RESUMO
Whereas proliferating cells enter M phase shortly after DNA replication, the first M phase of meiosis is preceded by an extended prophase in which homologous chromosomes undergo recombination. Exit from prophase I is controlled by the recombination checkpoint (RC), which, in yeast, represses the meiosis-specific transcription factor Ndt80 required for the expression of B-type cyclins and other M phase regulators. We show that an extended prophase I additionally requires the suppression of latent, mitotic cell-cycle controls by the anaphase-promoting complex (APC/C) and its meiosis-specific activator Ama1, which trigger the degradation of M phase regulators and Ndd1, a subunit of a mitotic transcription factor. ama1Δ mutants exit from prophase I prematurely and independently of the RC, which results in recombination defects and chromosome missegregation. Thus, control of prophase I by meiotic mechanisms depends on the suppression of the alternative, mitotic mechanisms by a meiosis-specific form of the APC/C.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Meiose , Prófase , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Metáfase , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Fuso Acromático , Fatores de Transcrição/metabolismoRESUMO
Meiosis differs from mitosis in that DNA replication is followed by the segregation of homologous chromosomes but not sister chromatids. This depends on the formation of interhomolog connections through crossover recombination and on the attachment of sister kinetochores to microtubules emanating from the same spindle pole. We show that in yeast, the Dbf4-dependent Cdc7 kinase (DDK) provides a link between premeiotic S phase, recombination, and monopolar attachment. Independently from its established role in initiating DNA replication, DDK promotes double-strand break formation, the first step of recombination, and the recruitment of the monopolin complex to kinetochores, which is essential for monopolar attachment. DDK regulates monopolin localization together with the polo-kinase Cdc5 bound to Spo13, probably through phosphorylation of the monopolin subunit Lrs4. Thus, activation of DDK both initiates DNA replication and commits meiotic cells to reductional chromosome segregation in the first division of meiosis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Meiose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Ciclo Celular , Cromossomos/ultraestrutura , Replicação do DNA , Deleção de Genes , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , Saccharomyces cerevisiaeRESUMO
By reporting the molar abundance of proteins, absolute quantification determines their stoichiometry in complexes, pathways, or networks. Typically, absolute quantification relies either on protein-specific isotopically labeled peptide standards or on a semiempirical calibration against the average abundance of peptides chosen from arbitrarily selected proteins. In contrast, a generic protein standard FUGIS (fully unlabeled generic internal standard) requires no isotopic labeling, chemical synthesis, or external calibration and is applicable to quantifying proteins of any organismal origin. The median intensity of the peptide peaks produced by the tryptic digestion of FUGIS is used as a single-point calibrant to determine the molar abundance of any codigested protein. Powered by FUGIS, median-based absolute quantification (MBAQ) outperformed other methods of untargeted proteome-wide absolute quantification.
Assuntos
Peptídeos , Proteoma , Calibragem , Marcação por Isótopo/métodos , Peptídeos/química , Padrões de ReferênciaRESUMO
Absolute (molar) quantification of clinically relevant proteins determines their reference values in liquid and solid biopsies. The FastCAT (for Fast-track QconCAT) method employs multiple short (<50 kDa), stable-isotope labeled chimeric proteins (CPs) composed of concatenated quantotypic (Q)-peptides representing the quantified proteins. Each CP also comprises scrambled sequences of reference (R)-peptides that relate its abundance to a single protein standard (bovine serum albumin, BSA). FastCAT not only alleviates the need to purify CP or use sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) but also improves the accuracy, precision, and dynamic range of the absolute quantification by grouping Q-peptides according to the expected abundance of the target proteins. We benchmarked FastCAT against the reference method of MS Western and tested it in the direct molar quantification of neurological markers in human cerebrospinal fluid at the low ng/mL level.
Assuntos
Proteínas , Proteômica , Eletroforese em Gel de Poliacrilamida , Humanos , Peptídeos/metabolismo , Proteômica/métodos , Padrões de ReferênciaRESUMO
Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR; Nannospalax galili) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved noncoding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated evolutionary changes in this element by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor replacement caused a more than 2-fold upregulation of Tdrd7 in the developing lens; however, increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, possibly explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.
Assuntos
Evolução Molecular , Cristalino/metabolismo , Ratos-Toupeira/genética , Elementos Reguladores de Transcrição/genética , Ribonucleoproteínas/genética , Animais , Feminino , Cristalino/crescimento & desenvolvimento , Masculino , Camundongos Transgênicos , Ribonucleoproteínas/metabolismoRESUMO
Absolute quantification of proteins elucidates the molecular composition, regulation and dynamics of multiprotein assemblies and networks. Here we report on a method termed MS Western that accurately determines the molar abundance of dozens of user-selected proteins at the subfemtomole level in whole cell or tissue lysates without metabolic or chemical labeling and without using specific antibodies. MS Western relies on GeLC-MS/MS and quantifies proteins by in-gel codigestion with an isotopically labeled QconCAT protein chimera composed of concatenated proteotypic peptides. It requires no purification of the chimera and relates the molar abundance of all proteotypic peptides to a single reference protein. In comparative experiments, MS Western outperformed immunofluorescence Western blotting by the protein detection specificity, linear dynamic range and sensitivity of protein quantification. To validate MS Western in an in vivo experiment, we quantified the molar content of zebrafish core histones H2A, H2B, H3 and H4 during ten stages of early embryogenesis. Accurate quantification (CV<10%) corroborated the anticipated histones equimolar stoichiometry and revealed an unexpected trend in their total abundance.
Assuntos
Proteômica/métodos , Animais , Western Blotting , Cromatografia Líquida , Embrião não Mamífero , Escherichia coli , Células HeLa , Histonas/química , Humanos , Proteínas/análise , Espectrometria de Massas em Tandem , Peixe-ZebraRESUMO
BACKGROUND: Baculovirus-mediated expression in insect cells is a powerful approach for protein production. However, many existing methods are time-consuming, offer limited options for protein tagging, and are unsuitable for secreted proteins requiring proteolytic maturation, such as TGF-ß family growth factors. RESULTS: To overcome the limitations of traditional baculovirus expression systems, we engineered "FlexiBAC". This system allows recombinant baculovirus formation inside insect cells and reduces the time between initial cloning and protein production to 13 days. FlexiBAC includes 143 shuttle vectors that append combinations of purification tags, fluorescent markers, proteolytic cleavage sites, trafficking signals, and chemical conjugation tags to the termini of the target protein. This system also overexpresses recombinant furin convertase to allow efficient proteolytic processing of secreted proteins. We demonstrate that FlexiBAC can be used to produce high levels of mature, active forms of TGF-ß family growth factors, such as Activin A, as well as other proteins that are typically difficult to reconstitute, such as proteins rich in coiled-coil, low complexity, and disordered domains. CONCLUSIONS: FlexiBAC is a protein expression system for production of both cytosolic proteins and secreted proteins that require proteolytic maturation. The design of FlexiBAC and its expansive complementary shuttle vector system reduces cloning steps and simplifies baculovirus production.
Assuntos
Baculoviridae/genética , Vetores Genéticos/genética , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Ativinas/biossíntese , Ativinas/genética , Animais , Expressão Gênica , Proteínas Recombinantes/metabolismo , Spodoptera/genética , Transfecção/métodos , Cultura de Vírus/métodosRESUMO
CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until recently suffered from low integration efficiencies despite traditional genetics being well established. Here, we present a protocol for efficient homology-directed knockin mutagenesis in all commonly used strains of Chlamydomonas. We describe steps for scarless integration of fusion tags and sequence modifications of almost all proteins without the need for a preceding mutant line. We further empower this genetic-editing approach by efficient crossing and highly robust screening protocols. For complete details on the use and execution of this protocol, please refer to Nievergelt et al. (2023).1.
Assuntos
Sistemas CRISPR-Cas , Chlamydomonas reinhardtii , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Mutagênese , GenomaRESUMO
CRISPR-Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now been primarily applied to targeted gene disruption, whereas scarless knockin transgenesis has generally been considered difficult in practice. We have developed an efficient homology-directed method for knockin mutagenesis in Chlamydomonas by delivering CRISPR-Cas ribonucleoproteins and a linear double-stranded DNA (dsDNA) donor into cells by electroporation. Our method allows scarless integration of fusion tags and sequence modifications of proteins without the need for a preceding mutant line. We also present methods for high-throughput crossing of transformants and a custom quantitative PCR (qPCR)-based high-throughput screening of mutants as well as meiotic progeny. We demonstrate how to use this pipeline to facilitate the generation of mutant lines without residual selectable markers by co-targeted insertion. Finally, we describe how insertional cassettes can be erroneously mutated during insertion and suggest strategies to select for lines that are modified as designed.
Assuntos
Chlamydomonas reinhardtii , Chlamydomonas , Sistemas CRISPR-Cas , Cultura , EletroporaçãoRESUMO
Cells exposed to starvation have to adjust their metabolism to conserve energy and protect themselves. Protein synthesis is one of the major energy-consuming processes and as such has to be tightly controlled. Many mechanistic details about how starved cells regulate the process of protein synthesis are still unknown. Here, we report that the essential translation initiation factor eIF2B forms filaments in starved budding yeast cells. We demonstrate that filamentation is triggered by starvation-induced acidification of the cytosol, which is caused by an influx of protons from the extracellular environment. We show that filament assembly by eIF2B is necessary for rapid and efficient downregulation of translation. Importantly, this mechanism does not require the kinase Gcn2. Furthermore, analysis of site-specific variants suggests that eIF2B assembly results in enzymatically inactive filaments that promote stress survival and fast recovery of cells from starvation. We propose that translation regulation through filament assembly is an efficient mechanism that allows yeast cells to adapt to fluctuating environments.
Assuntos
Citoesqueleto/metabolismo , Metabolismo Energético , Fator de Iniciação 2B em Eucariotos/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Estresse Fisiológico , Citosol/metabolismo , Concentração de Íons de Hidrogênio , Viabilidade Microbiana , Modelos Biológicos , Fosforilação , Leveduras/fisiologiaRESUMO
The human-specific gene ARHGAP11B is preferentially expressed in neural progenitors of fetal human neocortex and increases abundance and proliferation of basal progenitors (BPs), which have a key role in neocortex expansion. ARHGAP11B has therefore been implicated in the evolutionary expansion of the human neocortex, but its mode of action has been unknown. Here, we show that ARHGAP11B is imported into mitochondria, where it interacts with the adenine nucleotide translocase (ANT) and inhibits the mitochondrial permeability transition pore (mPTP). BP expansion by ARHGAP11B requires its presence in mitochondria, and pharmacological inhibition of ANT function or mPTP opening mimic BP expansion by ARHGAP11B. Searching for the underlying metabolic basis, we find that BP expansion by ARHGAP11B requires glutaminolysis, the conversion of glutamine to glutamate for the tricarboxylic acid (TCA) cycle. Hence, an ARHGAP11B-induced, mitochondria-based effect on BP metabolism that is a hallmark of highly mitotically active cells appears to underlie its role in neocortex expansion.
Assuntos
Proteínas Ativadoras de GTPase/metabolismo , Glutamina/metabolismo , Mitocôndrias/metabolismo , Neocórtex/metabolismo , Células-Tronco Neurais/metabolismo , Células 3T3 , Animais , Evolução Biológica , Proliferação de Células/genética , Ciclo do Ácido Cítrico , Proteínas Ativadoras de GTPase/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Translocases Mitocondriais de ADP e ATP/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Neocórtex/embriologia , Neurogênese/genéticaRESUMO
BACKGROUND: RNA interference coupled with videorecording of C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes. RESULTS: Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast cdc26Delta mutant. CONCLUSION: These data demonstrate that B0511.9 encodes the C. elegans APC/C subunit CDC-26.
Assuntos
Caenorhabditis elegans/genética , Polaridade Celular/genética , Genes cdc , Meiose/genética , Saccharomyces cerevisiae/genética , Ciclossomo-Complexo Promotor de Anáfase , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Embrião não Mamífero/citologia , Perfilação da Expressão Gênica , Teste de Complementação Genética , Vetores Genéticos , Fenótipo , Interferência de RNA , RNA de Helmintos/genética , Complexos Ubiquitina-Proteína Ligase/genética , Gravação em VídeoRESUMO
The anaphase-promoting complex (APC/C) is a large ubiquitin-protein ligase which controls progression through anaphase by triggering the degradation of cell cycle regulators such as securin and B-type cyclins. The APC/C is an unusually complex ligase containing at least 10 different, evolutionarily conserved components. In contrast to APC/C's role in cell cycle regulation little is known about the functions of individual subunits and how they might interact with each other. Here, we have analyzed Swm1/Apc13, a small subunit recently identified in the budding yeast complex. Database searches revealed proteins related to Swm1/Apc13 in various organisms including humans. Both the human and the fission yeast homologues are associated with APC/C subunits, and they complement the phenotype of an SWM1 deletion mutant of budding yeast. Swm1/Apc13 promotes the stable association with the APC/C of the essential subunits Cdc16 and Cdc27. Accordingly, Swm1/Apc13 is required for ubiquitin ligase activity in vitro and for the timely execution of APC/C-dependent cell cycle events in vivo.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Subunidades Proteicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Animais , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase , Ciclo Celular/fisiologia , Cromátides/metabolismo , DNA Polimerase III , Evolução Molecular , Teste de Complementação Genética , Humanos , Meiose/fisiologia , Dados de Sequência Molecular , Subunidades Proteicas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe , Alinhamento de Sequência , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína LigasesRESUMO
The size and position of mitotic spindles is determined by the lengths of their constituent microtubules. Regulation of microtubule length requires feedback to set the balance between growth and shrinkage. Whereas negative feedback mechanisms for microtubule length control, based on depolymerizing kinesins and severing proteins, have been studied extensively, positive feedback mechanisms are not known. Here, we report that the budding yeast kinesin Kip2 is a microtubule polymerase and catastrophe inhibitor in vitro that uses its processive motor activity as part of a feedback loop to further promote microtubule growth. Positive feedback arises because longer microtubules bind more motors, which walk to the ends where they reinforce growth and inhibit catastrophe. We propose that positive feedback, common in biochemical pathways to switch between signaling states, can also be used in a mechanical signaling pathway to switch between structural states, in this case between short and long polymers.
Assuntos
Retroalimentação Fisiológica , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND: Mechanoreceptors contain compliant elements, termed "gating springs," that transfer macroscopic stimuli impinging on the cells into microscopic stimuli that open the mechanosensitive channels. Evidence for gating springs comes from mechanical experiments; they have not been identified molecularly or ultrastructurally. RESULTS: We show that the filamentous structures that connect the plasma membrane to the microtubules are compliant structural elements in the mechanoreceptive organelle of fly campaniform receptors. These filaments colocalize with the ankyrin-repeat domain of the transient receptor potential (TRP) channel NOMPC. In addition, they resemble the purified ankyrin-repeat domain in size and shape. Most importantly, these filaments are nearly absent in nompC mutants and can be rescued by the nompC gene. Finally, mechanical modeling suggests that the filaments provide most of the compliance in the distal tip of the cell, thought to be the site of mechanotransduction. CONCLUSIONS: Our results provide strong evidence that the ankyrin-repeat domains of NOMPC structurally contribute to the membrane-microtubule connecting filaments. These filaments, as the most compliant element in the distal tip, are therefore good candidates for the gating springs.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Canais de Potencial de Receptor Transitório/genética , Sequência de Aminoácidos , Animais , Repetição de Anquirina , Citoesqueleto/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mecanorreceptores/metabolismo , Mecanotransdução Celular , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Microtúbulos/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
During meiosis, two rounds of chromosome segregation after a single round of DNA replication produce haploid gametes from diploid precursors. At meiosis I, maternal and paternal kinetochores are pulled toward opposite poles, and chiasmata holding bivalent chromosomes together are resolved by cleavage of cohesin's alpha-kleisin subunit (Rec8) along chromosome arms. This creates dyad chromosomes containing a pair of chromatids joined solely by cohesin at centromeres that had resisted cleavage. The discovery that centromeric Rec8 is protected from separase during meiosis I by shugoshin/MEI-S332 proteins that bind PP2A phosphatase suggests that phosphorylation either of separase or cohesin may be necessary for Rec8 cleavage. We show here that multiple phosphorylation sites within Rec8 as well as two different kinases, casein kinase 1delta/epsilon (CK1delta/epsilon) and Dbf4-dependent Cdc7 kinase (DDK), are required for Rec8 cleavage and meiosis I nuclear division. Rec8 with phosphomimetic mutations is no longer protected from separase at centromeres and is cleaved even when the two kinases are inhibited. Our data suggest that PP2A protects centromeric cohesion by opposing CK1delta/epsilon- and DDK-dependent phosphorylation of Rec8.
Assuntos
Caseína Quinase 1 épsilon/metabolismo , Caseína Quinase Idelta/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Endopeptidases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Caseína Quinase 1 épsilon/genética , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Caseína Quinase Idelta/genética , Proteínas de Ciclo Celular/genética , Centrômero/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Endopeptidases/genética , Genes Fúngicos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Meiose/genética , Meiose/fisiologia , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Separase , CoesinasRESUMO
LET-767 from Caenorhabditis elegans belongs to a family of short chain dehydrogenases/reductases and is homologous to 17beta-hydroxysterol dehydrogenases of type 3 and 3-ketoacyl-CoA reductases. Worms subjected to RNA interference (RNAi) of let-767 displayed multiple growth and developmental defects in the first generation and arrested in the second generation as L1 larvae. To determine the function of LET-767 in vivo, we exploited a biochemical complementation approach, in which let-767 (RNAi)-arrested larvae were rescued by feeding with compounds isolated from wild type worms. The arrest was only rescued by the addition of triacylglycerides extracted from worms but not from various natural sources, such as animal fats and plant oils. The mass spectrometric analyses showed alterations in the fatty acid content of triacylglycerides. Essential for the rescue were odd-numbered fatty acids with monomethyl branched chains. The rescue was improved when worms were additionally supplemented with long chain even-numbered fatty acids. Remarkably, let-767 completely rescued the yeast 3-ketoacyl-CoA reductase mutant (ybr159Delta). Because worm ceramides exclusively contain a monomethyl branched chain sphingoid base, we also investigated ceramides in let-767 (RNAi). Indeed, the amount of ceramides was greatly reduced, and unusual sphingoid bases were observed. Taken together, we conclude that LET-767 is a major 3-ketoacyl-CoA reductase in C. elegans required for the bulk production of monomethyl branched and long chain fatty acids, and the developmental arrest in let-767 (RNAi) worms is caused by the deficiency of the former.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Ácidos Graxos/metabolismo , Animais , Caenorhabditis elegans , Cromatografia em Camada Fina , Biologia Computacional , Lipídeos/química , Espectrometria de Massas , Modelos Biológicos , Modelos Químicos , Mutação , Fenótipo , Filogenia , Interferência de RNA , Triglicerídeos/químicaRESUMO
In meiosis, a single round of DNA replication is followed by two consecutive rounds of chromosome segregation, called meiosis I and II. Disjunction of maternal from paternal centromeres during meiosis I depends on the attachment of sister kinetochores to microtubules emanating from the same pole. In budding yeast, monopolar attachment requires recruitment to kinetochores of the monopolin complex. How monopolin promotes monopolar attachment was unclear, as its subunits are poorly conserved and lack similarities to proteins with known functions. We show here that the monopolin subunit Mam1 binds tightly to Hrr25, a highly conserved casein kinase 1 delta/epsilon (CK1delta/epsilon), and recruits it to meiosis I centromeres. Hrr25 kinase activity and Mam1 binding are both essential for monopolar attachment. Since CK1delta/epsilon activity is important for accurate chromosome segregation during meiosis I also in fission yeast, phosphorylation of kinetochore proteins by CK1delta/epsilon might be an evolutionary conserved process required for monopolar attachment.
Assuntos
Caseína Quinase I/metabolismo , Segregação de Cromossomos/fisiologia , Cinetocoros/metabolismo , Meiose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fuso Acromático/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Caseína Quinase I/genética , Centrômero/genética , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Replicação do DNA/fisiologia , Cinetocoros/ultraestrutura , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Fuso Acromático/genética , Fuso Acromático/ultraestruturaRESUMO
Cohesion established between sister chromatids during pre-meiotic DNA replication mediates two rounds of chromosome segregation. The first division is preceded by an extended prophase wherein homologous chromosomes undergo recombination. The persistence of cohesion during prophase is essential for recombination and both meiotic divisions. Here we show that Mnd2, a subunit of the anaphase-promoting complex (APC/C) from budding yeast, is essential to prevent premature destruction of cohesion in meiosis. During S- and prophase, Mnd2 prevents activation of the APC/C by a meiosis-specific activator called Ama1. In cells lacking Mnd2 the APC/C-Ama1 enzyme triggers degradation of Pds1, which causes premature sister chromatid separation due to unrestrained separase activity. In vitro, Mnd2 inhibits ubiquitination of Pds1 by APC/C-Ama1 but not by other APC/C holo-enzymes. We conclude that chromosome segregation in meiosis depends on the selective inhibition of a meiosis-specific form of the APC/C.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Meiose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Anáfase/genética , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Cromátides/genética , Cromátides/metabolismo , Segregação de Cromossomos/genética , Endopeptidases/metabolismo , Meiose/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Desnaturação Proteica/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Securina , Separase , Complexos Ubiquitina-Proteína Ligase/genéticaRESUMO
The WD repeat protein Cdc20 is essential for progression through mitosis because it is required to activate ubiquitin ligation by the anaphase-promoting complex (APC/C). Here we show in yeast that Cdc20 binds to the CCT chaperonin, which is known as a folding machine for actin and tubulin. The CCT is required for Cdc20's ability to bind and activate the APC/C. In vivo, CCT is essential for Cdc20-dependent cell cycle events such as sister chromatid separation and exit from mitosis. The chaperonin is also required for the function of the Cdc20-related protein Cdh1, which activates the APC/C during G1. We propose that folding of the Cdc20 family of APC/C activators is an essential and evolutionary conserved function of the CCT chaperonin.