TORC1 regulates the transcriptional response to glucose and developmental cycle via the Tap42-Sit4-Rrd1/2 pathway in Saccharomyces cerevisiae.

BACKGROUND: Target of Rapamycin Complex 1 (TORC1) is a highly conserved eukaryotic protein complex that couples the presence of growth factors and nutrients in the environment with cellular proliferation. TORC1 is primarily implicated in linking amino acid levels with cellular growth in yeast and mammals. Although glucose deprivation has been shown to cause TORC1 inactivation in yeast, the precise role of TORC1 in glucose signaling and the underlying mechanisms remain unclear. RESULTS: We demonstrate that the presence of glucose in the growth medium is both necessary and sufficient for TORC1 activation. TORC1 activity increases upon addition of glucose to yeast cells growing in a non-fermentable carbon source. Conversely, shifting yeast cells from glucose to a non-fermentable carbon source reduces TORC1 activity. Analysis of transcriptomic data revealed that glucose and TORC1 co-regulate about 27% (1668/6004) of yeast genes. We demonstrate that TORC1 orchestrates the expression of glucose-responsive genes mainly via the Tap42-Sit4-Rrd1/2 pathway. To confirm TORC1's function in glucose signaling, we tested its role in spore germination, a glucose-dependent developmental state transition in yeast. TORC1 regulates the glucose-responsive genes during spore germination and inhibition of TORC1 blocks spore germination. CONCLUSIONS: Our studies indicate that a regulatory loop that involves activation of TORC1 by glucose and regulation of glucose-responsive genes by TORC1, mediates nutritional control of growth and development in yeast.

Sit4p-mediated dephosphorylation of Atp2p regulates ATP synthase activity and mitochondrial function.

Sit4p is a type 2A-related protein phosphatase in Saccharomyces cerevisiae involved in a wide spectrum of cellular functions, including the glucose repression of mitochondrial transcription. Here we report that Sit4p is also involved in post-translational regulation of mitochondrial proteins and identified 9 potential targets. One of these, the ATP synthase (FoF1 complex) beta subunit Atp2p, was characterized and two phosphorylation sites, T124 and T317, were identified. Expression of Atp2p-T124 or T317 phosphoresistant versions in sit4Δ cells decreased Atp2p phosphorylation confirming these as Sit4p-regulated sites. Moreover, Sit4p and Atp2p interacted both physically and genetically. Mimicking phosphorylation at T124 or T317 increased Atp2p levels, resulting in higher abundance/activity of ATP synthase. Similar changes were observed in sit4Δ cells in which Atp2p is endogenously more phosphorylated. Expression of Atp2-T124 or T317 phosphomimetics also increased mitochondrial respiration and ATP levels and extended yeast lifespan. These results suggest that Sit4p-mediated dephosphorylation of Atp2p-T124/T317 downregulates Atp2p alongside with ATP synthase and mitochondrial function. Combination of transcriptional with post-translational regulation during fermentative growth may allow for a more efficient Sit4p repression of mitochondrial respiration.

The ceramide activated protein phosphatase Sit4 impairs sphingolipid dynamics, mitochondrial function and lifespan in a yeast model of Niemann-Pick type C1.

The Niemann-Pick type C is a rare neurodegenerative disease that results from loss-of-function point mutations in NPC1 or NPC2, which affect the homeostasis of sphingolipids and sterols in human cells. We have previously shown that yeast lacking Ncr1, the orthologue of human NPC1 protein, display a premature ageing phenotype and higher sensitivity to oxidative stress associated with mitochondrial dysfunctions and accumulation of long chain bases. In this study, a lipidomic analysis revealed specific changes in the levels of ceramide species in ncr1Δ cells, including decreases in dihydroceramides and increases in phytoceramides. Moreover, the activation of Sit4, a ceramide-activated protein phosphatase, increased in ncr1Δ cells. Deletion of SIT4 or CDC55, its regulatory subunit, increased the chronological lifespan and hydrogen peroxide resistance of ncr1Δ cells and suppressed its mitochondrial defects. Notably, Sch9 and Pkh1-mediated phosphorylation of Sch9 decreased significantly in ncr1Δsit4Δ cells. These results suggest that phytoceramide accumulation and Sit4-dependent signaling mediate the mitochondrial dysfunction and shortened lifespan in the yeast model of Niemann-Pick type C1, in part through modulation of the Pkh1-Sch9 pathway.

Ceramide signaling targets the PP2A-like protein phosphatase Sit4p to impair vacuolar function, vesicular trafficking and autophagy in Isc1p deficient cells.

The vacuoles play important roles in cellular homeostasis and their functions include the digestion of cytoplasmic material and organelles derived from autophagy. Conserved nutrient signaling pathways regulate vacuolar function and autophagy, ensuring normal cell and organismal development and aging. Recent evidence implicates sphingolipids in the modulation of these processes, but the impact of ceramide signaling on vacuolar dynamics and autophagy remains largely unknown. Here, we show that yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit vacuolar fragmentation and dysfunctions, namely decreased Pep4p-mediated proteolysis and V-ATPase activity, which impairs vacuolar acidification. Moreover, these phenotypes are suppressed by downregulation of the ceramide-activated protein phosphatase Sit4p. The isc1Δ cells also exhibit defective Cvt and vesicular trafficking in a Sit4p-dependent manner, ultimately contributing to a reduced autophagic flux. Importantly, these phenotypes are also suppressed by downregulation of the nutrient signaling kinase TORC1, which is known to inhibit Sit4p and autophagy, or Sch9p. These results support a model in which Sit4p functions downstream of Isc1p in a TORC1-independent, ceramide-dependent signaling branch that impairs vacuolar function and vesicular trafficking, leading to autophagic defects in yeast.

Elongator, a conserved complex required for wobble uridine modifications in eukaryotes.

Elongator is a 6 subunit protein complex highly conserved in eukaryotes. The role of this complex has been controversial as the pleiotropic phenotypes of Elongator mutants have implicated the complex in several cellular processes. However, in yeast there is convincing evidence that the primary and probably only role of this complex is in formation of the 5-methoxycarbonylmethyl (mcm(5)) and 5-carbamoylmethyl (ncm(5)) side chains on uridines at wobble position in tRNA. In this review we summarize the cellular processes that have been linked to the Elongator complex and discuss its role in tRNA modification and regulation of translation. We also describe additional gene products essential for formation of ncm(5) and mcm(5) side chains at U34 and their influence on Elongator activity.

Sit4 Genetically Interacts with Vps27 to Regulate Mitochondrial Function and Lifespan in Saccharomyces cerevisiae.

The Sit4 protein phosphatase plays a key role in orchestrating various cellular processes essential for maintaining cell viability during aging. We have previously shown that SIT4 deletion promotes vacuolar acidification, mitochondrial derepression, and oxidative stress resistance, increasing yeast chronological lifespan. In this study, we performed a proteomic analysis of isolated vacuoles and yeast genetic interaction analysis to unravel how Sit4 influences vacuolar and mitochondrial function. By employing high-resolution mass spectrometry, we show that sit4Δ vacuolar membranes were enriched in Vps27 and Hse1, two proteins that are part of the endosomal sorting complex required for transport-0. In addition, SIT4 exhibited a negative genetic interaction with VPS27, as sit4∆vps27∆ double mutants had a shortened lifespan compared to sit4∆ and vps27∆ single mutants. Our results also show that Vps27 did not increase sit4∆ lifespan by improving protein trafficking or vacuolar sorting pathways. However, Vps27 was critical for iron homeostasis and mitochondrial function in sit4∆ cells, as sit4∆vps27∆ double mutants exhibited high iron levels and impaired mitochondrial respiration. These findings show, for the first time, cross-talk between Sit4 and Vps27, providing new insights into the mechanisms governing chronological lifespan.

Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeast Saccharomyces cerevisiae.

Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent K(m) values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 µM, respectively, with V(max) values of 0.294 and 0.173 µmol mg⁻¹ min⁻¹, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the K(m) for TPP from 20 to 84 µM and for pyruvate from 2.3 to 4.6 mM, while the V(max) changed from 0.806 to 0.673 µmol mg⁻¹ min⁻¹. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate.

N- and C-terminal Gln3-Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization.

Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656-666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3-Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3-Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3-Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3-Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3-15-fold with three of them by 10-15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3-Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.

Sphingolipids and Inositol Phosphates Regulate the Tau Protein Phosphorylation Status in Humanized Yeast.

Hyperphosphorylation of protein tau is a hallmark of Alzheimer's disease (AD). Changes in energy and lipid metabolism have been correlated with the late onset of this neurological disorder. However, it is uncertain if metabolic dysregulation is a consequence of AD or one of the initiating factors of AD pathophysiology. Also, it is unclear whether variations in lipid metabolism regulate the phosphorylation state of tau. Here, we show that in humanized yeast, tau hyperphosphorylation is stimulated by glucose starvation in coincidence with the downregulation of Pho85, the yeast ortholog of CDK5. Changes in inositol phosphate (IP) signaling, which has a central role in energy metabolism, altered tau phosphorylation. Lack of inositol hexakisphosphate kinases Kcs1 and Vip1 (IP6 and IP7 kinases in mammals) increased tau hyperphosphorylation. Similar effects were found by mutation of IPK2 (inositol polyphosphate multikinase), or PLC1, the yeast phospholipase C gene. These effects may be explained by IP-mediated regulation of Pho85. Indeed, this appeared to be the case for plc1, ipk2, and kcs1. However, the effects of Vip1 on tau phosphorylation were independent of the presence of Pho85, suggesting additional mechanisms. Interestingly, kcs1 and vip1 strains, like pho85, displayed dysregulated sphingolipid (SL) metabolism. Moreover, genetic and pharmacological inhibition of SL biosynthesis stimulated the appearance of hyperphosphorylated forms of tau, while increased flux through the pathway reduced its abundance. Finally, we demonstrated that Sit4, the yeast ortholog of human PP2A protein phosphatase, is a downstream effector of SL signaling in mediating the tau phosphorylation state. Altogether, our results add new knowledge on the molecular effectors involved in tauopathies and identify new targets for pharmacological intervention.

PP2A-Like Protein Phosphatase (Sit4) Regulatory Subunits, Sap155 and Sap190, Regulate Candida albicans' Cell Growth, Morphogenesis, and Virulence.

PP2A-like phosphatases share high homology with PP2A enzymes and are composed of a catalytic subunit and a regulatory subunit. In Candida albicans, the PP2A-like catalytic subunit SIT4 regulates cell growth, morphogenesis, and virulence. However, the functions of its regulatory subunits remain unclear. Here, by homology analysis and co-IP experiments, we identified two regulatory subunits of SIT4 in C. albicans, SAP155 (orf19.642) and SAP190 (orf19.5160). We constructed sit4Δ/Δ, sap155Δ/Δ, sap190Δ/Δ, and sap155Δ/Δ sap190Δ/Δ mutants and found that deleting SAP155 had no apparent phenotypic consequence, while deleting SAP190 caused slow growth, hypersensitivity to cell wall stress, abnormal morphogenesis in response to serum or genotoxic stress (HU and MMS), less damage to macrophages, and attenuated virulence in mice. However, deleting both SAP155 and SAP190 caused significantly stronger defects, which was similar to deleting SIT4. Together, our results suggest that SAP190 is required for the function of SIT4 and that SAP155 can partially compensate for the loss of SAP190 in C. albicans. Given the vital role of these regulatory subunits of SIT4 in C. albicans physiology and virulence, they could serve as potential antifungal targets.

Sit4 and PP2A Dephosphorylate Nitrogen Catabolite Repression-Sensitive Gln3 When TorC1 Is Up- as Well as Downregulated.

Saccharomyces cerevisiae lives in boom and bust nutritional environments. Sophisticated regulatory systems have evolved to rapidly cope with these changes while preserving intracellular homeostasis. Target of Rapamycin Complex 1 (TorC1), is a serine/threonine kinase complex and a principle nitrogen-responsive regulator. TorC1 is activated by excess nitrogen and downregulated by limiting nitrogen. Two of TorC1's many downstream targets are Gln3 and Gat1-GATA-family transcription activators-whose localization and function are Nitrogen Catabolite Repression- (NCR-) sensitive. In nitrogen replete environments, TorC1 is activated, thereby inhibiting the PTap42-Sit4 and PTap42-PP2A (Pph21/Pph22-Tpd3, Pph21,22-Rts1/Cdc55) phosphatase complexes. Gln3 is phosphorylated, sequestered in the cytoplasm and NCR-sensitive transcription repressed. In nitrogen-limiting conditions, TorC1 is downregulated and PTap42-Sit4 and PTap42-PP2A are active. They dephosphorylate Gln3, which dissociates from Ure2, relocates to the nucleus, and activates transcription. A paradoxical observation, however, led us to suspect that Gln3 control was more complex than appreciated, i.e., Sit4 dephosphorylates Gln3 more in excess than in limiting nitrogen conditions. This paradox motivated us to reinvestigate the roles of these phosphatases in Gln3 regulation. We discovered that: (i) Sit4 and PP2A actively function both in conditions where TorC1 is activated as well as down-regulated; (ii) nuclear Gln3 is more highly phosphorylated than when it is sequestered in the cytoplasm; (iii) in nitrogen-replete conditions, Gln3 relocates from the nucleus to the cytoplasm, where it is dephosphorylated by Sit4 and PP2A; and (iv) in nitrogen excess and limiting conditions, Sit4, PP2A, and Ure2 are all required to maintain cytoplasmic Gln3 in its dephosphorylated form.

Protein Phosphatase Sit4 Affects Lipid Droplet Synthesis and Soraphen A Resistance Independent of Its Role in Regulating Elongator Dependent tRNA Modification.

The protein phosphatase Sit4 has been shown to be required for lipogenesis and resistance against the acetyl-CoA carboxylase inhibitor soraphen A. Since Sit4 is also required for biosynthesis of Elongator dependent tRNA modifications such as 5-methoxycarbonylmethyluridine (mcm5U), we investigated the relevance of tRNA modifications in lipogenesis and soraphen A response. While sit4 and Elongator (elp3) mutants copy defects in mcm5U formation and stress sensitivity, they do not share soraphen A sensitivity and low lipid droplet (LD) phenotypes. In contrast to sit4, we found elp3 mutants to display partial soraphen A resistance and a high LD phenotype. Screening a collection of tRNA modification mutants additionally identified the tRNA pseudo-uridine synthase gene DEG1 to be required for soraphen A sensitivity. Since deg1 and elp3 share high LD and soraphen A resistance phenotypes, these are likely caused by translational defects. In support of this notion, we observe overexpression of tRNAGlnUUG suppresses lipolysis defects of deg1 mutants. Hence, the sit4 mutation results in a composite defect including tRNA modification deficiency and loss of Snf1 kinase dephosphorylation, which induce opposite effects on LD regulation. Importantly, however, the Snf1 kinase regulatory defects of the phosphatase mutant dominate over effects on LD regulation imposed by loss of the tRNA modification alone.

TOR complex 1 regulates the yeast plasma membrane proton pump and pH and potassium homeostasis.

We have identified in yeast a connection between two master regulators of cell growth: a biochemical connection involving the TORC1 protein kinase (which activates protein synthesis, nutrient uptake, and anabolism) and a biophysical connection involving the plasma membrane proton-pumping H+ -ATPase Pma1 (which drives nutrient and K+ uptake and regulates pH homeostasis). Raising the temperature to nonpermissive values in a TOR thermosensitive mutant decreases Pma1 activity. Rapamycin, a TORC1 inhibitor, inhibits Pma1 dependent on its receptor Fpr1 and on the protein phosphatase Sit4, a TORC1 effector. Mutation of either Sit4 or Tco89, a nonessential subunit of TORC1, decreases proton efflux, K+ uptake, intracellular pH, cell growth, and tolerance to weak organic acids. Tco89 does not affect Pma1 activity but activates K+ transport.

The serine/threonine phosphatase DhSIT4 modulates cell cycle, salt tolerance and cell wall integrity in halo tolerant yeast Debaryomyces hansenii.

The highly conserved family of Phosphoprotein phosphatases (PPP) regulates several major physiological processes in yeast. However, very little is known about the PPP orthologs from the yeast species inhabiting extreme environmental niches. In the present study we have identified DhSIT4, a member of PPP6 class of serine threonine phosphatases from the halotolerant yeast Debaryomyces hansenii. Deletion of DhSIT4 in D. hansenii was not lethal but the mutant exhibited reduced growth due to its effect on the cell cycle. The knock out mutant Dhsit4Δ showed sensitivity towards Li+, Na+ and cell wall damaging agents. The expression of DhSit4p rescued salt, caffeine and calcofluor white sensitivity of Dhmpk1Δ strain and thereby indicating a genetic interaction of this phosphatase with the cell wall integrity pathway in this species. Our study also demonstrated the antagonistic roles of DhSit4p and DhPpz1p in maintaining the cell cycle and ion homeostasis in D. hansenii.

Saccharomyces cerevisiae TORC1 Controls Histone Acetylation by Signaling Through the Sit4/PP6 Phosphatase to Regulate Sirtuin Deacetylase Nuclear Accumulation.

The epigenome responds to changes in the extracellular environment, yet how this information is transmitted to the epigenetic regulatory machinery is unclear. Using a Saccharomyces cerevisiae yeast model, we demonstrate that target of rapamycin complex 1 (TORC1) signaling, which is activated by nitrogen metabolism and amino acid availability, promotes site-specific acetylation of histone H3 and H4 N-terminal tails by opposing the activity of the sirtuin deacetylases Hst3 and Hst4 TORC1 does so through suppression of the Tap42-regulated Sit4 (PP6) phosphatase complex, as sit4Δ rescues histone acetylation under TORC1-repressive conditions. We further demonstrate that TORC1 inhibition, and subsequent PP6 activation, causes a selective, rapid, nuclear accumulation of Hst4, which correlates with decreased histone acetylation. This increased Hst4 nuclear localization precedes an elevation in Hst4 protein expression, which is attributed to reduced protein turnover, suggesting that nutrient signaling through TORC1 may limit Hst4 nuclear accumulation to facilitate Hst4 degradation and maintain histone acetylation. This pathway is functionally relevant to TORC1 signaling since the stress sensitivity of a nonessential TORC1 mutant (tco89Δ) to hydroxyurea and arsenic can be reversed by combining tco89Δ with either hst3Δ, hst4Δ, or sit4Δ Surprisingly, while hst3Δ or hst4Δ rescues the sensitivity tco89Δ has to low concentrations of the TORC1 inhibitor rapamycin, sit4Δ fails to do so. These results suggest Sit4 provides an additional function necessary for TORC1-dependent cell growth and proliferation. Collectively, this study defines a novel mechanism by which TORC1 suppresses a PP6-regulated sirtuin deacetylase pathway to couple nutrient signaling to epigenetic regulation.

The ceramide-activated protein phosphatase Sit4p controls lifespan, mitochondrial function and cell cycle progression by regulating hexokinase 2 phosphorylation.

Sit4p is the catalytic subunit of a ceramide-activated PP2A-like phosphatase that regulates cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan in yeast. In this study, we show that hexokinase 2 (Hxk2p) is hyperphosphorylated in sit4Δ mutants grown in glucose medium by a Snf1p-independent mechanism and Hxk2p-S15A mutation suppresses phenotypes associated with SIT4 deletion, namely growth arrest at G1 phase, derepression of mitochondrial respiration, H2O2 resistance and lifespan extension. Consistently, the activation of Sit4p in isc1Δ mutants, which has been associated with premature aging, leads to Hxk2p hypophosphorylation, and the expression of Hxk2p-S15E increases the lifespan of isc1Δ cells. The overall results suggest that Hxk2p functions downstream of Sit4p in the control of cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan.

Ceramide signalling impinges on Sit4p and Hog1p to promote mitochondrial fission and mitophagy in Isc1p-deficient cells.

Mitochondria function as the powerhouses of the cell for energy conversion through the oxidative phosphorylation process. Accumulation of dysfunctional mitochondria promotes a bioenergetic crisis and cell death by apoptosis. Yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit mitochondrial dysfunction and shortened lifespan associated with the accumulation of specific ceramide species and activation of the PP2A-like protein phosphatase Sit4p and of the Hog1p kinase. Here, we show that isc1Δ cells display hyperactivation of mitophagy that is suppressed by downregulating Sit4p, Hog1p or the TORC1-Sch9p pathway. Notably, isc1Δ cells also have high levels of Dnm1p associated with unbalanced mitochondrial fission, leading to mitochondrial fragmentation, and DNM1 deletion suppressed the oxidative stress sensitivity and shortened lifespan of isc1Δ cells. Moreover, Isc1p and Dnm1p physically interact, suggesting a possible regulatory role for Isc1p in mitochondrial dynamics. Overall, our work demonstrates that Isc1p-mediated ceramide signalling regulates mitophagy and mitochondrial dynamics in yeast with impact on mitochondrial function and lifespan. Since ceramides have been implicated in ageing and diseases associated with mitochondrial dysfunction, our findings suggest that therapeutic strategies targeting ceramide signalling may improve mitochondrial function and human healthspan.

Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling.

Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41∆, sap190∆, ppm1∆, rrd1∆) and U34 modification defects (elp3∆, kti12∆, urm1∆, ncs2∆) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3∆ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3∆ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3∆, urm1∆) enhances nuclear import of and NCR gene activation (MEP2, GAP1) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TOR-sensitive NCR branch via Gln3 misregulation.