RESUMO
Certain proteins assemble into diverse complex states, each having a distinct and unique function in the cell. Target of rapamycin (Tor) complex 1 (TORC1) plays a central role in signalling pathways that allow cells to respond to the environment, including nutritional status signalling. TORC1 is widely recognised for its association with various diseases. The budding yeast Saccharomyces cerevisiae has two types of TORC1, Tor1-containing TORC1 and Tor2-containing TORC1, which comprise different constituent proteins but are considered to have the same function. Here, we computationally modelled the relevant complex structures and then, based on the structures, rationally engineered a Tor2 mutant that could form Tor complex 2 (TORC2) but not TORC1, resulting in a redesign of the complex states. Functional analysis of the Tor2 mutant revealed that the two types of TORC1 induce different phenotypes, with changes observed in rapamycin, caffeine and pH dependencies of cell growth, as well as in replicative and chronological lifespan. These findings uncovered by a general approach with huge potential - model structure-based engineering - are expected to provide further insights into various fields such as molecular evolution and lifespan.
Assuntos
Saccharomyces cerevisiae , Saccharomycetales , Saccharomyces cerevisiae/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina , Fenótipo , SirolimoRESUMO
The stress response is one of the most fundamental cellular processes. Although the molecular mechanisms underlying responses to a single stressor have been extensively studied, cellular responses to multiple stresses remain largely unknown. Here, we characterized fission yeast cellular responses to a novel stress inducer, non-thermal atmospheric-pressure plasma. Plasma irradiation generates ultraviolet radiation, electromagnetic fields and a variety of chemically reactive species simultaneously, and thus can impose multiple stresses on cells. We applied direct plasma irradiation to fission yeast and showed that strong plasma irradiation inhibited fission yeast growth. We demonstrated that mutants lacking sep1 and ace2, both of which encode transcription factors required for proper cell separation, were resistant to plasma irradiation. Sep1-target transcripts were downregulated by mild plasma irradiation. We also demonstrated that plasma irradiation inhibited the target of rapamycin kinase complex 1 (TORC1). These observations indicate that two pathways, namely the Sep1-Ace2 cell separation pathway and TORC1 pathway, operate when fission yeast cope with multiple stresses induced by plasma irradiation.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Raios Ultravioleta , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismoRESUMO
The fission yeast Schizosaccharomyces pombe ecl family genes respond to various starvation signals and induce appropriate intracellular responses, including the extension of chronological lifespan and induction of sexual differentiation. Herein, we propose that the colonization of hemocoel 1 (COH1) protein of Metarhizium robertsii, an insect-pathogenic fungus, is a functional homolog of S. pombe Ecl1 family proteins.
RESUMO
In the fission yeast Schizosaccharomyces pombe, the duration of survival in the stationary phase, termed the chronological lifespan (CLS), is affected by various environmental factors and the corresponding gene activities. The ecl family genes were identified in the genomic region encoding non-coding RNA as positive regulators of CLS in S. pombe, and subsequently shown to encode relatively short proteins. Several studies revealed that ecl family genes respond to various nutritional starvation conditions via different mechanisms, and they are additionally involved in stress resistance, autophagy, sexual differentiation, and cell cycle control. Recent studies reported that Ecl family proteins strongly suppress target of rapamycin complex 1, which is a conserved eukaryotic nutrient-sensing kinase complex that also regulates longevity in a variety of organisms. In this review, we introduce the regulatory mechanisms of Ecl family proteins and discuss their emerging findings.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Longevidade/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Ciclo Celular , Regulação Fúngica da Expressão Gênica/genéticaRESUMO
In Schizosaccharomyces pombe, ecl family genes are induced by several signals, such as starvation of various nutrients, including sulfur, amino acids and Mg2+, and environmental stress, including heat or oxidative stress. These genes mediate appropriate cellular responses and contribute to the maintenance of cell viability and induction of sexual differentiation. Although this yeast has three ecl family genes with overlapping functions, any environmental conditions that induce ecl3+ remain unidentified. We demonstrate that ecl3+ is induced by phosphate starvation, similar to its chromosomally neighboring genes, pho1+ and pho84+, which respectively encode an extracellular acid phosphatase and an inorganic phosphate transporter. ecl3+ expression was induced by the transcription factor Pho7 and affected by the cyclin-dependent kinase (CDK)-activating kinase Csk1. Phosphate starvation induced G1 arrest and sexual differentiation via ecl family genes. Biochemical analyses suggested that this G1 arrest was mediated by the stabilization of the CDK inhibitor Rum1, which was dependent on ecl family genes. This study shows that ecl family genes are required for appropriate responses to phosphate starvation and provides novel insights into the diversity and similarity of starvation responses.
Assuntos
Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Fosfatos/metabolismo , Diferenciação Sexual , Fatores de Transcrição/metabolismo , Regulação Fúngica da Expressão GênicaRESUMO
Nutrients including glucose, nitrogen, sulfur, zinc, and iron are involved in the regulation of chronological lifespan (CLS) of yeast, which serves as a model of the lifespan of differentiated cells of higher organisms. Herein, we show that magnesium (Mg2+ ) depletion extends CLS of the fission yeast Schizosaccharomyces pombe through a mechanism involving the Ecl1 gene family. We discovered that ecl1+ expression, which extends CLS, responds to Mg2+ depletion. Therefore, we investigated the underlying intracellular responses. In amino acid auxotrophic strains, Mg2+ depletion robustly induces ecl1+ expression through the activation of the general amino acid control (GAAC) pathway-the equivalent of the amino acid response of mammals. Polysome analysis indicated that the expression of Ecl1 family genes was required for regulating ribosome amount when cells were starved, suggesting that Ecl1 family gene products control the abundance of ribosomes, which contributes to longevity through the activation of the evolutionarily conserved GAAC pathway. The present study extends our understanding of the cellular response to Mg2+ depletion and its influence on the mechanism controlling longevity.
Assuntos
Aminoácidos/metabolismo , Magnésio/metabolismo , Proteínas Nucleares/fisiologia , Ribossomos/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Ciclo Celular , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Longevidade , Nutrientes/metabolismoRESUMO
Cold atmospheric plasma (CAP) has attracted much attention in the fields of biotechnology and medicine owing to its potential utility in clinical applications. Recently accumulating evidence has demonstrated that CAP influences protein structures. However, there remain open questions regarding the molecular mechanisms behind the CAP-induced structural perturbations of biomacromolecules. Here, we investigated the potential effects of CAP irradiation of amyloid ß (Aß), an amyloidogenic protein associated with Alzheimer's disease. Using nuclear magnetic resonance spectroscopy, we observed gradual spectral changes in Aß after a 10 s CAP pretreatment, which also suppressed its fibril formation, as revealed by thioflavin T assay. As per mass spectrometric analyses, these effects were attributed to selective oxidation of the methionine residue (Met) at position 35. Interestingly, this modification occurred when Aß was dissolved into a pre-irradiated buffer, indicating that some reactive species oxidize the Met residue. Our results strongly suggest that the H2O2 generated in the solution by CAP irradiation is responsible for Met oxidation, which inhibits Aß amyloid formation. The findings of the present study provide fundamental insights into plasma biology, giving clues for developing novel applications of CAP.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Gases em Plasma/farmacologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/ultraestrutura , Fluorescência , Espectroscopia de Ressonância Magnética , Metionina/metabolismo , Oxirredução , Agregados ProteicosRESUMO
Target of rapamycin (TOR) is a serine/threonine kinase that modulates cell growth and metabolism in response to environmental changes. Transfer RNA (tRNA) is an abundant and ubiquitous small non-coding RNA that is essential in the translation of mRNAs. Beyond its canonical role, it has been revealed that tRNAs have more diverse functions. TOR complex 1 (TORC1), which is one of the two TOR complexes, regulates tRNA synthesis by controlling RNA polymerase III. In addition to tRNA synthesis regulation, recent studies have revealed hidden connections between TORC1 and tRNA, which are both essential players in eukaryotic cellular activities. Here, we review the accumulating findings on the regulatory links between TORC1 and tRNA-particularly those links in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , RNA de Transferência/metabolismo , RNA não Traduzido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Regulação Fúngica da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Accurate target recognition in transcript degradation is crucial for regulation of gene expression. In the fission yeast Schizosaccharomyces pombe, a number of meiotic transcripts are recognized by a YTH-family RNA-binding protein, Mmi1, and selectively degraded by the nuclear exosome during mitotic growth. Mmi1 forms nuclear foci in mitotically growing cells, and the nuclear exosome colocalizes to such foci. However, it remains elusive how Mmi1 and the nuclear exosome are connected. Here, we show that a complex called MTREC (Mtl1-Red1 core) or NURS (nuclear RNA silencing) that consists of a zinc-finger protein, Red1, and an RNA helicase, Mtl1, is required for the recruitment of the nuclear exosome to Mmi1 foci. Physical interaction between Mmi1 and the nuclear exosome depends on Red1. Furthermore, a chimeric protein involving Mmi1 and Rrp6, which is a nuclear-specific component of the exosome, suppresses the ectopic expression phenotype of meiotic transcripts in red1Δ cells and mtl1 mutant cells. These data indicate that the primary function of MTREC/NURS in meiotic transcript elimination is to link Mmi1 to the nuclear exosome physically.
Assuntos
Proteínas de Transporte/metabolismo , Exossomos/metabolismo , Regulação Fúngica da Expressão Gênica , Meiose/genética , Interferência de RNA , Proteínas de Schizosaccharomyces pombe/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleases/genética , Ribonucleases/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Accurate and extensive regulation of meiotic gene expression is crucial to distinguish germ cells from somatic cells. In the fission yeast Schizosaccharomyces pombe, a YTH family RNA-binding protein, Mmi1, directs the nuclear exosome-mediated elimination of meiotic transcripts during vegetative proliferation. Mmi1 also induces the formation of facultative heterochromatin at a subset of its target genes. Here, we show that Mmi1 prevents the mistimed expression of meiotic proteins by tethering their mRNAs to the nuclear foci. Mmi1 interacts with itself with the assistance of a homolog of Enhancer of Rudimentary, Erh1. Mmi1 self-interaction is required for foci formation, target transcript elimination, their nuclear retention, and protein expression inhibition. We propose that nuclear foci formed by Mmi1 are not only the site of RNA degradation, but also of sequestration of meiotic transcripts from the translation machinery.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Regulação Fúngica da Expressão Gênica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Proteínas de Transporte/metabolismo , Ligação Proteica , Estabilidade de RNARESUMO
Target of rapamycin (TOR) kinase controls cell growth and metabolism in response to nutrient availability. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1) promotes vegetative growth and inhibits sexual differentiation in the presence of ample nutrients. Here, we report the isolation and characterization of mutants with similar phenotypes as TORC1 mutants, in that they initiate sexual differentiation even in nutrient-rich conditions. In most mutants identified, TORC1 activity is downregulated and the mutated genes are involved in tRNA expression or modification. Expression of tRNA precursors decreases when cells undergo sexual differentiation. Furthermore, overexpression of tRNA precursors prevents TORC1 downregulation upon nitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we propose that tRNA precursors operate in the S. pombe TORC1 pathway to switch growth mode from vegetative to reproductive.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/genética , RNA de Transferência/genética , Schizosaccharomyces/crescimento & desenvolvimento , Diferenciação Sexual/genética , Proliferação de Células/genética , Regulação Fúngica da Expressão Gênica , Nitrogênio/metabolismo , Nutrientes/genética , Nutrientes/metabolismo , Fosforilação , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Transdução de Sinais/genéticaRESUMO
Target of rapamycin (TOR) kinase controls cell metabolism and growth in response to environmental cues such as nutrients, growth factors, and stress. TOR kinase is widely conserved across eukaryotes. As in other organisms, the fission yeast Schizosaccharomyces pombe has two types of TOR complex, namely TOR complex 1 (TORC1) and TORC2. It is interesting that the two TOR complexes in S. pombe have opposite roles in sexual differentiation, which is induced by nutrient starvation. TORC1, which contains Tor2 as a catalytic subunit, promotes vegetative growth and represses sexual differentiation in nutrient-rich conditions, while TORC2 is required for the initiation of sexual differentiation. Multiple targets of TORC1 have been identified. Some of these, such as S6 kinase and an autophagy regulator Atg13, are known targets in other organisms. In addition, there is a novel group of TORC1 targets involved in the regulation of sexual differentiation. Here, we review recent findings on phosphorylation targets of TORC1 in S. pombe. Furthermore, we briefly report a novel S. pombe target of TORC1.
Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas Relacionadas à Autofagia/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Fosforilação , Proteínas Quinases S6 Ribossômicas/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Target of rapamycin (TOR) kinase regulates cell metabolism and growth, acting as a subunit of two multi-protein complexes, TORC1 and TORC2. Known TORC substrates are either kinases or general factors involved in growth control. Here, we show that fission yeast TORC1, which promotes vegetative growth and suppresses sexual development, can phosphorylate Mei2 (a specific factor involved in switching the cell fate) in vitro. Alanine substitutions at the nine Mei2 phosphorylation sites stabilize the protein and promote mating and meiosis in vivo. We found that Mei2 is polyubiquitylated in vivo in a TORC1-dependent manner. Based on these data, we propose that TORC1 contributes to the suppression of sexual development by phosphorylating Mei2, in addition to controlling the cellular metabolic status.
Assuntos
Complexos Multiproteicos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Serina-Treonina Quinases TOR/fisiologia , Ubiquitinação , Epistasia Genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Complexo de Endopeptidases do Proteassoma , Estabilidade Proteica , Proteólise , Schizosaccharomyces/crescimento & desenvolvimentoRESUMO
Rheb GTPase and the Tsc1-Tsc2 protein complex, which serves as a GTPase-activating protein for Rheb, have crucial roles in the regulation of cell growth in response to extracellular conditions. In Schizosaccharomyces pombe, Rheb and Tsc1-Tsc2 regulate cell cycle progression, the onset of meiosis and the uptake of amino acids. In cells lacking Tsc2 (Δtsc2), the amino acid transporter Aat1, which is normally expressed on the plasma membrane under starvation conditions, is confined to the Golgi. Here, we show that the loss of either pub1(+), encoding an E3 ubiquitin ligase, or any1(+), encoding a ß-arrestin-like protein, allows constitutive expression of Aat1 on the plasma membrane in Δtsc2 cells, suggesting that Pub1 and Any1 are required for localization of Aat1 to the Golgi. Subsequent analysis revealed that, in the Golgi, Pub1 and Any1 form a complex that ubiquitylates Aat1. Physical interaction of Pub1 and Any1 is more stable in Δtsc2 cells than in wild-type cells and is independent of Tor2 activity. These results indicate that the TSC-Rheb signaling pathway regulates the localization of amino acid transporters via Pub1 and Any1 in a Tor2-independent manner. Our study demonstrates that, unlike in budding yeast (in which Rsp5 and ARTs, a pair of proteins analogous to Pub1 and Any1, respectively, primarily act to reduce expression of the transporters on plasma membrane when nutrients are abundant), the primary role of fission yeast Pub1 and Any1 is to store the transporter in the Golgi under nutrient-rich conditions.
Assuntos
Arrestinas/metabolismo , Carbono-Nitrogênio Ligases/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Sistemas de Transporte de Aminoácidos Básicos/biossíntese , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Arrestinas/deficiência , Arrestinas/genética , Carbono-Nitrogênio Ligases/deficiência , Carbono-Nitrogênio Ligases/genética , Ciclo Celular , Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Meiose , Complexos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais , beta-ArrestinasRESUMO
Target of rapamycin (TOR), an evolutionarily conserved serine/threonine protein kinase, plays pivotal roles in several important cellular processes in eukaryotes. In the fission yeast Schizosaccharomyces pombe, TOR complex 1 (TORC1), which includes Tor2 as a catalytic subunit, manages the switch between cell proliferation and differentiation by sensing nutrient availability. However, little is known about the direct target of TORC1 that plays key roles in nutrient-dependent TORC1 signaling in fission yeast. Here we report that in fission yeast, three AGC kinase family members, named Psk1, Sck1 and Sck2, which exhibit high homology with human S6K1, are phosphorylated under nutrient-rich conditions and are dephosphorylated by starvation conditions. Among these, Psk1 is necessary for phosphorylation of ribosomal protein S6. Furthermore, Psk1 phosphorylation is regulated by TORC1 in nutrient-dependent and rapamycin-sensitive manners in vivo. Three conserved regulatory motifs (the activation loop, the hydrophobic and the turn motifs) in Psk1 are phosphorylated and these modifications are required for Psk1 activity. In particular, phosphorylation of the hydrophobic motif is catalyzed by TORC1 in vivo and in vitro. Ksg1, a homolog of PDK1, is also important for Psk1 phosphorylation in the activation loop and for its activity. The TORC1 components Pop3, Toc1 and Tco89, are dispensable for Psk1 regulation, but disruption of pop3(+) causes an increase in the sensitivity of TORC1 to rapamycin. Taken together, these results provide convincing evidence that TORC1/Psk1/Rps6 constitutes a nutrient-dependent signaling pathway in fission yeast.
Assuntos
Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Schizosaccharomyces/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Quinases S6 Ribossômicas/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/genéticaRESUMO
The number of root nodules developing on legume roots after rhizobial infection is controlled by the plant shoot through autoregulation and mutational inactivation of this mechanism leads to hypernodulation. We have characterised the Pisum sativum (pea) Sym28 locus involved in autoregulation and shown that it encodes a protein similar to the Arabidopsis CLAVATA2 (CLV2) protein. Inactivation of the PsClv2 gene in four independent sym28 mutant alleles, carrying premature stop codons, results in hypernodulation of the root and changes to the shoot architecture. In the reproductive phase sym28 shoots develops additional flowers, the stem fasciates, and the normal phyllotaxis is perturbed. Mutational substitution of an amino acid in one leucine rich repeat of the corresponding Lotus japonicus LjCLV2 protein results in increased nodulation. Similarly, down-regulation of the Lotus Clv2 gene by RNAi mediated reduction of the transcript level also resulted in increased nodulation. Gene expression analysis of LjClv2 and Lotus hypernodulation aberrant root formation Har1 (previously shown to regulate nodule numbers) indicated they have overlapping organ expression patterns. However, we were unable to demonstrate a direct protein-protein interaction between LjCLV2 and LjHAR1 proteins in contrast to the situation between equivalent proteins in Arabidopsis. LjHAR1 was localised to the plasma membrane using a YFP fusion whereas LjCLV2-YFP localised to the endoplasmic reticulum when transiently expressed in Nicotiana benthamiana leaves. This finding is the most likely explanation for the lack of interaction between these two proteins.
Assuntos
Genes de Plantas , Lotus/genética , Lotus/fisiologia , Pisum sativum/genética , Pisum sativum/fisiologia , Nodulação/genética , Nodulação/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , DNA de Plantas/genética , Homeostase/genética , Homeostase/fisiologia , Lotus/crescimento & desenvolvimento , Modelos Biológicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pisum sativum/crescimento & desenvolvimento , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Interferência de RNA , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Nicotiana/genética , Nicotiana/fisiologiaRESUMO
Fission yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this yeast, as in budding yeast and mammals. Fission yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type fission yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this yeast. It appears that fission yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.
Assuntos
Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Transdução de Sinais , Animais , Fase G1 , Schizosaccharomyces/citologia , Schizosaccharomyces/efeitos dos fármacos , Sirolimo/farmacologiaRESUMO
Rheb is a unique member of the Ras superfamily GTP-binding proteins. We as well as others previously have shown that Rheb is a critical component of the TSC/TOR signaling pathway. In fission yeast, Rheb is encoded by the rhb1 gene. Rhb1p is essential for growth and directly interacts with Tor2p. In this article, we report identification of 22 single amino acid changes in the Tor2 protein that enable growth in the absence of Rhb1p. These mutants also exhibit decreased mating efficiency. Interestingly, the mutations are located in the C-terminal half of the Tor2 protein, clustering mainly within the FAT and kinase domains. We noted some differences in the effect of a mutation in the FAT domain (L1310P) and in the kinase domain (E2221K) on growth and mating. Although the Tor2p mutations bypass Rhb1p's requirement for growth, they are incapable of suppressing Rhb1p's requirement for resistance to stress and toxic amino acids, pointing to multiple functions of Rhb1p. In mammalian systems, we find that mammalian target of rapamycin (mTOR) carrying analogous mutations (L1460P or E2419K), although sensitive to rapamycin, exhibits constitutive activation even when the cells are starved for nutrients. These mutations do not show significant difference in their ability to form complexes with Raptor, Rictor, or mLST8. Furthermore, we present evidence that mutant mTOR can complex with wild-type mTOR and that this heterodimer is active in nutrient-starved cells.
Assuntos
Fosfatidilinositol 3-Quinases/genética , Mutação Puntual/genética , Proteínas Quinases/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Transdução de Sinais/genética , Sequência de Aminoácidos , Dimerização , GTP Fosfo-Hidrolases/genética , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Schizosaccharomyces/crescimento & desenvolvimento , Especificidade da Espécie , Serina-Treonina Quinases TORRESUMO
Fission yeast has two TOR (target of rapamycin) kinases, namely Tor1 and Tor2. Tor1 is required for survival under stressed conditions, proper G(1) arrest, and sexual development. In contrast, Tor2 is essential for growth. To analyze the functions of Tor2, we constructed two temperature-sensitive tor2 mutants. Interestingly, at the restrictive temperature, these mutants mimicked nitrogen starvation by arresting the cell cycle in G(1) phase and initiating sexual development. Microarray analysis indicated that expression of nitrogen starvation-responsive genes was induced extensively when Tor2 function was suppressed, suggesting that Tor2 normally mediates a signal from the nitrogen source. As with mammalian and budding yeast TOR, we find that fission yeast TOR also forms multiprotein complexes analogous to TORC1 and TORC2. The raptor homologue, Mip1, likely forms a complex predominantly with Tor2, producing TORC1. The rictor/Avo3 homologue, Ste20, and the Avo1 homologue, Sin1, appear to form TORC2 mainly with Tor1 but may also bind Tor2. The Lst8 homologue, Wat1, binds to both Tor1 and Tor2. Our analysis shows, with respect to promotion of G(1) arrest and sexual development, that the loss of Tor1 (TORC2) and the loss of Tor2 (TORC1) exhibit opposite effects. This highlights an intriguing functional relationship among TOR kinase complexes in the fission yeast Schizosaccharomyces pombe.