Gtr/Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane

Abstract

TORC1 is a central regulator of cell growth in response to amino acids. The role of the evolutionarily conserved Gtr/Rag pathway in the regulation of TORC1 is well-established. Recent genetic studies suggest that an additional regulatory pathway, depending on the activity of Pib2, plays a role in TORC1 activation independently of the Gtr/Rag pathway. However, the interplay between the Pib2 pathway and the Gtr/Rag pathway remains unclear. In this study, we show that Pib2 and Gtr/Ego form distinct complexes with TORC1 in a mutually exclusive manner, implying dedicated functional relationships between TORC1 and Pib2 or Gtr/Rag in response to specific amino acids. Furthermore, simultaneous depletion of Pib2 and the Gtr/Ego system abolishes TORC1 activity and completely compromises the vacuolar localization of TORC1. Thus, the amino acid-dependent activation of TORC1 is achieved through the Pib2 and Gtr/Ego pathways alone. Finally, we show that glutamine induces a dose-dependent increase in Pib2-TORC1 complex formation, and that glutamine binds directly to the Pib2 complex. These data provide strong preliminary evidence for Pib2 functioning as a putative glutamine sensor in the regulation of TORC1.

Spatially Distinct Pools of TORC1 Balance Protein Homeostasis

“…To assess whether TORC1 may also phosphorylate Vps27 in vivo, we performed quantitative phosphoproteomic analyses by stable isotope labeling by amino acids in cell culture (SILAC) (Ong et al, 2002) using extracts from cycloheximide-treated and untreated yeast cells. Addition of cycloheximide activates TORC1, as was shown by increased phosphorylation of Sch9 Urban et al, 2007). These assays retrieved the same 5 Vps27 phosphopeptides that we obtained in our in vitro assays and allowed us to pinpoint unequivocally 7 residues, of which minimally 2, Ser 155 and Thr 159 , seemed to be regulated by TORC1 in vivo ( Figure S4).…”

“…This interaction is likely also mediated by the ENEALL di-leucine motif in Ego1, because it not only matches reasonably well with the canonical GGA-binding motif in mammals (DXXLL) (Bonifacino and Traub, 2003) but also is required for Ego1-GFP sorting to both vacuoles and endosomes ( Figure 1E). On vacuoles, EGOC teams up with Pib2, a FYVE domain-containing protein that is required for amino acidmediated activation of TORC1 (Dubouloz et al, 2005;Kim and Cunningham, 2015;Michel et al, 2017;Tanigawa and Maeda, 2017;Varlakhanova et al, 2017), to ensure proper membrane tethering of TORC1 (Ukai et al, 2018). Because Pib2 200 -mCherry almost perfectly colocalized with Ego1-GFP, GFP-Gtr1, and GFP-Tor1 on both vacuoles and endosomes ( Figures 2J-2L), we wondered whether the failure of GFP-Tor1 to assemble on endosomes in gga1/2D cells ( Figure 2C) could be explained by the concurrent absence of the EGOC and Pib2 on endosomes of these cells (Ego1 per se is not required for this process) (Figure S2E).…”

“…See also Figure S2. The ET reporter consists of the EEA1 FYVE domain, which preferentially docks onto PI3P-rich endosomes (Burd and Emr, 1998;Hayakawa et al, 2004), fused to GFP and the C-terminal part of Sch9 that harbors the TORC1 target residue Thr 737 (Urban et al, 2007). In WT cells growing exponentially on synthetic dextrose media containing proline as a nitrogen source (synthetic dextrose-proline), this construct colocalizes with the endosomal marker Vps27-mCherry but is hardly detectable on vacuolar membranes (pictures on the right in A).…”

“…TORC1 adjusts growth to amino acid availability by balancing the equilibrium between protein synthesis and degradation. In yeast, TORC1 stimulates protein expression globally by activating ribosome biogenesis and protein translation through the AGC-family kinase Sch9, which, analogous to S6 kinases (S6K1/2) in mammals, is directly phosphorylated by TORC1 at the vacuolar membrane (Jin et al, 2014;Urban et al, 2007). Conversely, TORC1 inhibits bulk protein degradation through phosphorylation of Atg13 (at an elusive location) to prevent its association with Atg1 and consequently inhibit the induction of macroautophagy (Kamada et al, 2010).…”

Indirect monitoring of TORC1 signalling pathway reveals molecular diversity among different yeast strains

“…Furthermore, constitutive activity of EGOC fails to suppress the TORC1 signalling defect under ammonium deprivation (Binda et al, 2009). Thus, there is an alternative mechanism of TORC1 activation independent of EGOC in which the participating proteins have not been fully determined (Chantranupong, Wolfson, & Sabatini, 2015;Gonzalez & Hall, 2017), with only suggested actors such as Pib2 protein (Kim & Cunningham, 2015;Michel et al, 2017;Tanigawa & Maeda, 2017;Ukai et al, 2018;Varlakhanova, Mihalevic, Bernstein, & Ford, 2017).…”