An In Vitro TORC1 Kinase Assay That Recapitulates the Gtr-Independent Glutamine-Responsive TORC1 Activation Mechanism on Yeast Vacuoles

Abstract

Evolutionarily conserved target of rapamycin (TOR) complex 1 (TORC1) responds to nutrients, especially amino acids, to promote cell growth. In the yeast Saccharomyces cerevisiae, various nitrogen sources activate TORC1 with different efficiencies, although the mechanism remains elusive. Leucine, and perhaps other amino acids, was reported to activate TORC1 via the heterodimeric small GTPases Gtr1-Gtr2, the orthologues of the mammalian Rag GTPases. More recently, an alternative Gtr-independent TORC1 activation mechanism that may respond to glutamine was reported, although its molecular mechanism is not clear. In studying the nutrient-responsive TORC1 activation mechanism, the lack of an in vitro assay hinders associating particular nutrient compounds with the TORC1 activation status, whereas no in vitro assay that shows nutrient responsiveness has been reported. In this study, we have developed a new in vitro TORC1 kinase assay that reproduces, for the first time, the nutrient-responsive TORC1 activation. This in vitro TORC1 assay recapitulates the previously predicted Gtr-independent glutamine-responsive TORC1 activation mechanism. Using this system, we found that this mechanism specifically responds to L-glutamine, resides on the vacuolar membranes, and involves a previously uncharacterized Vps34-Vps15 phosphatidylinositol (PI) 3-kinase complex and the PI-3-phosphate [PI(3)P]-binding FYVE domain-containing vacuolar protein Pib2. Thus, this system was proved to be useful for dissecting the glutamine-responsive TORC1 activation mechanism.KEYWORDS Saccharomyces cerevisiae, TOR kinase, TORC1, Vps34, glutamine, in vitro kinase assay T he target of rapamycin (TOR) is an evolutionarily conserved protein kinase that regulates cell growth as the catalytic component of rapamycin-sensitive TOR complex 1 (TORC1) (1, 2). TORC1 is activated by nutrients, particularly amino acids, in all tested eukaryotes, and active TORC1 promotes cell growth by activating anabolic processes, including synthesis of protein, lipids, and nucleotides and by inhibiting catabolic processes, such as autophagy. In mammals, TORC1 consists of mammalian/ mechanistic TOR (mTOR), raptor, mammalian Lst8 (mLst8), DEP domain-containing mTOR-interacting protein (DEPTOR), and PRAS40, whereas in the yeast Saccharomyces cerevisiae, TORC1 consists of Tor1 or Tor2, the raptor orthologue Kog1, the mLst8 orthologue Lst8, and Tco89. Recent studies have revealed an evolutionarily conserved amino acid-responsive TORC1 activation mechanism, in which heterodimeric small GTPases, RagA or RagB (RagA/B)-RagC/D in mammals and Gtr1-Gtr2 in yeast, play a central role (3)(4)(5). The mammalian Rag heterodimer is anchored to the lysosomal membrane through the Ragulator complex. An amino acid stimulus induces conversion of RagA/B from the GDP-bound state to the GTP-bound state, and the GTP-bound form directly binds to TORC1 (3, 4), thereby recruiting TORC1 to the lysosomal membrane Citation Tanigawa M, Maeda T. 2017. An in vitro TORC1 kinase assay that recapitulates the...

Spatially Distinct Pools of TORC1 Balance Protein Homeostasis

“…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).…”

Multilayered Control of Protein Turnover by TORC1 and Atg1

“…Analysis of the Atg1-dependent phosphoproteome revealed a similarly complex network of interactions specifically with the TORC1 signaling branch. For instance, Atg1 may feedback regulate TORC1 by (directly or indirectly) (1) impinging on Seh1 and Sea4, two subunits of the SEACAT complex that controls TORC1 through the Rag GTPases (Panchaud et al, 2013); (2) regulating Ser 327 phosphorylation within the TORC1 subunit Tco89; and/or (3) controlling the Ser 445 /Ser 449 phosphorylation within the PI3-kinase Vps34 that is key for TORC1 and autophagy activation (Reidick et al, 2017;Tanigawa and Maeda, 2017) (Figure 3). Lastly, Atg1 also converges with TORC1 on Gcn2.…”

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).…”