The essence of yeast quiescence

Like all microorganisms, yeast cells spend most of their natural lifetime in a reversible, quiescent state that is primarily induced by limitation for essential nutrients. Substantial progress has been made in defining the features of quiescent cells and the nutrient-signaling pathways that shape these features. A view that emerges from the wealth of new data is that yeast cells dynamically configure the quiescent state in response to nutritional challenges by using a set of key nutrient-signaling pathways, which (1) regulate pathway-specific effectors, (2) converge on a few regulatory nodes that bundle multiple inputs to communicate unified, graded responses, and (3) mutually modulate their competences to transmit signals. Here, I present an overview of our current understanding of the architecture of these pathways, focusing on how the corresponding core signaling protein kinases (i.e. PKA, TORC1, Snf1, and Pho85) are wired to ensure an adequate response to nutrient starvation, which enables cells to tide over decades, if not centuries, of famin

Quantification of mRNA stability of stress-responsive yeast genes following conditional excision of open reading frames

Eukaryotic cells rapidly adjust the levels of mRNAs in response to environmental stress primarily by controlling transcription and mRNA turnover. How different stress conditions influence the fate of stress-responsive mRNAs, however, is relatively poorly understood. This is largely due to the fact that mRNA half-life assays are traditionally based on interventions (e.g., temperature-shifts using temperature-sensitive RNA polymerase II alleles or treatment with general transcription inhibitory drugs), which, rather than blocking, specifically induce transcription of stress-responsive genes. To study the half-lives of the latter suite of mRNAs, we developed and describe here a minimally perturbing alternative method, coined CEO, which is based on discontinuance of transcription following the conditional excision of open reading frames. Using CEO, we confirm that the target of rapamycin complex I (TORC1), a nutrient-activated, central stimulator of eukaryotic cell growth, favors the decay of mRNAs that depend on the stress- and/or nutrient-regulated transcription factors Msn2/4 and Gis1 for their transcription. We further demonstrate that TORC1 controls the stability of these mRNAs via the Rim15-Igo1/2-PP2ACdc55 effector branch, which reportedly also controls Gis1 promoter recruitment. These data pinpoint PP2ACdc55 as a central node in homo-directional coordination of transcription and post-transcriptional mRNA stabilization of a specific array of nutrient-regulated genes

TORC1 controls G1-S cell cycle transition in yeast via Mpk1 and the greatwall kinase pathway

The target of rapamycin complex 1 (TORC1) pathway couples nutrient, energy and hormonal signals with eukaryotic cell growth and division. In yeast, TORC1 coordinates growth with G₁–S cell cycle progression, also coined as START, by favouring the expression of G₁ cyclins that activate cyclin-dependent protein kinases (CDKs) and by destabilizing the CDK inhibitor Sic1. Following TORC1 downregulation by rapamycin treatment or nutrient limitation, clearance of G₁ cyclins and C-terminal phosphorylation of Sic1 by unknown protein kinases are both required for Sic1 to escape ubiquitin-dependent proteolysis prompted by its flagging via the SCFCdc4 (Skp1/Cul1/F-box protein) ubiquitin ligase complex. Here we show that the stabilizing phosphorylation event within the C-terminus of Sic1 requires stimulation of the mitogen-activated protein kinase, Mpk1, and inhibition of the Cdc55 protein phosphatase 2A (PP2ACdc55) by greatwall kinase-activated endosulfines. Thus, Mpk1 and the greatwall kinase pathway serve TORC1 to coordinate the phosphorylation status of Sic1 and consequently START with nutrient availability

The architecture of the Rag GTPase signaling network

The evolutionarily conserved target of rapamycin complex 1 (TORC1) couples an array of intra- and extracellular stimuli to cell growth, proliferation and metabolism, and its deregulation is associated with various human pathologies such as immunodeficiency, epilepsy, and cancer. Among the diverse stimuli impinging on TORC1, amino acids represent essential input signals, but how they control TORC1 has long remained a mystery. The recent discovery of the Rag GTPases, which assemble as heterodimeric complexes on vacuolar/lysosomal membranes, as central elements of an amino acid signaling network upstream of TORC1 in yeast, flies, and mammalian cells represented a breakthrough in this field. Here, we review the architecture of the Rag GTPase signaling network with a special focus on structural aspects of the Rag GTPases and their regulators in yeast and highlight both the evolutionary conservation and divergence of the mechanisms that control Rag GTPases

A spatially and functionally distinct pool of TORC1 defines signaling endosomes in yeast

The evolutionarily conserved target of rapamycin complex 1 (TORC1) regulates cell growth in a homeostatic manner by tuning anabolic and catabolic processes in response to nutritional and hormonal cues. Interestingly, rather than being localized at the plasma membrane as perhaps expected for an integrator of extracellular signals, TORC1 mainly localizes at vacuolar (in yeast) and lysosomal (in more complex eukaryotes) membranes where it seems optimally placed to sense both the nutrient status within the cytoplasm and the vacuolar/lysosomal compartment. How TORC1 controls downstream targets that are distant from the vacuole/lysosome, is currently poorly understood. In this context, we recently identified and characterized 2 spatially and functionally distinct pools of TORC1 in the budding yeast Saccharomyces cerevisiae: one at the vacuole that promotes protein synthesis, and another one at endosomes that inhibits protein degradation. Thus, our findings highlight the presence of spatially separated pools of TORC1 that are commissioned with functionally specific tasks within cells. In addition, they pinpoint the existence of signaling endosomes in yeast, which raises numerous new questions that are warranted to direct future research in this area

TORC1 specifically inhibits microautophagy through ESCRT-0

Nutrient starvation induces the degradation of specific plasma membrane proteins through the multivesicular body (MVB) sorting pathway and of vacuolar membrane proteins through microautophagy. Both of these processes require the gateway protein Vps27, which recognizes ubiquitinated cargo proteins at phosphatidylinositol 3-phosphate-rich membranes as part of a heterodimeric complex coined endosomal sorting complex required for transport 0. The target of rapamycin complex 1 (TORC1), a nutrient-activated central regulator of cell growth, directly phosphorylates Vps27 to antagonize its function in microautophagy, but whether this also serves to restrain MVB sorting at endosomes is still an open question. Here, we show that TORC1 inhibits both the MVB pathway-driven turnover of the plasma membrane-resident high-affinity methionine permease Mup1 and the inositol transporter Itr1 and the microautophagy-dependent degradation of the vacuolar membrane-associated v- ATPase subunit Vph1. Using a Vps277D variant that mimics the TORC1- phosphorylated state of Vps27, we further show that cargo sorting of Vph1 at the vacuolar membrane, but not of Mup1 and Itr1 at endosomes, is sensitive to the TORC1-controlled modifications of Vps27. Thus, TORC1 specifically modulates microautophagy through phosphorylation of Vps27, but controls MVB sorting through alternative mechanisms

Leucyl-tRNA synthetase controls TORC1 via the EGO complex

The target of rapamycin complex 1 (TORC1) is an essential regulator of eukaryotic cell growth that responds to growth factors, energy levels, and amino acids. The mechanisms through which the preeminent amino acid leucine signals to the TORC1-regulatory Rag GTPases, which activate TORC1 within the yeast EGO complex (EGOC) or the structurally related mammalian Rag-Ragulator complex, remain elusive. We find that the leucyl-tRNA synthetase (LeuRS) Cdc60 interacts with the Rag GTPase Gtr1 of the EGOC in a leucine-dependent manner. This interaction is necessary and sufficient to mediate leucine signaling to TORC1 and is disrupted by the engagement of Cdc60 in editing mischarged tRNALeu. Thus, the EGOC-TORC1 signaling module samples, via the LeuRS-intrinsic editing domain, the fidelity of tRNALeu aminoacylation as a proxy for leucine availability

Amino Acids Stimulate TORC1 through Lst4-Lst7, a GTPase-Activating Protein Complex for the Rag Family GTPase Gtr2

Rag GTPases assemble into heterodimeric complexes consisting of RagA or RagB and RagC or RagD in higher eukaryotes, or Gtr1 and Gtr2 in yeast, to relay amino acid signals toward the growth-regulating target of rapamycin complex 1 (TORC1). The TORC1-stimulating state of Rag GTPase heterodimers, containing GTP- and GDP-loaded RagA/B/Gtr1 and RagC/D/Gtr2, respectively, is maintained in part by the FNIP-Folliculin RagC/D GAP complex in mammalian cells. Here, we report the existence of a similar Lst4-Lst7 complex in yeast that functions as a GAP for Gtr2 and that clusters at the vacuolar membrane in amino acid-starved cells. Refeeding of amino acids, such as glutamine, stimulated the Lst4-Lst7 complex to transiently bind and act on Gtr2, thereby entailing TORC1 activation and Lst4-Lst7 dispersal from the vacuolar membrane. Given the remarkable functional conservation of the RagC/D/Gtr2 GAP complexes, our findings could be relevant for understanding the glutamine addiction of mTORC1-dependent cancers

Feedback inhibition of the Rag GTPase GAP complex Lst4-Lst7 safeguards TORC1 from hyperactivation by amino acid signals

Amino acids stimulate the eukaryotic target of rapamycin complex 1 (TORC1), and hence growth, through the Rag GTPases and their regulators. Among these, the yeast Lst4-Lst7 Rag GTPase GAP complex clusters, as we previously reported, at the vacuolar membrane upon amino acid starvation. In response to amino acid refeeding, it activates the Rag GTPase-TORC1 branch and is then dispersed from the vacuolar surface. Here, we show that the latter effect is driven by TORC1 itself, which directly phosphorylates several residues within the intra-DENN loop of Lst4 that, only in its non-phosphorylated state, tethers the Lst4-Lst7 complex to the vacuolar membrane. An Lst4 variant disrupting this feedback inhibition mechanism causes TORC1 hyperactivation and proliferation defects in cells grown on poor nitrogen sources. Thus, we identify Lst4 as a TORC1 target and key node of a homeostatic mechanism that adjusts TORC1 activity to the availability of amino acids

The evolutionary conserved BER1 gene is involved in microtubule stability in yeast

In yeast, microtubules are dynamic filaments necessary for spindle and nucleus positioning, as well as for proper chromosome segregation. We identify a function for the yeast gene BER1 (Benomyl REsistant 1) in microtubule stability. BER1 belongs to an evolutionary conserved gene family whose founding member Sensitivity to Red light Reduced is involved in red-light perception and circadian rhythms in Arabidopsis. Here, we present data showing that the ber1Δ mutant is affected in microtubule stability, particularly in presence of microtubule-depolymerising drugs. The pattern of synthetic lethal interactions obtained with the ber1Δ mutant suggests that Ber1 may function in N-terminal protein acetylation. Our work thus suggests that microtubule stability might be regulated through this post-translational modification on yet-to-be determined protein