RESUMO
Peroxisome proliferation occurs by at least two routes, division of existing peroxisomes and de novo biogenesis from the endoplasmic reticulum (ER). The proteins and molecular mechanisms governing peroxisome emergence from the ER are poorly characterized. In this study, we report that two integral membrane peroxins (proteins required for peroxisome biogenesis) in Saccharomyces cerevisiae, Pex29 and Pex30, reside in distinct regions of the ER and associate with Rtn1 and Yop1, reticulon family members that contribute to ER morphology, to govern peroxisome emergence from the ER. In vivo and in vitro analyses reveal that peroxisome proliferation is therefore not restricted to the peroxisome but begins at the level of the ER.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Peroxissomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Retículo Endoplasmático/genética , Proteínas de Membrana/genética , Proteínas de Membrana Transportadoras/genética , Peroxissomos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Systems scale models provide the foundation for an effective iterative cycle between hypothesis generation, experiment and model refinement. Such models also enable predictions facilitating the understanding of biological complexity and the control of biological systems. Here, we demonstrate the reconstruction of a globally predictive gene regulatory model from public data: a model that can drive rational experiment design and reveal new regulatory mechanisms underlying responses to novel environments. Specifically, using â¼ 1500 publically available genome-wide transcriptome data sets from Saccharomyces cerevisiae, we have reconstructed an environment and gene regulatory influence network that accurately predicts regulatory mechanisms and gene expression changes on exposure of cells to completely novel environments. Focusing on transcriptional networks that induce peroxisomes biogenesis, the model-guided experiments allow us to expand a core regulatory network to include novel transcriptional influences and linkage across signaling and transcription. Thus, the approach and model provides a multi-scalar picture of gene dynamics and are powerful resources for exploiting extant data to rationally guide experimentation. The techniques outlined here are generally applicable to any biological system, which is especially important when experimental systems are challenging and samples are difficult and expensive to obtain-a common problem in laboratory animal and human studies.
Assuntos
Redes Reguladoras de Genes , Biologia de Sistemas/métodos , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genéticaRESUMO
The continued need to improve therapeutic recombinant protein productivity has led to ongoing assessment of appropriate strategies in the biopharmaceutical industry to establish robust processes with optimized critical variables, that is, viable cell density (VCD) and specific productivity (product per cell, qP). Even though high VCD is a positive factor for titer, uncontrolled proliferation beyond a certain cell mass is also undesirable. To enable efficient process development to achieve consistent and predictable growth arrest while maintaining VCD, as well as improving qP, without negative impacts on product quality from clone to clone, we identified an approach that directly targets the cell cycle G1-checkpoint by selectively inhibiting the function of cyclin dependent kinases (CDK) 4/6 with a small molecule compound. Results from studies on multiple recombinant Chinese hamster ovary (CHO) cell lines demonstrate that the selective inhibitor can mediate a complete and sustained G0/G1 arrest without impacting G2/M phase. Cell proliferation is consistently and rapidly controlled in all recombinant cell lines at one concentration of this inhibitor throughout the production processes with specific productivities increased up to 110 pg/cell/day. Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted. In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature. Microarray analysis showed major differences in expression of regulatory genes of the glycosylation and cell cycle signaling pathways between these different growth control methods. Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.
Assuntos
Técnicas de Cultura de Células/métodos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Animais , Reatores Biológicos , Células CHO , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Proteínas Recombinantes/análiseRESUMO
Cellular control of protein activities by modulation of their abundance or compartmentalization is not easily measured on a large scale. We developed and applied a method to globally interrogate these processes that is widely useful for systems-level analyses of dynamic cellular responses in many cell types. The approach involves subcellular fractionation followed by comprehensive proteomic analysis of the fractions, which is enabled by a data-independent acquisition mass spectrometry approach that samples every available mass to charge channel systematically to maximize sensitivity. Next, various fraction-enrichment ratios are measured for all detected proteins across different environmental conditions and used to group proteins into clusters reflecting changes in compartmentalization and relative conditional abundance. Application of the approach to characterize the response of yeast proteins to fatty acid exposure revealed dynamics of peroxisomes and novel dynamics of MCC/eisosomes, specialized plasma membrane domains comprised of membrane compartment occupied by Can1 (MCC) and eisosome subdomains. It also led to the identification of Fat3, a fatty acid transport protein of the plasma membrane, previously annotated as Ykl187.
Assuntos
Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fracionamento Celular , Meios de Cultura , Glucose/metabolismo , Metabolismo dos Lipídeos , Microscopia de Fluorescência , Anotação de Sequência Molecular , Ácido Oleico/metabolismo , Organelas/química , Organelas/metabolismo , Transporte Proteico , Proteoma/química , Proteômica , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/química , Frações Subcelulares/químicaRESUMO
High-field asymmetric waveform ion mobility spectrometry (FAIMS) is an atmospheric pressure ion mobility technique that can be used to reduce sample complexity and increase dynamic range in tandem mass spectrometry experiments. FAIMS fractionates ions in the gas-phase according to characteristic differences in mobilities in electric fields of different strengths. Undesired ion species such as solvated clusters and singly charged chemical background ions can be prevented from reaching the mass analyzer, thus decreasing chemical noise. To date, there has been limited success using the commercially available Thermo Fisher FAIMS device with both standard ESI and nanoLC-MS. We have modified a Thermo Fisher electrospray source to accommodate a fused silica pulled tip capillary column for nanospray ionization, which will enable standard laboratories access to FAIMS technology. Our modified source allows easily obtainable stable spray at flow rates of 300 nL/min when coupled with FAIMS. The modified electrospray source allows the use of sheath gas, which provides a fivefold increase in signal obtained when nanoLC is coupled to FAIMS. In this work, nanoLC-FAIMS-MS and nanoLC-MS were compared by analyzing a tryptic digest of a 1:1 mixture of SILAC-labeled haploid and diploid yeast to demonstrate the performance of nanoLC-FAIMS-MS, at different compensation voltages, for post-column fractionation of complex protein digests. The effective dynamic range more than doubled when FAIMS was used. In total, 10,377 unique stripped peptides and 1649 unique proteins with SILAC ratios were identified from the combined nanoLC-FAIMS-MS experiments, compared with 6908 unique stripped peptides and 1003 unique proteins with SILAC ratios identified from the combined nanoLC-MS experiments. This work demonstrates how a commercially available FAIMS device can be combined with nanoLC to improve proteome coverage in shotgun and targeted type proteomics experiments.
Assuntos
Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida/métodos , Nanotecnologia/métodos , Proteoma , Saccharomyces cerevisiae , Espectrometria de Massas em Tandem/métodosRESUMO
Positive feedback is a common mechanism enabling biological systems to respond to stimuli in a switch-like manner. Such systems are often characterized by the requisite formation of a heterodimer where only one of the pair is subject to feedback. This ASymmetric Self-UpREgulation (ASSURE) motif is central to many biological systems, including cholesterol homeostasis (LXRα/RXRα), adipocyte differentiation (PPARγ/RXRα), development and differentiation (RAR/RXR), myogenesis (MyoD/E12) and cellular antiviral defense (IRF3/IRF7). To understand why this motif is so prevalent, we examined its properties in an evolutionarily conserved transcriptional regulatory network in yeast (Oaf1p/Pip2p). We demonstrate that the asymmetry in positive feedback confers a competitive advantage and allows the system to robustly increase its responsiveness while precisely tuning the response to a consistent level in the presence of varying stimuli. This study reveals evolutionary advantages for the ASSURE motif, and mechanisms for control, that are relevant to pharmacologic intervention and synthetic biology applications.
Assuntos
Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Modelos Moleculares , Saccharomyces cerevisiae/genética , Biologia de Sistemas/métodos , Citometria de Fluxo , Redes Reguladoras de Genes , Saccharomyces cerevisiae/crescimento & desenvolvimento , Transdução de Sinais , Regulação para CimaRESUMO
Phosphorylation of proteins is a key posttranslational modification in cellular signaling, regulating many aspects of cellular responses. We used a quantitative, integrated, phosphoproteomics approach to characterize the cellular responses of the yeast Saccharomyces cerevisiae to the fatty acid oleic acid, a molecule with broad human health implications and a potent inducer of peroxisomes. A combination of cryolysis and urea solubilization was used to minimize the opportunity for reorientation of the phosphoproteome, and hydrophilic interaction liquid chromatography and IMAC chemistries were used to fractionate and enrich for phosphopeptides. Using these approaches, numerous phosphorylated peptides specific to oleate-induced and glucose-repressed conditions were identified and mapped to known signaling pathways. These include several transcription factors, two of which, Pip2p and Cst6p, must be phosphorylated for the normal transcriptional response of fatty acid-responsive loci encoding peroxisomal proteins. The phosphoproteome data were integrated with results from genome-wide assays studying the effects of signaling molecule deletions and known protein-protein interactions to generate a putative fatty acid-responsive signaling network. In this network, the most highly connected nodes are those with the largest effects on cellular responses to oleic acid. These properties are consistent with a scale-free topology, demonstrating that scale-free properties are conserved in condition-specific networks.
Assuntos
Peroxissomos , Fosfoproteínas/metabolismo , Proteômica , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Espectrometria de Massas , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Chz1p is a histone chaperone that interacts physically and functionally with the histone variant Htz1p, which has been implicated in establishing and maintaining boundaries between transcriptionally inactive heterochromatin and active euchromatin. To investigate the role of Chz1p in chromatin organization, we performed genome-wide expression arrays and chromatin immunoprecipitations of SIR complex components and modified histones in a CHZ1 deletion strain. Deletion of CHZ1 led to reduced ubiquitination of subtelomere-associated H2B, reduced subtelomeric H3K79 di-methylation, and increased binding of Sir3p, and Sir4p at telomere-distal euchromatin regions, correlating with decreased gene expression in subtelomeric regions. This anti-silencing defect appears to be mediated by enhanced association of de-ubiquitinase Ubp10p with subtelomeric DNA, as detected by chromatin immunoprecipitation analysis. In support of this, we show that deletion of UBP10 can antagonize the subtelomeric silencing phenotype of Deltachz1. Taken together, the results demonstrate a novel role for Chz1p in epigenetic regulation, through H2B de-ubiquitination by Ubp10p.
Assuntos
Regulação Fúngica da Expressão Gênica , Chaperonas de Histonas/fisiologia , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Ubiquitinação , Deleção de Genes , Inativação Gênica , Chaperonas de Histonas/genética , Histonas/química , Metilação , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Telômero/metabolismo , Transcrição Gênica , Ubiquitina Tiolesterase/metabolismoRESUMO
We have combined classical subcellular fractionation with large-scale quantitative mass spectrometry to identify proteins that enrich specifically with peroxisomes of Saccharomyces cerevisiae. In two complementary experiments, isotope-coded affinity tags and tandem mass spectrometry were used to quantify the relative enrichment of proteins during the purification of peroxisomes. Mathematical modeling of the data from 306 quantified proteins led to a prioritized list of 70 candidates whose enrichment scores indicated a high likelihood of them being peroxisomal. Among these proteins, eight novel peroxisome-associated proteins were identified. The top novel peroxisomal candidate was the small GTPase Rho1p. Although Rho1p has been shown to be tethered to membranes of the secretory pathway, we show that it is specifically recruited to peroxisomes upon their induction in a process dependent on its interaction with the peroxisome membrane protein Pex25p. Rho1p regulates the assembly state of actin on the peroxisome membrane, thereby controlling peroxisome membrane dynamics and biogenesis.
Assuntos
Actinas/metabolismo , Membranas Intracelulares/metabolismo , Peroxissomos/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/análise , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Teóricos , Peroxinas , Peroxissomos/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
In transcriptional regulatory networks, the coincident binding of a combination of factors to regulate a gene implies the existence of complex mechanisms to control both the gene expression profile and specificity of the response. Unraveling this complexity is a major challenge to biologists. Here, a novel network topology-based clustering approach was applied to condition-specific genome-wide chromatin localization and expression data to characterize a dynamic transcriptional regulatory network responsive to the fatty acid oleate. A network of four (predicted) regulators of the response (Oaf1p, Pip2p, Adr1p and Oaf3p) was investigated. By analyzing trends in the network structure, we found that two groups of multi-input motifs form in response to oleate, each controlling distinct functional classes of genes. This functionality is contributed in part by Oaf1p, which is a component of both types of multi-input motifs and has two different regulatory activities depending on its binding context. The dynamic cooperation between Oaf1p and Pip2p appears to temporally synchronize the two different responses. Together, these data suggest a network mechanism involving dynamic combinatorial control for coordinating transcriptional responses.
Assuntos
Ácidos Graxos/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Redes Reguladoras de Genes , Saccharomyces cerevisiae/genética , Transcrição Gênica , Regulação para Baixo/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Dynamic control of peroxisome proliferation is integral to the peroxisome's many functions. The endoplasmic reticulum (ER) serves as a source of preperoxisomal vesicles (PPVs) that mature into peroxisomes during de novo peroxisome biogenesis and support growth and division of existing peroxisomes. However, the mechanism of PPV formation and release from the ER remains poorly understood. In this study, we show that endosomal sorting complexes required for transport (ESCRT)-III are required to release PPVs budding from the ER into the cytosol. Absence of ESCRT-III proteins impedes de novo peroxisome formation and results in an aberrant peroxisome population in vivo. Using a cell-free PPV budding assay, we show that ESCRT-III proteins Vps20 and Snf7 are necessary to release PPVs from the ER. ESCRT-III is therefore a positive effector of membrane scission for vesicles budding both away from and toward the cytosol. These findings have important implications for the evolutionary timing of emergence of peroxisomes and the rest of the internal membrane architecture of the eukaryotic cell.
Assuntos
Retículo Endoplasmático/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Peroxissomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Citoplasmáticas/efeitos dos fármacos , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Testes Genéticos , Ácido Oleico/farmacologia , Biogênese de Organelas , Peroxissomos/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Non-enzymatic glycation is a challenging post-translational modification to characterize due to the structural heterogeneity it generates in proteins. Glycation has become increasingly recognized as an important product quality attribute to monitor, particularly for the biotechnology sector, which produces recombinant proteins under conditions that are amenable to protein glycation. The elucidation of sites of glycation can be problematic using conventional collision-induced dissociation (CID)-based mass spectrometry because of the predominance of neutral loss ions. A method to characterize glycation using an IgG1 monoclonal antibody (mAb) as a model is reported here. The sugars present on this mAb were derivatized using sodium borohydride chemistry to stabilize the linkage and identified using CID-based MS(2) mass spectrometry and spectral search engines. Quantification of specific glycation sites was then done using a targeted MS(1) based approach, which allowed the identification of a glycation hot spot in the heavy chain complementarity-determining region 3 of the mAb. This targeted approach provided a path forward to developing a structural understanding of the propensity of sites to become glycated on mAbs. Through structural analysis we propose a model in which the number and 3-dimensional distances of carboxylic acid amino acyl residues create a favorable environment for glycation to occur.
Assuntos
Anticorpos Monoclonais Murinos/química , Imunoglobulina G/química , Processamento de Proteína Pós-Traducional , Anticorpos Monoclonais Murinos/metabolismo , Glicosilação , Imunoglobulina G/metabolismo , Espectrometria de MassasRESUMO
PURPOSE: To determine the genetic and biochemical defects that underlie Axenfeld-Rieger malformations, identify the pathogenic mutation causing these malformations, and understand how these mutations alter protein function. METHODS: FOXC1 was amplified from a proband with Axenfeld-Rieger malformations and the proband's mother. PCR products were sequenced to identify the pathogenic mutation. Site-directed mutagenesis was used to introduce this mutation into the FOXC1 cDNA. A synthetic mutation at the same position was also introduced, and both natural and synthetic proteins were tested for their ability to localize to the nucleus, bind DNA, and transactivate gene expression. RESULTS: A novel missense mutation (L86F) was identified in FOXC1 in this family. The mutation is located in alpha-helix 1 of the forkhead domain. Biochemical assays showed that the L86F mutation does not affect nuclear localization of FOXC1, but reduces DNA binding and significantly reduces transactivation. The severity of the disruption to FOXC1 protein activity does not appear to correspond well with the severity of the phenotype in the patient. Analogous studies using a L86P, a known alpha-helix breaker, severely disrupts FOXC1 function, revealing the importance of helix 1 in FOXC1 structure and function. CONCLUSIONS: A novel mutation in helix 1 of the FOXC1 forkhead domain has been identified and the importance of position 86 in FOXC1 activity demonstrated. These studies also identified the role of helix 1 in FOXC1 function and provide further evidence for the lack of strong genotype-phenotype correlation in FOXC1 pathogenesis. Normal development appears to be dependent on tight upper and lower thresholds of FOXC1 activity.
Assuntos
Segmento Anterior do Olho/anormalidades , Proteínas de Ligação a DNA , Anormalidades do Olho/genética , Mutação de Sentido Incorreto , Fatores de Transcrição/genética , Adulto , Animais , Células COS , Chlorocebus aethiops , Análise Mutacional de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Fatores de Transcrição Forkhead , Células HeLa , Humanos , Masculino , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
PURPOSE: To determine the biochemical defects that underlie Axenfeld-Rieger malformations, to determine a functional role for wing 2 in FOXC1, and to understand how mutations in this region disrupt FOXC1 function. METHODS: Sequencing DNA from patients with Axenfeld-Rieger malformation resulted in the identification of two novel missense mutations (G165R and R169P) in wing 2 of FOXC1. Site-directed mutagenesis was used to introduce these mutations, as well as previously reported mutation (M161K), into the FOXC1 cDNA. These FOXC1 mutants were evaluated to determine their ability to localize to the nucleus, bind DNA and activate gene expression. RESULTS: Two novel missense mutations were identified in unrelated patients, in wing 2 of the FOXC1 forkhead domain. Because there had been no previous biochemical analysis, the mutation M161K was also investigated. All three mutant proteins localized correctly to the nucleus. The G165R mutation maintained wild-type levels of DNA binding; however, both the M161K and R169P mutations displayed reduced DNA binding ability. Biochemical analysis showed that all three mutations disrupt FOXC1's transactivation ability. CONCLUSIONS: Biochemical analysis of mutations G165R and R169P and of a previously reported mutation, M161K, demonstrate the functional significance of wing 2. M161K and R169P disrupt DNA binding of FOXC1, consistent with the hypothesis that wing 2 is necessary for DNA binding. The results also suggest that wing 2 plays a role in gene activation. These results provide the first insights into how mutations in wing 2 disrupt FOXC1 function.
Assuntos
Segmento Anterior do Olho/anormalidades , Proteínas de Ligação a DNA/fisiologia , Anormalidades do Olho/genética , Glaucoma/genética , Iris/anormalidades , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Pré-Escolar , Chlorocebus aethiops , Proteínas de Ligação a DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Fatores de Transcrição Forkhead , Células HeLa , Humanos , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Plasmídeos , Conformação Proteica , Fatores de Transcrição/químicaRESUMO
Here, we present a detailed method for generating a dynamic transcriptional regulatory network from large-scale chromatin immunoprecipitation data, and functional analysis of participating factors through the identification and characterization of significantly overrepresented multi-input motifs in the network. This is done by visualizing interactive data using a network analysis tool, such as Cytoscape, clustering DNA targets of the transcription factors based on their network topologies, and statistically analyzing each cluster based on its size and properties of its members. These analyses yield testable predictions about the conditional and cooperative functions of the factors. This is a versatile approach that allows the visualization of network architecture on a genome-wide level and is applicable to understanding combinatorial control mechanisms of DNA-binding regulators that conditionally cooperate in a wide variety of biological models.
Assuntos
Redes Reguladoras de Genes/genética , Fatores de Transcrição/genética , Imunoprecipitação da Cromatina , Análise por Conglomerados , Interpretação Estatística de Dados , Bases de Dados Genéticas , Humanos , Reação em Cadeia da PolimeraseRESUMO
The histone chaperones play an important role in chromatin assembly and disassembly during replication and transcription. We have assessed the global roles of histone chaperones in Saccharomyces cerevisiae. Microarray transcriptional analyzes indicate that histone chaperones have their own specific target genes, and various histone chaperones have partially overlapping functions during transcriptional regulation. The histone deacetylase inhibitor TSA and histone chaperones Asf1, Vps75 and Rtt106 can function in parallel pathways to regulate transcription. Moreover, TSA can specifically antagonize histone chaperone Chz1-mediated telomere anti-silencing. This study demonstrates that a mutual cross-talk mechanism exists between histone chaperones and histone deacetylation in transcriptional regulation.
Assuntos
Perfilação da Expressão Gênica , Chaperonas de Histonas/antagonistas & inibidores , Ácidos Hidroxâmicos/farmacologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Telômero/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Histona Desacetilases , Transcrição GênicaRESUMO
Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.
Assuntos
Técnicas Citológicas/métodos , Técnicas Genéticas , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Fuso Acromático/química , Biologia de Sistemas/métodos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/genéticaRESUMO
Stable incorporation of labeled amino acids in cell culture is a simple approach to label proteins in vivo for mass spectrometric quantification. Full incorporation of isotopically heavy amino acids facilitates accurate quantification of proteins from different cultures, yet analysis methods for determination of incorporation are cumbersome and time-consuming. We present QTIPS, Quantification by Total Identified Peptides for SILAC, a straightforward, accurate method to determine the level of heavy amino acid incorporation throughout a population of peptides detected by mass spectrometry. Using QTIPS, we show that the incorporation of heavy amino acids in baker's yeast is unaffected by the use of prototrophic strains, indicating that auxotrophy is not a requirement for SILAC experiments in this organism. This method has general utility for multiple applications where isotopic labeling is used for quantification in mass spectrometry.
Assuntos
Aminoácidos/química , Marcação por Isótopo/métodos , Isótopos/química , Fragmentos de Peptídeos/química , Sequência de Aminoácidos , Aminoácidos/metabolismo , Espectrometria de Massas , Redes e Vias Metabólicas , Dados de Sequência Molecular , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismoRESUMO
Peroxisomes are intracellular organelles that house a number of diverse metabolic processes, notably those required for beta-oxidation of fatty acids. Peroxisomes biogenesis can be induced by the presence of peroxisome proliferators, including fatty acids, which activate complex cellular programs that underlie the induction process. Here, we used multi-parameter quantitative phenotype analyses of an arrayed mutant collection of yeast cells induced to proliferate peroxisomes, to establish a comprehensive inventory of genes required for peroxisome induction and function. The assays employed include growth in the presence of fatty acids, and confocal imaging and flow cytometry through the induction process. In addition to the classical phenotypes associated with loss of peroxisomal functions, these studies identified 169 genes required for robust signaling, transcription, normal peroxisomal development and morphologies, and transmission of peroxisomes to daughter cells. These gene products are localized throughout the cell, and many have indirect connections to peroxisome function. By integration with extant data sets, we present a total of 211 genes linked to peroxisome biogenesis and highlight the complex networks through which information flows during peroxisome biogenesis and function.
Assuntos
Genômica , Peroxissomos/metabolismo , Biologia Computacional , Bases de Dados Factuais , Citometria de Fluxo , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis. Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics. Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.