The Ccr4-Not complex regulates TORC1 signaling and mitochondrial metabolism by promoting vacuole V-ATPase activity

Abstract

The Ccr4-Not complex functions as an effector of multiple signaling pathways that control gene transcription and mRNA turnover. Consequently, Ccr4-Not contributes to a diverse array of processes, which includes a significant role in cell metabolism. Yet a mechanistic understanding of how it contributes to metabolism is lacking. Herein, we provide evidence that Ccr4-Not activates nutrient signaling through the essential target of rapamycin complex 1 (TORC1) pathway. Ccr4-Not disruption reduces global TORC1 signaling, and it also upregulates expression of the cell wall integrity (CWI) pathway terminal kinase Mpk1. Although CWI signaling represses TORC1 signaling, we find that Ccr4-Not loss inhibits TORC1 independently of CWI activation. Instead, we demonstrate that Ccr4-Not promotes the function of the vacuole V-ATPase, which interacts with the Gtr1 GTPase-containing EGO complex to stimulate TORC1 in response to nutrient sufficiency. Bypassing the V-ATPase requirement in TORC1 activation using a constitutively active Gtr1 mutant fully restores TORC1 signaling in Ccr4-Not deficient cells. Transcriptome analysis and functional studies revealed that loss of the Ccr4 subunit activates the TORC1 repressed retrograde signaling pathway to upregulate mitochondrial activity. Blocking this mitochondrial upregulation in Ccr4-Not deficient cells further represses TORC1 signaling, and it causes synergistic deficiencies in mitochondrial-dependent metabolism. These data support a model whereby Ccr4-Not loss impairs V-ATPase dependent TORC1 activation that forces cells to enhance mitochondrial metabolism to sustain a minimal level of TORC1 signaling necessary for cell growth and proliferation. Therefore, Ccr4-Not plays an integral role in nutrient signaling and cell metabolism by promoting V-ATPase dependent TORC1 activation.

Expression of a Truncated Yeast Ccc1 Vacuolar Transporter Increases the Accumulation of Endogenous Iron

“…For instance, CCC1 overexpression could cause a decrease in vacuolar lumen acidification, since Ccc1 seems to be a H + /Fe 2+ antiporter [12]. In this sense, recent data demonstrated that the loss of vacuolar acidification leads to oxidative damage of mitochondrial Fe/S cluster biosynthesis components and activities and the development of an age-related mitochondrial dysfunction [39,40]. Importantly, Ccc1 overexpression toxicity was not observed in high iron media (Figures 2 and 3).…”

Transcription by the Three RNA Polymerases under the Control of the TOR Signaling Pathway in Saccharomyces cerevisiae

“…Genetic approaches have identified some Ccr4-Not mutants as being rapamycin-sensitive, which indicates the relation between this complex and the TOR pathway [ 56 , 57 ]. In fact, Ccr4-Not may act upstream of TOR through a mechanism that involves V-ATPase [ 58 ]. Ccr4-Not associates with both RNA pol I and rDNA, which indicates a role in RNA pol I regulation [ 59 ].…”

Ccr4-Not ubiquitin ligase signaling regulates ribosomal protein homeostasis and inhibits 40S ribosomal autophagy

“…Additional Not4 studies have revealed that it controls translation (23)(24)(25), proteostasis (26)(27)(28), and endolysosomal-dependent nutrient signaling (29), but its role in these processes remains incompletely understood. Specifically, Not4 monoubiquitinates both the ribosomal protein Rps7a and the Egd1 and Egd2 (Egd1/2) subunits that make up the nascent polypeptide associated complex (NAC) involved in co-translational quality control (23,25).…”

“…Macroautophagy regulates protein quality control so Ccr4-Not, and specifically Not4, may contribute to nutrient stress responses and protein quality control through macroautophagy regulation. Ccr4-Not also activates nutrient signaling through the TORC1 pathway by promoting the function of the vacuole V-ATPase H + pump that both acidifies the vacuole and activates TORC1 and PKA signaling in response to nutrients (29,(35)(36)(37). Ccr4-Not mutants exhibit synthetic sick or lethal genetic interactions with endolysosomal pathway effectors, including V-ATPase mutants and vacuolar protein sorting (Vps factors) (8,(38)(39)(40).…”

“…The role individual Ccr4-Not subunits have on the transcriptome has been described previously (29,(41)(42)(43), yet the effect that Not4 ligase signaling has on the proteome remains poorly understood.…”