RESUMO
Chromatin endogenous cleavage coupled with high-throughput sequencing (ChEC-seq) is a profiling method for protein-DNA interactions that can detect binding locations in vivo, does not require antibodies or fixation, and provides genome-wide coverage at near nucleotide resolution.The core of this method is an MNase fusion of the target protein, which allows it, when triggered by calcium exposure, to cut DNA at its binding sites and to generate small DNA fragments that can be readily separated from the rest of the genome and sequenced.Improvements since the original protocol have increased the ease, lowered the costs, and multiplied the throughput of this method to enable a scale and resolution of experiments not available with traditional methods such as ChIP-seq. This method describes each step from the initial creation and verification of the MNase-tagged yeast strains, over the ChEC MNase activation and small fragment purification procedure to the sequencing library preparation. It also briefly touches on the bioinformatic steps necessary to create meaningful genome-wide binding profiles.
Assuntos
Genoma Fúngico , Sequenciamento de Nucleotídeos em Larga Escala , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Cromatina/genética , Cromatina/metabolismo , Sítios de Ligação , Análise de Sequência de DNA/métodos , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genética , Biologia Computacional/métodosRESUMO
Contact-sites are specialized zones of proximity between two organelles, essential for organelle communication and coordination. The formation of contacts between the Endoplasmic Reticulum (ER), and other organelles, relies on a unique membrane environment enriched in sterols. However, how these sterol-rich domains are formed and maintained had not been understood. We found that the yeast membrane protein Yet3, the homolog of human BAP31, is localized to multiple ER contact sites. We show that Yet3 interacts with all the enzymes of the post-squalene ergosterol biosynthesis pathway and recruits them to create sterol-rich domains. Increasing sterol levels at ER contacts causes its depletion from the plasma membrane leading to a compensatory reaction and altered cell metabolism. Our data shows that Yet3 provides on-demand sterols at contacts thus shaping organellar structure and function. A molecular understanding of this protein's functions gives new insights into the role of BAP31 in development and pathology.
RESUMO
DNA packaging within chromatin depends on histone chaperones and remodelers that form and position nucleosomes. Cells express multiple such chromatin regulators with overlapping in-vitro activities. Defining specific in-vivo activities requires monitoring histone dynamics during regulator depletion, which has been technically challenging. We have recently generated histone-exchange sensors in Saccharomyces cerevisiae, which we now use to define the contributions of 15 regulators to histone dynamics genome-wide. While replication-independent exchange in unperturbed cells maps to promoters, regulator depletions primarily affected gene bodies. Depletion of Spt6, Spt16 or Chd1 sharply increased nucleosome replacement sequentially at the beginning, middle or end of highly expressed gene bodies. They further triggered re-localization of chaperones to affected gene body regions, which compensated for nucleosome loss during transcription complex passage, but concurred with extensive TF binding in gene bodies. We provide a unified quantitative screen highlighting regulator roles in retaining nucleosome binding during transcription and preserving genomic packaging.
Assuntos
Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , DNA/genética , DNA/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eviction of histones from nucleosomes and their exchange with newly synthesized or alternative variants is a central epigenetic determinant. Here, we define the genome-wide occupancy and exchange pattern of canonical and non-canonical histone variants in mouse embryonic stem cells by genetically encoded exchange sensors. While exchange of all measured variants scales with transcription, we describe variant-specific associations with transcription elongation and Polycomb binding. We found considerable exchange of H3.1 and H2B variants in heterochromatin and repeat elements, contrasting the occupancy and little exchange of H3.3 in these regions. This unexpected association between H3.3 occupancy and exchange of canonical variants is also evident in active promoters and enhancers, and further validated by reduced H3.1 dynamics following depletion of H3.3-specific chaperone, HIRA. Finally, analyzing transgenic mice harboring H3.1 or H3.3 sensors demonstrates the vast potential of this system for studying histone exchange and its impact on gene expression regulation in vivo.
Assuntos
Histonas , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Histonas/genética , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/genética , Sequências Reguladoras de Ácido Nucleico , Regulação da Expressão GênicaRESUMO
Cellular decision making often builds on ultrasensitive MAPK pathways. The phosphorylation mechanism of MAP kinase has so far been described as either distributive or processive, with distributive mechanisms generating ultrasensitivity in theoretical analyses. However, the in vivo mechanism of MAP kinase phosphorylation and its activation dynamics remain unclear. Here, we characterize the regulation of the MAP kinase Hog1 in Saccharomyces cerevisiae via topologically different ODE models, parameterized on multimodal activation data. Interestingly, our best fitting model switches between distributive and processive phosphorylation behavior regulated via a positive feedback loop composed of an affinity and a catalytic component targeting the MAP kinase-kinase Pbs2. Indeed, we show that Hog1 directly phosphorylates Pbs2 on serine 248 (S248), that cells expressing a non-phosphorylatable (S248A) or phosphomimetic (S248E) mutant show behavior that is consistent with simulations of disrupted or constitutively active affinity feedback and that Pbs2-S248E shows significantly increased affinity to Hog1 in vitro. Simulations further suggest that this mixed Hog1 activation mechanism is required for full sensitivity to stimuli and to ensure robustness to different perturbations.
Assuntos
Proteínas de Saccharomyces cerevisiae , Fosforilação , Retroalimentação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
DNA replication perturbs chromatin by triggering the eviction, replacement, and incorporation of nucleosomes. How this dynamic is orchestrated in time and space is poorly understood. Here, we apply a genetically encoded sensor for histone exchange to follow the time-resolved histone H3 exchange profile in budding yeast cells undergoing slow synchronous replication in nucleotide-limiting conditions. We find that new histones are incorporated not only behind, but also ahead of the replication fork. We provide evidence that Rtt109, the S-phase-induced acetyltransferase, stabilizes nucleosomes behind the fork but promotes H3 replacement ahead of the fork. Increased replacement ahead of the fork is independent of the primary Rtt109 acetylation target H3K56 and rather results from Vps75-dependent Rtt109 activity toward the H3 N terminus. Our results suggest that, at least under nucleotide-limiting conditions, selective incorporation of differentially modified H3s behind and ahead of the replication fork results in opposing effects on histone exchange, likely reflecting the distinct challenges for genome stability at these different regions.
Assuntos
Replicação do DNA , Histona Acetiltransferases , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Acetilação , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/genética , Histonas/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Nucleossomos/genética , Nucleossomos/metabolismo , Nucleotídeos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Histone exchange between histones carrying position-specific marks and histones bearing general marks is important for gene regulation, but understanding of histone exchange remains incomplete. To overcome the poor time resolution of conventional pulse-chase histone labeling, we present a genetically encoded histone exchange timer sensitive to the duration that two tagged histone subunits co-reside at an individual genomic locus. We apply these sensors to map genome-wide patterns of histone exchange in yeast using single samples. Comparing H3 exchange in cycling and G1-arrested cells suggests that replication-independent H3 exchange occurs at several hundred nucleosomes (<1% of all nucleosomes) per minute, with a maximal rate at histone promoters. We observed substantial differences between the two nucleosome core subcomplexes: H2A-H2B subcomplexes undergo rapid transcription-dependent replacement within coding regions, whereas H3-H4 replacement occurs predominantly within promoter nucleosomes, in association with gene activation or repression. Our timers allow the in vivo study of histone exchange dynamics with minute time scale resolution.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Montagem e Desmontagem da Cromatina , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The wrapping of DNA around histone octamers challenges processes that use DNA as their template. In vitro, DNA replication through chromatin depends on histone modifiers, raising the possibility that cells modify histones to optimize fork progression. Rtt109 is an acetyl transferase that acetylates histone H3 before its DNA incorporation on the K56 and N-terminal residues. We previously reported that, in budding yeast, a wave of histone H3 K9 acetylation progresses â¼3-5 kb ahead of the replication fork. Whether this wave contributes to replication dynamics remained unknown. Here, we show that the replication fork velocity increases following deletion of RTT109, the gene encoding the enzyme required for the prereplication H3 acetylation wave. By using histone H3 mutants, we find that Rtt109-dependent N-terminal acetylation regulates fork velocity, whereas K56 acetylation contributes to replication dynamics only when N-terminal acetylation is compromised. We propose that acetylation of newly synthesized histones slows replication by promoting replacement of nucleosomes evicted by the incoming fork, thereby protecting genome integrity.
Assuntos
Replicação do DNA , Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Histonas/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
Cell growth is driven by the synthesis of proteins, genes, and other cellular components. Defining processes that limit biosynthesis rates is fundamental for understanding the determinants of cell physiology. Here, we analyze the consequences of engineering cells to express extremely high levels of mCherry proteins, as a tool to define limiting processes that fail to adapt upon increasing biosynthetic demands. Protein-burdened cells were transcriptionally and phenotypically similar to mutants of the Mediator, a transcription coactivator complex. However, our binding data suggest that the Mediator was not depleted from endogenous promoters. Burdened cells showed an overall increase in the abundance of the majority of endogenous transcripts, except for highly expressed genes. Our results, supported by mathematical modeling, suggest that wild-type cells transcribe highly expressed genes at the maximal possible rate, as defined by the transcription machinery's physical properties. We discuss the possible cellular benefit of maximal transcription rates to allow a coordinated optimization of cell size and cell growth.
Assuntos
Fatores de Transcrição , Transcrição Gênica , Ciclo Celular , Proliferação de Células , Regiões Promotoras Genéticas , Fatores de Transcrição/genéticaRESUMO
The evolutionary pressure imposed by phage predation on bacteria and archaea has resulted in the development of effective anti-phage defence mechanisms, including restriction-modification and CRISPR-Cas systems. Here, we report on a new defence system, DISARM (defence island system associated with restriction-modification), which is widespread in bacteria and archaea. DISARM is composed of five genes, including a DNA methylase and four other genes annotated as a helicase domain, a phospholipase D (PLD) domain, a DUF1998 domain and a gene of unknown function. Engineering the Bacillus paralicheniformis 9945a DISARM system into Bacillus subtilis has rendered the engineered bacteria protected against phages from all three major families of tailed double-stranded DNA phages. Using a series of gene deletions, we show that four of the five genes are essential for DISARM-mediated defence, with the fifth (PLD) being redundant for defence against some of the phages. We further show that DISARM restricts incoming phage DNA and that the B. paralicheniformis DISARM methylase modifies host CCWGG motifs as a marker of self DNA akin to restriction-modification systems. Our results suggest that DISARM is a new type of multi-gene restriction-modification module, expanding the arsenal of defence systems known to be at the disposal of prokaryotes against their viruses.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/virologia , Proteínas de Bactérias/metabolismo , Bacteriófagos/fisiologia , Enzimas de Restrição-Modificação do DNA/genética , Família Multigênica/genética , Proteínas de Bactérias/genética , Bacteriófagos/classificação , Bacteriófagos/crescimento & desenvolvimento , Clonagem Molecular , Biologia Computacional , Genoma Bacteriano/genética , Ilhas Genômicas , Metiltransferases/genética , Modelos Genéticos , Deleção de Sequência , Replicação ViralRESUMO
Growing cells coordinate protein translation with metabolic rates. Central to this coordination is ribosome production. Ribosomes drive cell growth, but translation of ribosomal proteins competes with production of non-ribosomal proteins. Theory shows that cell growth is maximized when all expressed ribosomes are constantly translating. To examine whether budding yeast function at this limit of full ribosomal usage, we profiled the proteomes of cells growing in different environments. We find that cells produce excess ribosomal proteins, amounting to a constant ≈8% of the proteome. Accordingly, ≈25% of ribosomal proteins expressed in rapidly growing cells does not contribute to translation. Further, this fraction increases as growth rate decreases and these excess ribosomal proteins are employed when translation demands unexpectedly increase. We suggest that steadily growing cells prepare for conditions that demand increased translation by producing excess ribosomes, at the expense of lower steady-state growth rate.
Assuntos
Regulação Fúngica da Expressão Gênica , Proteoma/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Perfilação da Expressão Gênica , Biossíntese de Peptídeos Independentes de Ácido Nucleico/genética , Biossíntese de Proteínas , Proteoma/metabolismo , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Ribossomos/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mutation rate balances the need to protect genome integrity with the advantage of evolutionary innovations. Microorganisms increase their mutation rate when stressed, perhaps addressing the growing need for evolutionary innovation. Such a strategy, however, is only beneficial under moderate stresses that allow cells to divide and realize their mutagenic potential. In contrast, severe stresses rapidly kill the majority of the population with the exception of a small minority of cells that are in a phenotypically distinct state termed persistence. Although persisters were discovered many decades ago, the stochastic event triggering persistence is poorly understood. We report that spontaneous DNA damage triggers persistence in Saccharomyces cerevisiae by activating the general stress response, providing protection against a range of harsh stress and drug environments. We further show that the persister subpopulation carries an increased load of genetic variants in the form of insertions, deletions or large structural variations, which are unrelated to their stress survival. This coupling of DNA damage to phenotypic persistence may increase genetic diversity specifically in severe stress conditions, where diversity is beneficial but the ability to generate de novo mutations is limited.
RESUMO
Upon environmental changes or extracellular signals, cells are subjected to marked changes in gene expression. Dealing with high levels of transcription during replication is critical to prevent collisions between the transcription and replication pathways and avoid recombination events. In response to osmostress, hundreds of stress-responsive genes are rapidly induced by the stress-activated protein kinase (SAPK) Hog1 (ref. 6), even during S phase. Here we show in Saccharomyces cerevisae that a single signalling molecule, Hog1, coordinates both replication and transcription upon osmostress. Hog1 interacts with and phosphorylates Mrc1, a component of the replication complex. Phosphorylation occurs at different sites to those targeted by Mec1 upon DNA damage. Mrc1 phosphorylation by Hog1 delays early and late origin firing by preventing Cdc45 loading, as well as slowing down replication-complex progression. Regulation of Mrc1 by Hog1 is completely independent of Mec1 and Rad53. Cells carrying a non-phosphorylatable allele of MRC1 (mrc1(3A)) do not delay replication upon stress and show a marked increase in transcription-associated recombination, genomic instability and Rad52 foci. In contrast, mrc1(3A) induces Rad53 and survival in the presence of hydroxyurea or methyl methanesulphonate. Therefore, Hog1 and Mrc1 define a novel S-phase checkpoint independent of the DNA-damage checkpoint that permits eukaryotic cells to prevent conflicts between DNA replication and transcription, which would otherwise lead to genomic instability when both phenomena are temporally coincident.
Assuntos
Replicação do DNA , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Alelos , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Pressão Osmótica , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Recombinação Genética , Origem de Replicação/genética , Fase S , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Estresse Fisiológico , Especificidade por Substrato , Fatores de TempoRESUMO
In anaphase, sister chromatids separate abruptly and are then segregated by the mitotic spindle. The protease separase triggers sister separation by cleaving the Scc1/Mcd1 subunit of the cohesin ring that holds sisters together. Polo-kinase phosphorylation of Scc1 promotes its cleavage, but the underlying regulatory circuits are unclear. We developed a separase biosensor in Saccharomyces cerevisiae that provides a quantitative indicator of cohesin cleavage in single cells. Separase is abruptly activated and cleaves most cohesin within 1 min, after which anaphase begins. Cohesin near centromeres and telomeres is cleaved at the same rate and time. Protein phosphatase PP2A(Cdc55) inhibits cohesin cleavage by counteracting polo-kinase phosphorylation of Scc1. In early anaphase, the previously described separase inhibition of PP2A(Cdc55) promotes cohesin cleavage. Thus, separase acts directly on Scc1 and also indirectly, through inhibition of PP2A(Cdc55), to stimulate cohesin cleavage, providing a feedforward loop that may contribute to a robust and timely anaphase.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Segregação de Cromossomos/fisiologia , Endopeptidases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimologia , Técnicas Biossensoriais , Endopeptidases/genética , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência/métodos , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Separase , CoesinasRESUMO
Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress activates the Hog1 SAPK, which modulates cell cycle progression at G1 and G2 by the phosphorylation of elements of the cell cycle machinery, such as Sic1 and Hsl1, and by down-regulation of G1 and G2 cyclins. Here, we show that upon stress, Hog1 also modulates S phase progression. The control of S phase is independent of the S phase DNA damage checkpoint and of the previously characterized Hog1 cell cycle targets Sic1 and Hsl1. Hog1 uses at least two distinct mechanisms in its control over S phase progression. At early S phase, the SAPK prevents firing of replication origins by delaying the accumulation of the S phase cyclins Clb5 and Clb6. In addition, Hog1 prevents S phase progression when activated later in S phase or cells containing a genetic bypass for cyclin-dependent kinase activity. Hog1 interacts with components of the replication complex and delays phosphorylation of the Dpb2 subunit of the DNA polymerase. The two mechanisms of Hog1 action lead to delayed firing of origins and prolonged replication, respectively. The Hog1-dependent delay of replication could be important to allow Hog1 to induce gene expression before replication.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2 , Ciclina B/metabolismo , Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA , Citometria de Fluxo , Fase G1 , Fase G2 , Imunoprecipitação , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Pressão Osmótica , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
Control of cell cycle progression by stress-activated protein kinases (SAPKs) is essential for cell adaptation to extracellular stimuli. Exposure of yeast to osmostress leads to activation of the Hog1 SAPK, which controls cell cycle at G1 by the targeting of Sic1. Here, we show that survival to osmostress also requires regulation of G2 progression. Activated Hog1 interacts and directly phosphorylates a residue within the Hsl7-docking site of the Hsl1 checkpoint kinase, which results in delocalization of Hsl7 from the septin ring and leads to Swe1 accumulation. Upon Hog1 activation, cells containing a nonphosphorylatable Hsl1 by Hog1 are unable to promote Hsl7 delocalization, fail to arrest at G2 and become sensitive to osmostress. Together, we present a novel mechanism that regulates the Hsl1-Hsl7 complex to integrate stress signals to mediate cell cycle arrest and, demonstrate that a single MAPK coordinately modulates different cell cycle checkpoints to improve cell survival upon stress.
Assuntos
Sobrevivência Celular , Fase G2/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ativação Enzimática , Estabilidade Enzimática , Proteínas Quinases Ativadas por Mitógeno/genética , Concentração Osmolar , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteína-Arginina N-Metiltransferases , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Constitutively active mutants that acquired intrinsic activity and escaped regulation, serve as powerful tools for revealing the biochemical, biological and pathological functions of proteins. Such mutants are not available for mitogen-activated protein kinases (MAPKs). It is not known how to mimic the unusual mode of MAPK activation and to enforce, by mutations, their active conformation. In this review we describe the strategies employed in attempts to overcome this obstacle. We focus on a recent breakthrough with the p38 family that suggests that active variants of all MAPKs will soon be available.
Assuntos
Variação Genética , Proteínas Quinases Ativadas por Mitógeno/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Drosophila/enzimologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ativação Enzimática , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mutagênese , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Mitogen-activated protein kinases (MAPKs) play key roles in differentiation, growth, proliferation, and apoptosis. Although MAPKs have been extensively studied, the precise function, specific substrates, and target genes of each MAPK are not known. These issues could be addressed by sole activation of a given MAPK, e.g., through the use of constitutively active MAPK enzymes. We have recently reported the isolation of eight hyperactive mutants of the Saccharomyces cerevisiae MAPK Hog1, each of which bears a distinct single point mutation. These mutants acquired high intrinsic catalytic activity but did not impose the full biological potential of the Hog1 pathway. Here we describe our attempt to obtain a MAPK that is more active than the previous mutants both catalytically and biologically. We combined two different activating point mutations in the same gene and found that two of the resulting double mutants acquired unusual properties. These alleles, HOG1(D170A,F318L) and HOG1(D170A,F318S), induced a severe growth inhibition and had to be studied through an inducible expression system. This growth inhibition correlated with very high spontaneous (in the absence of any stimulation) catalytic activity and strong induction of Hog1 target genes. Furthermore, analysis of the phosphorylation status of these active alleles shows that their acquired intrinsic activity is independent of either phospho-Thr174 or phospho-Tyr176. Through fluorescence-activated cell sorting analysis, we show that the effect on cell growth inhibition is not a result of cell death. This study provides the first example of a MAPK that is intrinsically activated by mutations and induces a strong biological effect.