible assembly/disassembly of higher-level structure may be a new paradigm for the regulation of protein kinases.

Reorganization of budding yeast cytoplasm upon energy depletion

“…Filaments were mostly aligned in regular rows in the bundle, with a between-row spacing of ~13 nm and within-row spacing of ~26 nm ( Figure 5C; Figure 6A). Diameter, periodicity and spacing of eIF2B filaments in the bundle differed from the ones of previously characterized enzymatic polymers, including those made of other metabolic enzymes such as CtpS, Gln1 and TORC1 (Barry et al, 2014;Petrovska et al, 2014;Prouteau et al, 2017). In stressed cells, we also observed filaments with a similar morphology and subunit repeat (~6 nm) to the ones described for Gln1 (Petrovska et al, 2014), with smaller diameter, shorter between-filament spacing, and smoother longitudinal striations than eIF2B filaments ( Figure 3, F and H -white arrows).…”

“…This condition lasted until replenishment of ATP, when the fluorescence signal promptly diffused again, and cells quickly re-entered the cell cycle (Supplemental Figure S3, D and E). The reversibility of the condensation process has also been described for self-assemblies made by CtpS (Barry et al, 2014), Gln1 (Petrovska et al 2014) and TORC1 (Prouteau et al, 2017), with clear regulatory functions for all the three enzymes.…”

“…However, so far only Gln1, CtpS and TORC1 enzymes have been shown to self-assemble into highly ordered polymers in such stressful condition by TEM (Petrovska et al, 2014;Barry et al, 2014;Prouteau et al, 2017). It is not known whether other enzymes assemble into polymers and whether this is a common occurrence in the cytoplasm of energy-depleted cells.…”

“…Live-cell imaging has shown that several fluorescently tagged enzymes form assemblies in the cytoplasm of dormant cells (Narayanaswamy et al, 2009;Noree et al, 2010;Liu, 2010;Petrovska et al, 2014;Riback et al, 2017;Prouteau et al, 2017). Electron microscopy imaging of three of these metabolic enzymes, cytidine triphosphate synthetase (CtpS, Barry et al, 2014), glutamine synthetase (Gln1, Petrovska et al, 2014) and target of rapamycin complex 1 (TORC1, Prouteau et al, 2017), revealed that they assemble into filaments and that the filaments organize in bundles, visible in fluorescence microscopy. In all three cases, the polymerization process is reversible, and it is coupled to the inactivation of the enzymatic activities.…”

Modeling and Analysis of the Macronutrient Signaling Network in Budding Yeast

“…PKA activates PDE, which converts cAMP to AMP. In our [71,72]. Finally, the relationship between Sch9 and PKA is confounded by the complex and overlapping regulation of their many shared downstream targets [19].…”