Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae

Abstract

The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.

Role of Mitochondrial Retrograde Pathway in Regulating Ethanol-Inducible Filamentous Growth in Yeast

“…TOR plays an important role in nutrient-regulated responses in yeast (Heitman et al, 1991) and is a master regulator of nitrogen control (Beck and Hall, 1999; Cardenas et al, 1999; Bruckner et al, 2011; Kingsbury et al, 2015). TOR signaling also links nitrogen quality to the activity of the Rtg1p and Rtg3p transcription factors (Komeili et al, 2000).…”

Metabolism of Preimplantation Embryo Development

“…Although the rate of O 2 consumption and OXPHOS is primarily induced by the need for ATP (ie, ATP/ADP ratio), it is thought that amino acids can also modulate OXPHOS activity. For energy production, amino acids enhance OXPHOS by increasing the TCA flux, as metabolites from some amino acids are catabolized to either pyruvate or Ac-CoA which can feed into the TCA cycle (Kingsbury, Sen, & Cardenas, 2015;Nelson, Lehninger, & Cox, 2008). Some amino acids are also metabolized into components of the TCA cycle through a process known as anaplerosis (Owen, Kalhan, & Hanson, 2002).…”

“…Activation of mTOR by amino acids, especially glutamine and branched-chain amino acids such as leucine, leads to an increase in OXPHOS activity as well as in ribosome synthesis for biomass accumulation (Fan et al, 2013;Kingsbury et al, 2015;Laplante & Sabatini, 2012;Schieke et al, 2006;Tokunaga, Yoshino, & Yonezawa, 2004). Glutamine and leucine may both directly activate mTOR through distinct pathways ( Jewell et al, 2015), or glutamine may indirectly modulate leucine activation of mTOR through glutamine/leucine antiporter in which glutamine's cellular export is coupled to leucine import (Nicklin et al, 2009).…”

Phosphate is the third nutrient monitored by TOR in Candida albicans and provides a target for fungal-specific indirect TOR inhibition

“…Seeking a molecular link between Pho84 and TORC1 activity, we considered the possibility that Gtr1 may connect Pho84 to TORC1. GTR1 was first described for its functional and physical proximity to S. cerevisiae PHO84 (32,33), and its product later was characterized as a component of the TORC1-activating EGO complex (12,13,34,35). We found that the P-S6 response of gtr1 −/− cells to phosphate refeeding was blunted ( Fig.…”

“…2E). A GTR1 mutant encoding constitutively GTP-bound Gtr1 Q67L , homologous to S. cerevisiae Gtr1 Q65L (12,35,36), was then constructed and overexpressed from the ACT1 promoter. This GTR1-GTP allele suppressed the TORC1 signaling defect of pho84 −/− cells apparently equally to the overexpressed WT GTR1 (Fig.…”