RESUMO
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder resulting from motor neuron death. Approximately 10% of cases are familial (FALS), typically with a dominant inheritance mode. Despite numerous advances in recent years, nearly 50% of FALS cases have unknown genetic aetiology. Here we show that mutations within the profilin 1 (PFN1) gene can cause FALS. PFN1 is crucial for the conversion of monomeric (G)-actin to filamentous (F)-actin. Exome sequencing of two large ALS families showed different mutations within the PFN1 gene. Further sequence analysis identified 4 mutations in 7 out of 274 FALS cases. Cells expressing PFN1 mutants contain ubiquitinated, insoluble aggregates that in many cases contain the ALS-associated protein TDP-43. PFN1 mutants also display decreased bound actin levels and can inhibit axon outgrowth. Furthermore, primary motor neurons expressing mutant PFN1 display smaller growth cones with a reduced F/G-actin ratio. These observations further document that cytoskeletal pathway alterations contribute to ALS pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Predisposição Genética para Doença/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Profilinas/genética , Profilinas/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/metabolismo , Animais , Axônios/metabolismo , Axônios/patologia , Células Cultivadas , Exoma/genética , Feminino , Cones de Crescimento/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Judeus/genética , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Proteínas Mutantes/genética , Linhagem , Conformação Proteica , Ubiquitinação , População Branca/genéticaRESUMO
The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.
Assuntos
Núcleo Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Carioferinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transporte Ativo do Núcleo Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , Glucose/metabolismo , Carioferinas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Transporte de RNA , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In all eukaryotes, transcribed precursor tRNAs are maturated by processing and modification processes in nucleus and are transported to the cytoplasm. The cytoplasmic export protein (Cex1p) captures mature tRNAs from the nuclear export receptor (Los1p) on the cytoplasmic side of the nuclear pore complex, and it delivers them to eukaryotic elongation factor 1α. This conserved Cex1p function is essential for the quality control of mature tRNAs to ensure accurate translation. However, the structural basis of how Cex1p recognizes tRNAs and shuttles them to the translational apparatus remains unclear. Here, we solved the 2.2 Å resolution crystal structure of Saccharomyces cerevisiae Cex1p with C-terminal 197 disordered residues truncated. Cex1p adopts an elongated architecture, consisting of N-terminal kinase-like and a C-terminal α-helical HEAT repeat domains. Structure-based biochemical analyses suggested that Cex1p binds tRNAs on its inner side, using the positively charged HEAT repeat surface and the C-terminal disordered region. The N-terminal kinase-like domain acts as a scaffold to interact with the Ran-exportin (Los1p·Gsp1p) machinery. These results provide the structural basis of Los1p·Gsp1p·Cex1p·tRNA complex formation, thus clarifying the dynamic mechanism of tRNA shuttling from exportin to the translational apparatus.
Assuntos
Modelos Moleculares , Proteínas de Transporte Nucleocitoplasmático/química , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Cristalografia por Raios X , Citoplasma/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Nuclear tRNA export plays an essential role in key cellular processes such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage and development. Like other nuclear export processes, assembly of the nuclear tRNA export complex in the nucleus is dependent on Ran-GTP/Gsp1p-GTP, and dissociation of the export receptor-tRNA-Ran-GTP/Gsp1p-GTP complex in the cytoplasm requires RanBP1/Yrb1p and RanGAP/Rna1p to activate the GTPase activity of Ran-GTP/Gsp1p-GTP. The Saccharomyces cerevisiae Cex1p and Human Scyl1 have also been proposed to participate in unloading of the tRNA export receptors at the cytoplasmic face of the nuclear pore complex (NPC). Here, we provide evidence suggesting that Cex1p is required for activation of the GTPase activity of Gsp1p and dissociation of the receptor-tRNA-Gsp1p export complex in S. cerevisiae. The data suggest that Cex1p recruits Rna1p from the cytoplasm to the NPC and facilitates Rna1p activation of the GTPase activity of Gsp1p by enabling Rna1p to gain access to Gsp1p-GTP bound to the export receptor tRNA complex. It is possible that this tRNA unloading mechanism is conserved in evolutionarily diverse organisms and that other Gsp1p-GTP-dependent export processes use a pathway-specific component to recruit Rna1p to the NPC.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA/fisiologia , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas Ativadoras de GTPase/genética , Guanosina Trifosfato/metabolismo , Carioferinas/metabolismo , Modelos Biológicos , Mutação/fisiologia , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Ligação Proteica/fisiologia , RNA Mensageiro/metabolismo , RNA de Transferência de Glicina/metabolismo , RNA de Transferência de Tirosina/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Utp8p is an essential nucleolar protein that channels aminoacyl-tRNAs from aminoacyl-tRNA synthetases in the nucleolus to the nuclear tRNA export receptors located in the nucleoplasm and nuclear pore complex in Saccharomyces cerevisiae. Utp8p is also part of the U3 snoRNA-associated protein complex involved in 18S rRNA biogenesis in the nucleolus. We report that Utp22p, which is another member of the U3 snoRNA-associated protein complex, is also an intranuclear component of the nuclear tRNA export machinery. Depletion of Utp22p results in nuclear retention of mature tRNAs derived from intron-containing and intronless precursors. Moreover, Utp22p copurifies with the nuclear tRNA export receptor Los1p, the aminoacyl-tRNA synthetase Tys1p and Utp8p, but not with the RanGTPase Gsp1p and the nuclear tRNA export receptor Msn5p. Utp22p interacts directly with Utp8p and Los1p in a tRNA-independent manner in vitro. Utp22p also interacts directly with Tys1p, but this binding is stimulated when Tys1p is bound to tRNA. However, Utp22p, unlike Utp8p, does not bind tRNA saturably. These data suggest that Utp22p recruits Utp8p to aminoacyl-tRNA synthetases in the nucleolus to collect aminoacyl-tRNA and then accompanies the Utp8p-tRNA complex to deliver the aminoacyl-tRNAs to Los1p but not Msn5p. It is possible that Nrap/Nol6, the mammalian orthologue of Utp22p, plays a role in channelling aminoacyl-tRNA to the nuclear tRNA export receptor exportin-t.
Assuntos
Nucléolo Celular/metabolismo , Carioferinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Carioferinas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Transporte de RNA , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The mono-ADP-ribosyltransferase toxins are bacterial virulence factors that contribute to many disease states in plants, animals, and humans. These toxins function as enzymes that target various host proteins and covalently attach an ADP-ribose moiety that alters target protein function. We tested compounds from a virtual screen of commercially available compounds combined with a directed poly(ADP-ribose) polymerase (PARP) inhibitor library and found several compounds that bind tightly and inhibit toxins from Pseudomonas aeruginosa and Vibrio cholerae. The most efficacious compounds completely protected human lung epithelial cells against the cytotoxicity of these bacterial virulence factors. Moreover, we determined high-resolution crystal structures of the best inhibitors in complex with cholix toxin to reveal important criteria for inhibitor binding and mechanism of action. These results provide new insight into development of antivirulence compounds for treating many bacterial diseases.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Antibacterianos/farmacologia , Fatores de Ribosilação do ADP/antagonistas & inibidores , Antibacterianos/efeitos adversos , Toxinas Bacterianas/antagonistas & inibidores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Inibidores de Poli(ADP-Ribose) Polimerases , Pseudomonas aeruginosa/enzimologiaRESUMO
Eukaryotic cells adapt to changes in nutrient levels by regulating key processes, such as gene transcription, ribosome biogenesis, and protein translation. Several studies have shown that nuclear export of tRNAs is also regulated in Saccharomyces cerevisiae and rat hepatoma H4IIE cells during nutrient stress. However, recent studies suggest that nutrient stress does not affect nuclear tRNA export in several mammalian cell lines, including rat hepatoma H4IIE. Furthermore, in contrast to previous studies, data reported more recently established that nuclear export of mature tRNAs derived from intron-containing pre-tRNAs, but not mature tRNAs made from intronless precursors, is affected by nutrient stress in several species of Saccharomyces, but not in the yeast Kluyveromyces lactis . Here, we provide evidence suggesting that Schizosaccharomyces pombe, like mammalian cells and K. lactis, but unlike Saccharomyces, do not directly regulate nuclear export of mature tRNAs made from intron-containing pre-tRNAs in response to nutrient stress. These studies collectively suggest that regulation of nuclear export of spliced tRNAs to the cytoplasm in response to nutrient availability may be limited to the genus Saccharomyces, which unlike other yeasts and higher eukaryotes produce energy for fermentative growth using respiration-independent pathways by downregulating the citric acid cycle and the electron transport chain.
Assuntos
Splicing de RNA , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Transporte Ativo do Núcleo Celular , Aminoácidos/metabolismo , Western Blotting , Núcleo Celular/metabolismo , Meios de Cultura/metabolismo , Citoplasma/metabolismo , Nitrogênio/metabolismo , Fosforilação , Especificidade da EspécieRESUMO
Utp8p is an essential nucleolar component of the nuclear tRNA export machinery in Saccharomyces cerevisiae. It is thought to act at a step between tRNA maturation/aminoacylation and translocation of the tRNA across the nuclear pore complex. To understand the function of Utp8p in nuclear tRNA export, a comprehensive affinity purification analysis was conducted to identify proteins that interact with Utp8p in vivo. In addition to finding proteins that have been shown previously to copurify with Utp8p, a number of new interactions were identified. These interactions include aminoacyl-tRNA synthetases, the RanGTPase Gsp1p, and nuclear tRNA export receptors such as Los1p and Msn5p. Characterization of the interaction of Utp8p with a subset of the newly identified proteins suggests that Utp8p most likely transfer tRNAs to the nuclear tRNA export receptors by using a channeling mechanism.
Assuntos
Nucléolo Celular/metabolismo , Complexos Multiproteicos/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Nucléolo Celular/enzimologia , Cromatografia de Afinidade , Espectrometria de Massas , Mutação/genética , Ligação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Receptores de Superfície Celular/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Tirosina-tRNA Ligase/metabolismoRESUMO
Nuclear tRNA export in Saccharomyces cerevisiae has been proposed to involve three pathways, designated Los1p-dependent, Los1p-independent nuclear aminoacylation-dependent, and Los1p- and nuclear aminoacylation-independent. Here, a comprehensive biochemical analysis was performed to identify tRNAs exported by the aminoacylation-dependent and -independent pathways of S. cerevisiae. Interestingly, the major tRNA species of at least 19 families were found in the aminoacylated form in the nucleus. tRNAs known to be exported by the export receptor Los1p were also aminoacylated in the nucleus of both wild-type and mutant Los1p strains. FISH (fluorescence in situ hybridization) analyses showed that tRNA(Tyr) co-localizes with the U18 small nucleolar RNA in the nucleolus of a tyrosyl-tRNA synthetase mutant strain defective in nuclear tRNA(Tyr) export because of a block in nuclear tRNA(Tyr) aminoacylation. tRNA(Tyr) was also found in the nucleolus of a utp8 mutant strain defective in nuclear tRNA export but not nuclear tRNA aminoacylation. These results strongly suggest that the nuclear aminoacylation-dependent pathway is principally responsible for tRNA export in S. cerevisiae and that Los1p is an export receptor of this pathway. It is also likely that in mammalian cells tRNAs are mainly exported from the nucleus by the nuclear aminoacylation-dependent pathway. In addition, the data are consistent with the idea that nuclear aminoacylation is used as a quality control mechanism for ensuring nuclear export of only mature and functional tRNAs, and that this quality assurance step occurs in the nucleolus.
Assuntos
Núcleo Celular/metabolismo , Aminoacil-RNA de Transferência/metabolismo , RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Nucléolo Celular/química , Núcleo Celular/química , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Aminoacil-RNA de Transferência/análise , RNA de Transferência de Tirosina/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de SinaisRESUMO
The yeast two-hybrid system was used to identify peptide inhibitors of exotoxin A of Pseudomonas aeruginosa with the goal of using these to design peptide-based drugs against the toxin. A random peptide library consisting of 10(7) peptides ranging in length from 16 to 63 residues was screened for peptides that interact with the C-domain of exotoxin A. From the 10(7) transformants screened, three unique peptides of 63, 61 and 25 amino acids in length were found to specifically interact with the enzyme domain. The genes encoding these peptides were cloned and expressed as fusion proteins with the maltose-binding protein. In vitro inhibition measurements indicated that two of the peptides were modest inhibitors of toxin enzyme activity. These peptides now provide the basis for the development of more potent inhibitors, which will serve as lead inhibitors for evolution of potent peptide-based therapeutics.
Assuntos
ADP Ribose Transferases/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Exotoxinas/antagonistas & inibidores , Pseudomonas aeruginosa/genética , Fatores de Virulência/antagonistas & inibidores , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Biblioteca de Peptídeos , Peptídeos/genética , Peptídeos/isolamento & purificação , Pseudomonas aeruginosa/crescimento & desenvolvimento , Técnicas do Sistema de Duplo-Híbrido , Leveduras , Exotoxina A de Pseudomonas aeruginosaRESUMO
Nuclear-cytoplasmic tRNA transport involves multiple pathways that are segregated by the involvement of distinct proteins. The tRNA export process begins in the nucleolus, where the functionality of newly produced tRNAs are tested by aminoacylation, and ends with the delivery of the exported aminoacyl tRNAs to the eukaryotic elongation factor eEF-1A for utilization in protein synthesis in the cytoplasm. Recent studies have identified a number of proteins that participate in nuclear tRNA export in both yeast and mammals. However, genetic and biochemical evidence suggest that additional components, which have yet to be identified, also participate in nuclear-cytoplasmic tRNA trafficking. Here we review key strategies that have led to the identification and characterization of proteins that are involved in the nuclear tRNA export process in yeasts and mammals. The approaches described will greatly facilitate the identification and delineation of the roles of new proteins involved in nuclear export of tRNAs to the cytoplasm.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Região Organizadora do Nucléolo/genética , Transporte de RNA/fisiologia , RNA de Transferência/genética , Aminoacilação de RNA de Transferência/fisiologia , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Genes Reporter/genética , Genes Supressores , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Carioferinas/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas Nucleares/genética , Fator 1 de Elongação de Peptídeos/genética , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-Híbrido , beta Carioferinas/metabolismoRESUMO
NTF2 is a cytosolic protein responsible for nuclear import of Ran, a small Ras-like GTPase involved in a number of critical cellular processes, including cell cycle regulation, chromatin organization during mitosis, reformation of the nuclear envelope following mitosis, and controlling the directionality of nucleocytoplasmic transport. Herein, we provide evidence for the first time that translocation of the mammalian NTF2 from the nucleus to the cytoplasm to collect Ran in the GDP form is subjected to regulation. Treatment of mammalian cells with polysorbitan monolaurate was found to inhibit nuclear export of tRNA and proteins, which are processes dependent on RanGTP in the nucleus, but not nuclear import of proteins. Inhibition of the export processes by polysorbitan monolaurate is specific and reversible, and is caused by accumulation of Ran in the cytoplasm because of a block in translocation of NTF2 to the cytoplasm. Nuclear import of Ran and the nuclear export processes are restored in polysorbitan monolaurate treated cells overproducing NTF2. Moreover, increased phosphorylation of a phospho-tyrosine protein and several phospho-threonine proteins was observed in polysorbitan monolaurate treated cells. Collectively, these findings suggest that nucleocytoplasmic translocation of NTF2 is regulated in mammalian cells, and may involve a tyrosine and/or threonine kinase-dependent signal transduction mechanism(s).
Assuntos
Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas da Gravidez/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Ácido Desoxicólico/farmacologia , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Sinais de Exportação Nuclear , Poro Nuclear/efeitos dos fármacos , Poro Nuclear/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotreonina/metabolismo , Fosfotirosina/metabolismo , Polissorbatos/farmacologia , Transporte Proteico/efeitos dos fármacos , Transporte de RNA/efeitos dos fármacos , RNA Mensageiro/metabolismo , RNA de Transferência/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirolimo/farmacologia , Proteína ran de Ligação ao GTP/antagonistas & inibidoresRESUMO
Cells respond to nutrient stress by regulating gene transcription and various key metabolic processes, including ribosome biogenesis and protein synthesis. Several studies have shown that yeasts and mammalian cells also regulate export of tRNAs from the nucleus to the cytosol in response to nutrient stress. However, nuclear export of tRNA in mammalian cells during nutrient stress is controversial, as it has been recently shown that nuclear-cytoplasmic transport of tRNAs in several mammalian cell lines is not affected by nutrient deprivation. Furthermore, contrary to previous studies, data reported recently indicate that nuclear export of mature tRNAs derived from intron-containing precursor tRNAs, but not tRNAs made from intronless precursors, is affected by nutrient availability in several Saccharomyces species, although not in Kluyveromyces lactis and Schizosaccharomyces pombe. Here, we report that plants, like mammals and some yeasts, but unlike Saccharomyces, do not directly regulate nuclear export of tRNA in response to nutrient stress, indicating that this process is not entirely conserved among evolutionarily diverse organisms.
Assuntos
Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Nicotiana/metabolismo , RNA de Transferência/metabolismo , Transporte Ativo do Núcleo Celular , Arabidopsis/citologia , Nitrogênio/metabolismo , Saccharomyces/metabolismo , Sacarose/metabolismo , Nicotiana/citologiaRESUMO
Intracellular trafficking of tRNA was long thought to be a one-way trip from the site of biogenesis in the nucleus to the translation machinery in the cytoplasm. This view has recently been challenged, however, by the discovery that tRNA can move retrograde from the cytoplasm back to the nucleus in Saccharomyces cerevisiae and rat hepatoma H4IIE cells during nutrient stress and in S. cerevisiae after intron-containing pre-tRNAs are spliced in the cytoplasm. Contrary to studies reported, we present data suggesting that nutrient stress does not cause retrograde transport of cytoplasmic tRNAs to the nucleus in rat hepatoma H4IIE cells, human HeLa and HEK293 cells, and the yeasts Kluyveromyces lactis and S. cerevisiae. However, the efficiency of nuclear re-export of retrograded spliced tRNA was severely affected in S. cerevisiae and two other Saccharomyces species deprived of nutrient. Collectively, the data suggest that nutrient stress does not cause nuclear import of cytoplasmic tRNA; instead, nutrient stress specifically regulates nuclear re-export of retrograded spliced tRNAs but not nuclear export of tRNAs made from intronless pre-tRNAs in Saccharomyces species. Furthermore, we provide evidence suggesting that Mtr10p and the Gsp1pGTP/Gsp1pGDP cycle are not involved in nuclear tRNA import in S. cerevisiae during nutrient stress.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Evolução Biológica , Núcleo Celular/genética , Citoplasma/genética , RNA de Transferência/metabolismo , Estresse Fisiológico , Aminoácidos/deficiência , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Kluyveromyces/citologia , Kluyveromyces/genética , Kluyveromyces/metabolismo , RNA de Transferência/genética , Ratos , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.
Assuntos
Poro Nuclear/metabolismo , RNA de Transferência/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transporte Vesicular , Animais , Células COS , Extratos Celulares , Chlorocebus aethiops , Códon sem Sentido/genética , Proteínas de Ligação a DNA , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Carioferinas/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fator 1 de Elongação de Peptídeos/isolamento & purificação , Fator 1 de Elongação de Peptídeos/metabolismo , Ligação Proteica , Aminoacil-RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Supressão Genética , Fatores de Transcrição/isolamento & purificação , Aminoacilação de RNA de Transferência/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
In Saccharomyces cerevisiae intron-containing pre-tRNAs are exported from the nucleus to the cytoplasm for removal of the introns, and the spliced tRNAs are returned to the nucleus for reasons that are not understood. The re-imported spliced tRNAs are then subjected to aminoacylation in the nucleolus to ensure that they are functional prior to re-export to the cytoplasm. Previous studies have shown that re-imported spliced tRNAs and mature tRNAs made entirely in the nucleus from intronless precursors are retained in the nucleus of S. cerevisiae in response to glucose, amino acid, nitrogen or inorganic phosphate deprivation. Contrary to these studies, we recently reported that starvation of S. cerevisiae of amino acids or nitrogen results in nuclear accumulation of re-imported spliced tRNAs, but not tRNAs made from intronless precursors. This finding suggests that separate pathways are used for nuclear export of retrogradely transported spliced tRNAs and tRNAs made from intronless pre-tRNAs. In addition, the data support the conclusion that the nuclear re-export pathway for retrogradely transported spliced tRNAs, but not the pathway responsible for nuclear export of tRNAs derived from intronless precursors is regulated during amino acid or nitrogen starvation. This regulation appears to occur at a step after the re-imported spliced tRNAs have undergone aminoacylation quality assurance and, in part, involves the TORC1 signalling pathway. Moreover, it was established that Utp9p is an intranuclear component that only facilitates nuclear re-export of retrogradely transported spliced tRNAs by the ß-karyopherin Msn5p. Utp9p acts in concert with Utp8p, a key player in nuclear tRNA export in S. cerevisiae, to translocate aminoacylated re-imported spliced tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex. This pathway, however, is not the only one responsible for nuclear re-export of retrogradely transported spliced tRNAs.
Assuntos
RNA de Transferência/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Humanos , Splicing de RNA , RNA de Transferência/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Utp9p is a nucleolar protein that is part of a subcomplex containing several U3 snoRNA-associated proteins including Utp8p, which is a protein that shuttles aminoacyl-tRNAs from the nucleolus to the nuclear tRNA export receptors Los1p and Msn5p in Saccharomyces cerevisiae. Here we show that Utp9p is also an intranuclear component of the Msn5p-mediated nuclear tRNA export pathway. Depletion of Utp9p caused nuclear accumulation of mature tRNAs derived from intron-containing precursors, but not tRNAs made from intronless pre-tRNAs. Utp9p binds tRNA directly and saturably, and copurifies with Utp8p, Gsp1p, and Msn5p, but not with Los1p or aminoacyl-tRNA synthetases. Utp9p interacts directly with Utp8p, Gsp1p, and Msn5p in vitro. Furthermore, Gsp1p forms a complex with Msn5p and Utp9p in a tRNA-dependent manner. However, Utp9p does not shuttle between the nucleus and the cytoplasm. Because tRNA splicing occurs in the cytoplasm and the spliced tRNAs are retrograded back to the nucleus, we propose that Utp9p facilitates nuclear reexport of retrograded tRNAs. Moreover, the data suggest that Utp9p together with Utp8p translocate aminoacyl-tRNAs from the nucleolus to Msn5p and assist with formation of the Msn5p-tRNA-Gsp1p-GTP export complex.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Carioferinas/metabolismo , Proteínas Nucleares/metabolismo , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Animais , Núcleo Celular/metabolismo , Hibridização in Situ Fluorescente , Íntrons , Carioferinas/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA de Transferência/genética , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Utp8p is an essential 80 kDa intranuclear tRNA chaperone that transports tRNAs from the nucleolus to the nuclear tRNA export receptors in Saccharomyces cerevisiae. To help understand the mechanism of Utp8p function, predictive tools were used to derive a partial model of the tertiary structure of Utp8p. Secondary structure prediction, supported by circular dichroism measurements, indicated that Utp8p is divided into 2 domains: the N-terminal beta sheet and the C-terminal alpha helical domain. Tertiary structure prediction was more challenging, because the amino acid sequence of Utp8p is not directly homologous to any known protein structure. The tertiary structures predicted by threading and fold recognition had generally modest scores, but for the C-terminal domain, threading and fold recognition consistently pointed to an alpha-alpha superhelix. Because of the sequence diversity of this fold type, no single structural template was an ideal fit to the Utp8p sequence. Instead, a composite template was constructed from 3 different alpha-alpha superhelix structures that gave the best matches to different portions of the C-terminal domain sequence. In the resulting model, the most conserved sequences grouped in a tight cluster of positive charges on a protein that is otherwise predominantly negative, suggesting that the positive-charge cleft may be the tRNA-binding site. Mutations of conserved positive residues in the proposed binding site resulted in a reduction in the affinity of Utp8p for tRNA both in vivo and in vitro. Models were also derived for the 10 fungal homologues of Utp8p, and the localization of the positive charges on the conserved surface was found in all cases. Taken together, these data suggest that the positive-charge cleft of the C-terminal domain of Utp8p is involved in tRNA-binding.
Assuntos
Nucléolo Celular/metabolismo , Modelos Moleculares , RNA de Transferência/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Arginina/genética , Sítios de Ligação , Sequência Conservada , Lisina/genética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
The emergence of bacterial antibiotic resistance poses a significant challenge in the pursuit of novel therapeutics, making new strategies for drug discovery imperative. We have developed a yeast growth-defect phenotypic screen to help solve this current dilemma. This approach facilitates the identification and characterization of a new diphtheria toxin (DT) group, ADP-ribosyltransferase toxins from pathogenic bacteria. In addition, this assay utilizes Saccharomyces cerevisiae, a reliable model for bacterial toxin expression, to streamline the identification and characterization of new inhibitors against this group of bacterial toxins that may be useful for antimicrobial therapies. We show that a mutant of the elongation factor 2 target protein in yeast, G701R, confers resistance to all DT group toxins and recovers the growth-defect phenotype in yeast. We also demonstrate the ability of a potent small-molecule toxin inhibitor, 1,8-naphthalimide (NAP), to alleviate the growth defect caused by toxin expression in yeast. Moreover, we determined the crystal structure of the NAP inhibitor-toxin complex at near-atomic resolution to provide insight into the inhibitory mechanism. Finally, the NAP inhibitor shows therapeutic protective effects against toxin invasion of mammalian cells, including human lung cells.
Assuntos
Bioensaio/métodos , Toxina Diftérica/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , ADP Ribose Transferases/química , ADP Ribose Transferases/genética , ADP Ribose Transferases/metabolismo , ADP Ribose Transferases/farmacologia , Linhagem Celular , Toxina Diftérica/química , Toxina Diftérica/genética , Toxina Diftérica/metabolismo , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismoRESUMO
The Saccharomyces cerevisiae Yor112wp, which we named Cex1p, was identified using a yeast tRNA three-hybrid interaction approach and an in vivo nuclear tRNA export assay as a cytoplasmic component of the nuclear tRNA export machinery. Cex1p binds tRNA saturably, and associates with the nuclear pore complex by interacting directly with Nup116p. Cex1p co-purifies with the nuclear tRNA export receptors Los1p and Msn5p, the eukaryotic elongation factor eEF-1A, which delivers aminoacylated tRNAs to the ribosome, and the RanGTPase Gsp1p, but not with Cca1p, a tRNA maturation enzyme that facilitates translocation of non-aminoacylated tRNAs across the nuclear pore complex. Depletion of Cex1p and eEF-1A or Los1p significantly reduced the efficiency of nuclear tRNA export. Cex1p interacts with Los1p but not with eEF-1A in vitro. These findings suggest that Cex1p is a component of the nuclear aminoacylation-dependent tRNA export pathway in S. cerevisiae. They also suggest that Cex1p collects aminoacyl-tRNAs from the nuclear export receptors at the cytoplasmic side of the nuclear pore complex, and transfers them to eEF-1A using a channelling mechanism.