RESUMO
The conserved Gcn2 protein kinase mediates cellular adaptations to amino acid limitation through translational control of gene expression that is exclusively executed by phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α). Using quantitative phosphoproteomics, however, we discovered that Gcn2 targets auxiliary effectors to modulate translation. Accordingly, Gcn2 also phosphorylates the ß-subunit of the trimeric eIF2 G protein complex to promote its association with eIF5, which prevents spontaneous nucleotide exchange on eIF2 and thereby restricts the recycling of the initiator methionyl-tRNA-bound eIF2-GDP ternary complex in amino-acid-starved cells. This mechanism contributes to the inhibition of translation initiation in parallel to the sequestration of the nucleotide exchange factor eIF2B by phosphorylated eIF2α. Gcn2 further phosphorylates Gcn20 to antagonize, in an inhibitory feedback loop, the formation of the Gcn2-stimulatory Gcn1-Gcn20 complex. Thus, Gcn2 plays a substantially more intricate role in controlling translation initiation than hitherto appreciated.
Assuntos
Aminoácidos/deficiência , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/metabolismo , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Fosforilação , Proteínas Serina-Treonina Quinases/genética , RNA de Transferência de Metionina/genética , RNA de Transferência de Metionina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The eukaryotic TORC1 kinase is a homeostatic controller of growth that integrates nutritional cues and mediates signals primarily from the surface of lysosomes or vacuoles. Amino acids activate TORC1 via the Rag GTPases that combine into structurally conserved multi-protein complexes such as the EGO complex (EGOC) in yeast. Here we show that Ego1, which mediates membrane-anchoring of EGOC via lipid modifications that it acquires while traveling through the trans-Golgi network, is separately sorted to vacuoles and perivacuolar endosomes. At both surfaces, it assembles EGOCs, which regulate spatially distinct pools of TORC1 that impinge on functionally divergent effectors: vacuolar TORC1 predominantly targets Sch9 to promote protein synthesis, whereas endosomal TORC1 phosphorylates Atg13 and Vps27 to inhibit macroautophagy and ESCRT-driven microautophagy, respectively. Thus, the coordination of three key regulatory nodes in protein synthesis and degradation critically relies on a division of labor between spatially sequestered populations of TORC1.
Assuntos
Proteostase , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/enzimologia , Endossomos/genética , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Transcrição/genética , Vacúolos/enzimologia , Vacúolos/genéticaRESUMO
Yeast cells maintain an intricate network of nutrient signaling pathways enabling them to integrate information on the availability of different nutrients and adjust their metabolism and growth accordingly. Cells that are no longer capable of integrating this information, or that are unable to make the necessary adaptations, will cease growth and eventually die. Here, we studied the molecular basis underlying the synthetic lethality caused by loss of the protein kinase Sch9, a key player in amino acid signaling and proximal effector of the conserved growth-regulatory TORC1 complex, when combined with either loss of the cyclin-dependent kinase (CDK) Pho85 or loss of its inhibitor Pho81, which both have pivotal roles in phosphate sensing and cell cycle regulation. We demonstrate that it is specifically the CDK-cyclin pair Pho85-Pho80 or the partially redundant CDK-cyclin pairs Pho85-Pcl6/Pcl7 that become essential for growth when Sch9 is absent. Interestingly, the respective three CDK-cyclin pairs regulate the activity and distribution of the phosphatidylinositol-3 phosphate 5-kinase Fab1 on endosomes and vacuoles, where it generates phosphatidylinositol-3,5 bisphosphate that serves to recruit both TORC1 and its substrate Sch9. In addition, Pho85-Pho80 directly phosphorylates Sch9 at Ser726, and to a lesser extent at Thr723, thereby priming Sch9 for its subsequent phosphorylation and activation by TORC1. The TORC1-Sch9 signaling branch therefore integrates Pho85-mediated information at different levels. In this context, we also discovered that loss of the transcription factor Pho4 rescued the synthetic lethality caused by loss of Pho85 and Sch9, indicating that both signaling pathways also converge on Pho4, which appears to be wired to a feedback loop involving the high-affinity phosphate transporter Pho84 that fine-tunes Sch9-mediated responses.
Assuntos
Quinases Ciclina-Dependentes , Proteínas de Saccharomyces cerevisiae , Quinases Ciclina-Dependentes/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Repressoras/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ciclinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fosfatos/metabolismo , Fosfatidilinositóis/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
The Saccharomyces cerevisiae casein kinase protein Yck3 is a central regulator at the vacuole that phosphorylates several proteins involved in membrane trafficking. Here, we set out to identify novel substrates of this protein. We found that endogenously tagged Yck3 localized not only at the vacuole, but also on endosomes. To disable Yck3 function, we generated a kinase-deficient mutant and thus identified the I-BAR-protein Ivy1 as a novel Yck3 substrate. Ivy1 localized to both endosomes and vacuoles, and Yck3 controlled this localization. A phosphomimetic Ivy1-SD mutant was found primarily on vacuoles, whereas its non-phosphorylatable SA variant strongly localized to endosomes, similar to what was observed upon deletion of Yck3. In vitro analysis revealed that Yck3-mediated phosphorylation strongly promoted Ivy1 recruitment to liposomes carrying the Rab7-like protein Ypt7. Modeling of Ivy1 with Ypt7 identified binding sites for Ypt7 and a positively charged patch, which were both required for Ivy1 localization. Strikingly, Ivy1 mutations in either site resulted in more cells with multilobed vacuoles, suggesting a partial defect in its membrane biogenesis. Our data thus indicate that Yck3-mediated phosphorylation controls both localization and function of Ivy1 in endolysosomal biogenesis.
Assuntos
Proteínas de Saccharomyces cerevisiae , Vacúolos , Vacúolos/metabolismo , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Caseína Quinases/metabolismoRESUMO
Indole-3-acetic acid (IAA) is the most common, naturally occurring phytohormone that regulates cell division, differentiation, and senescence in plants. The capacity to synthesize IAA is also widespread among plant-associated bacterial and fungal species, which may use IAA as an effector molecule to define their relationships with plants or to coordinate their physiological behavior through cell-cell communication. Fungi, including many species that do not entertain a plant-associated life style, are also able to synthesize IAA, but the physiological role of IAA in these fungi has largely remained enigmatic. Interestingly, in this context, growth of the budding yeast Saccharomyces cerevisiae is sensitive to extracellular IAA. Here, we use a combination of various genetic approaches including chemical-genetic profiling, SAturated Transposon Analysis in Yeast (SATAY), and genetic epistasis analyses to identify the mode-of-action by which IAA inhibits growth in yeast. Surprisingly, these analyses pinpointed the target of rapamycin complex 1 (TORC1), a central regulator of eukaryotic cell growth, as the major growth-limiting target of IAA. Our biochemical analyses further demonstrate that IAA inhibits TORC1 both in vivo and in vitro. Intriguingly, we also show that yeast cells are able to synthesize IAA and specifically accumulate IAA upon entry into stationary phase. Our data therefore suggest that IAA contributes to proper entry of yeast cells into a quiescent state by acting as a metabolic inhibitor of TORC1.
Assuntos
Fungos/efeitos dos fármacos , Fungos/enzimologia , Ácidos Indolacéticos/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Elementos de DNA Transponíveis , Relação Dose-Resposta a Droga , Ativação Enzimática , Fungos/genética , Ácidos Indolacéticos/química , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Inibidores de Proteínas Quinases/química , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
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 tRNA(Leu). Thus, the EGOC-TORC1 signaling module samples, via the LeuRS-intrinsic editing domain, the fidelity of tRNA(Leu) aminoacylation as a proxy for leucine availability.
Assuntos
Leucina-tRNA Ligase/metabolismo , Leucina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismo , Leucina/genética , Leucina-tRNA Ligase/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.
Assuntos
Envelhecimento/genética , Glucose/metabolismo , Longevidade/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , ATPases Vacuolares Próton-Translocadoras/genética , Concentração de Íons de Hidrogênio , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Vacúolos/genéticaRESUMO
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.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microautofagia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Microautofagia/genética , Modelos Biológicos , Mutação , Fosforilação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic cell proliferation is controlled by growth factors and essential nutrients, in the absence of which cells may enter into a quiescent (G(0)) state. In yeast, nitrogen and/or carbon limitation causes downregulation of the conserved TORC1 and PKA signaling pathways and, consequently, activation of the PAS kinase Rim15, which orchestrates G(0) program initiation and ensures proper life span by controlling distal readouts, including the expression of specific genes. Here, we report that Rim15 coordinates transcription with posttranscriptional mRNA protection by phosphorylating the paralogous Igo1 and Igo2 proteins. This event, which stimulates Igo proteins to associate with the mRNA decapping activator Dhh1, shelters newly expressed mRNAs from degradation via the 5'-3' mRNA decay pathway, thereby enabling their proper translation during initiation of the G(0) program. These results delineate a likely conserved mechanism by which nutrient limitation leads to stabilization of specific mRNAs that are critical for cell differentiation and life span.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Fase de Repouso do Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas , Carbono/metabolismo , Proteínas de Ciclo Celular/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Glucose/deficiência , Proteínas de Choque Térmico/genética , Mutação , Nitrogênio/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Tempo , Transcrição GênicaRESUMO
Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show that Ivy1, the yeast homolog of mammalian missing-in-metastasis (MIM), is a vacuolar effector of Ypt7-GTP and interacts with the EGO/ragulator complex, an activator of the target of rapamycin kinase complex 1 (TORC1) on vacuoles. Loss of Ivy1 does not affect EGO vacuolar localization and function. In combination with the deletion of individual subunits of the V-ATPase, however, we observed reduced TORC1 activity and massive enlargement of the vacuole surface. Consistent with this, Ivy1 localizes to invaginations at the vacuole surface and on liposomes in a phosphoinositide- and Ypt7-GTP-controlled manner, which suggests a role in microautophagy. Our data, thus, reveal that Ivy1 is a novel regulator of vacuole membrane homeostasis with connections to TORC1 signaling.
Assuntos
Proteínas de Transporte/metabolismo , Homeostase , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Autofagia , Endocitose , Alvo Mecanístico do Complexo 1 de Rapamicina , Modelos Biológicos , Complexos Multiproteicos , Fosfatidilinositóis/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/ultraestrutura , Transdução de Sinais , Serina-Treonina Quinases TOR , Vacúolos/ultraestruturaRESUMO
The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1(GTP), which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1(GDP), caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Membranas Intracelulares/enzimologia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Vacúolos/enzimologia , Proteínas Adaptadoras de Transporte Vesicular/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/metabolismo , Cicloeximida/farmacologia , Proteínas de Ligação a DNA/metabolismo , Endossomos/enzimologia , Fatores de Troca do Nucleotídeo Guanina/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos , Mutação , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Sirolimo/farmacologia , Fatores de Tempo , Fatores de Transcrição/metabolismo , Vacúolos/efeitos dos fármacosRESUMO
The eukaryotic TORC1 kinase integrates and links nutritional, energy, and hormonal signals to cell growth and homeostasis, and its deregulation is associated with human diseases including neurodegeneration, cancer, and metabolic syndrome. Quantification of TORC1 activities in various genetic settings and defined physiological conditions generally relies on the assessment of the phosphorylation level of residues in TORC1 targets. Here we show that two commonly used TORC1 effectors in yeast, namely Sch9 and Rps6, exhibit distinct phosphorylation patterns in response to rapamycin treatment or changes in nitrogen availability, indicating that the choice of TORC1 proxies introduces a bias in decoding TORC1 activity.
RESUMO
Organelles of the endomembrane system contain Rab GTPases as identity markers. Their localization is determined by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). It remains largely unclear how these regulators are specifically targeted to organelles and how their activity is regulated. Here, we focus on the GAP Gyp7, which acts on the Rab7-like Ypt7 protein in yeast, and surprisingly observe the protein exclusively in puncta proximal to the vacuole. Mistargeting of Gyp7 to the vacuole strongly affects vacuole morphology, suggesting that endosomal localization is needed for function. In agreement, efficient endolysosomal transport requires Gyp7. In vitro assays reveal that Gyp7 requires a distinct lipid environment for membrane binding and activity. Overexpression of Gyp7 concentrates Ypt7 in late endosomes and results in resistance to rapamycin, an inhibitor of the target of rapamycin complex 1 (TORC1), suggesting that these late endosomes are signaling endosomes. We postulate that Gyp7 is part of regulatory machinery involved in late endosome function.
Assuntos
Endossomos , Proteínas de Saccharomyces cerevisiae , Proteínas rab de Ligação ao GTP , Proteínas Ativadoras de ras GTPase , Transporte Biológico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Vacúolos , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Caloric restriction and intermittent fasting prolong the lifespan and healthspan of model organisms and improve human health. The natural polyamine spermidine has been similarly linked to autophagy enhancement, geroprotection and reduced incidence of cardiovascular and neurodegenerative diseases across species borders. Here, we asked whether the cellular and physiological consequences of caloric restriction and fasting depend on polyamine metabolism. We report that spermidine levels increased upon distinct regimens of fasting or caloric restriction in yeast, flies, mice and human volunteers. Genetic or pharmacological blockade of endogenous spermidine synthesis reduced fasting-induced autophagy in yeast, nematodes and human cells. Furthermore, perturbing the polyamine pathway in vivo abrogated the lifespan- and healthspan-extending effects, as well as the cardioprotective and anti-arthritic consequences of fasting. Mechanistically, spermidine mediated these effects via autophagy induction and hypusination of the translation regulator eIF5A. In summary, the polyamine-hypusination axis emerges as a phylogenetically conserved metabolic control hub for fasting-mediated autophagy enhancement and longevity.
RESUMO
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-PP2A(Cdc55) effector branch, which reportedly also controls Gis1 promoter recruitment. These data pinpoint PP2A(Cdc55) as a central node in homo-directional coordination of transcription and post-transcriptional mRNA stabilization of a specific array of nutrient-regulated genes.
Assuntos
Fases de Leitura Aberta , Estabilidade de RNA , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Estresse Fisiológico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Meia-Vida , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , RNA Fúngico/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Yeast cells are equipped with different nutrient signaling pathways that enable them to sense the availability of various nutrients and adjust metabolism and growth accordingly. These pathways are part of an intricate network since most of them are cross-regulated and subject to feedback regulation at different levels. In yeast, a central role is played by Sch9, a protein kinase that functions as a proximal effector of the conserved growth-regulatory TORC1 complex to mediate information on the availability of free amino acids. However, recent studies established that Sch9 is more than a TORC1-effector as its activity is tuned by several other kinases. This allows Sch9 to function as an integrator that aligns different input signals to achieve accuracy in metabolic responses and stress-related molecular adaptations. In this review, we highlight the latest findings on the structure and regulation of Sch9, as well as its role as a nutrient-responsive hub that impacts on growth and longevity of yeast cells. Given that most key players impinging on Sch9 are well-conserved, we also discuss how studies on Sch9 can be instrumental to further elucidate mechanisms underpinning healthy aging in mammalians.
RESUMO
The AMP-activated protein kinase (AMPK) and the target of rapamycin complex 1 (TORC1) are central kinase modules of two opposing signaling pathways that control eukaryotic cell growth and metabolism in response to the availability of energy and nutrients. Accordingly, energy depletion activates AMPK to inhibit growth, while nutrients and high energy levels activate TORC1 to promote growth. Both in mammals and lower eukaryotes such as yeast, the AMPK and TORC1 pathways are wired to each other at different levels, which ensures homeostatic control of growth and metabolism. In this context, a previous study (Hughes Hallett et al., 2015) reported that AMPK in yeast, that is Snf1, prevents the transient TORC1 reactivation during the early phase following acute glucose starvation, but the underlying mechanism has remained elusive. Using a combination of unbiased mass spectrometry (MS)-based phosphoproteomics, genetic, biochemical, and physiological experiments, we show here that Snf1 temporally maintains TORC1 inactive in glucose-starved cells primarily through the TORC1-regulatory protein Pib2. Our data, therefore, extend the function of Pib2 to a hub that integrates both glucose and, as reported earlier, glutamine signals to control TORC1. We further demonstrate that Snf1 phosphorylates the TORC1 effector kinase Sch9 within its N-terminal region and thereby antagonizes the phosphorylation of a C-terminal TORC1-target residue within Sch9 itself that is critical for its activity. The consequences of Snf1-mediated phosphorylation of Pib2 and Sch9 are physiologically additive and sufficient to explain the role of Snf1 in short-term inhibition of TORC1 in acutely glucose-starved cells.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Mamíferos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding.
Assuntos
Acetil-CoA Carboxilase , Malonil Coenzima A , Animais , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Malonil Coenzima A/metabolismo , Serina-Treonina Quinases TOR/genética , Ácidos Graxos/metabolismo , Mamíferos/metabolismo , Trifosfato de AdenosinaRESUMO
Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are "auto- activating", even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of "canonical" mTORC1 substrates, such as S6K. By using HeLa and HK-2 cell lines, human induced pluripotent stem cell-derived cardiomyocytes and patient-derived primary fibroblasts, we show that RRAGD auto-activating mutations lead to inhibition of TFEB and TFE3 nuclear translocation and transcriptional activity, which impairs the response to lysosomal and mitochondrial injury. These data suggest that inhibition of MiT/TFE factors plays a key role in kidney tubulopathy and cardiomyopathy syndrome.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Células-Tronco Pluripotentes Induzidas , Humanos , Autofagia/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células HeLa , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/metabolismo , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , MutaçãoRESUMO
TORC1-dependent phosphorylation of Saccharomyces cerevisiae Sch9 was dramatically reduced upon exposure to a protonophore or in respiration-incompetent ρ(0) cells but not in respiration-incompetent pet mutants, providing important insight into the molecular mechanisms governing interorganellar signaling in general and retrograde signaling in particular.