Materiais e Métodos <ol><li> Cepas de leveduras <ul><li> Todas as cepas utilizadas neste estudo são baseados no fundo BY4742. Da estirpe do controlo SILAC (Mat a, his3, leu2, ura3, lys2 e arg4:: G418) foi feita por meio de cruzamentos arg4 tensão exclusão (Mat a, his3, ura3, leu2 e arg4:: G418) para BY4742 (Mat alfa, his3, leu2, ura3 e lys2), e as haplóides meióticas foram obtidas por dissecção tétrade. Portanto, a tensão de controle é um auxotroph de lisina e arginina. A cepa isca foi feita por PCR mediada por recombinação homóloga usando o vetor pBS1479. Portanto, a tag epítopo utilizado neste estudo é a purificação de afinidade tandem (TAP) tag (3). </li></ul></li><li> SILAC: <ul><li> Da estirpe do controlo SILAC foi cultivado em meios de abandono mínimo suplementado com 98% L-lisina-4 ,4,5,5-D4 (0,03 g / L, Cambridge Isotope) e 98% L-arginina-13 C 6 (0,036 g / L, Cambridge Isotope). </li><li> A cepa isca foi cultivada em meio rico regular com normais isotópica aminoácidos. </li></ul></li><li> Primeira etapa de purificação TAP [Este protocolo é adaptado a partir do Cold Spring Harbor curso de laboratório manual (4)] ** A noite antes da sua purificação, argamassa pré-chill e pilão em freezer -80 ° C. Além disso, pré-chill um copo em -20 ° C ** Tornar esses buffers direito antes de usar (para 1 L de cultura): <ul><li> 30 ml NP-40 com 25 de buffer PIC mL (1 / 2000), 50 ul da TDT 1M </li><li> 20 ml de tampão NP-40 com 1 / 200 PIC, 50 ul de 1M TDT </li><li> 10 ml de TEV-C buffer com 1 / 2000 PIC, 10 ml de 1 M DTT </li></ul></li></ol> SILAC e preparação <ol><li> Grow 1L cada um dos tensão isca eo controle de tensão em separado para = 1,0-1,3 OD 600. </li><li> Despeje a cultura em tubos de centrífuga de 500ml Nalgene. Equilibrar a carga e células de pelotização em 2580X g. </li><li> Decantar mídia e ressuspender pellet com 50ml de frio dH 2 O durante a transferência das células para tubos de centrífuga de 50ml. Células da pelota. ** Você pode congelar o pellet a -80 ° C ** </li><li> ** Importante ** coincidir com a cultura estirpe de controlo para a cultura tensão isca em 1:1 em peso pellet seco. </li><li> Ressuspender cada pellet celular em 10ml de NP-40 buffer com Cocktail 1 / / 2000 Inibidor da Protease (PIC). </li><li> Misturar as duas culturas de células de decantação um tubo diretamente para o outro. Grinding </li><li> Despeje o líquido em N 2 a argamassa pré-refrigeradas e permitir que ele evapore completamente. Adicionar 2 ml de células para a argamassa em movimento circular. Despeje um pouco de N 2 líquido para congelar as células. Moer as células até as células se tornam pó fino. Repita o processo até que todas as células são chão. ** Não deixe que as células para descongelar. Add N 2 líquido quando necessário ** </li><li> Transferir o pó para um copo gelado e descongele em temperatura ambiente. Quando as bordas começam a derreter, adicione 20 ml de tampão NP-40 com 1 / 200 PIC. </li><li> Transferência de lisado bruto para tubos de 40 ml Nalgene. Giram em 39.000 xgfor 30 min. </li><li> Enquanto espera, tomar 300 ml de contas Sepharose 2B (GE). Lave as contas em 300 l de tampão NP-40 (1 / 2, 000 PIC) 3 vezes. Volte a suspender as contas em 300 mL de buffer para que eles sejam, numa suspensão de 1:1. Pré-limpeza lisado celular </li><li> Transfira o sobrenadante para um novo tubo e adicionar as contas 2B Sepharose. Incubar em uma plataforma rotativa, em câmara fria por 30 min. </li><li> Enquanto espera, tomar 300 ml de IgG Sepharose 6 contas (GE). Lave as contas em 300 l de tampão NP-40 (1 / 2, 000 PIC) 3 vezes. Volte a suspender as contas em 300 _l tampão para que eles sejam, numa suspensão de 1:1. </li><li> Pellet as contas. Transfira o sobrenadante para um novo tubo. ** Salve uma alíquota do lisado celular para Western Blotting tarde ** Ligação a contas de IgG sepharose </li><li> Remova as contas 2B Sepharose por centrifugação. Adicionar as contas IgG (previamente preparado em 1:1 slurry). Separar os conteúdos em dois tubos para a ligação mais eficiente. Incubar em uma plataforma rotativa, em câmara fria por 2 horas. </li><li> Girar as esferas. ** Salve uma alíquota da facção unbound ** </li><li> Lavar contas com 10 ml de tampão NP-40 (02/01, 000 PIC). </li><li> Lavar contas com 3 ml TEV-C buffer (02/01, 000 PIC). ** Salve uma alíquota das esferas. Isso irá refletir a eficiência da ligação ** Eluição de contas de IgG por TEV clivagem da protease </li><li> Adicionar 5 mL de AcTEV para 1ml de TEV-C-tampão (com 1 / 2, 000 PIC). Depois de misturar, separar o conteúdo em dois tubos de 1,5 ml para mais Eppendorf mistura eficiente. Incubar em uma plataforma rotativa no quarto frio durante a noite. </li><li> Spin para baixo as contas e transferir o eluato para um tubo de 1,5 ml de frescor. Lave as contas com 0,5 ml adicional de TEV-C tampão </li><li> Combine o elaute em um tubo. Você deve ter 1,5 ml de eluído no total.** Salve uma alíquota do eluato TEV ** Ácido tricloroacético precipatation (TCA) (Para a análise de espectrometria de massa-base, recomendamos o uso de EtOH / precipitação de acetato) </li><li> Ajustar as alíquotas de 25% TCA com 100% de TCA. Para fazer isso, separar o eluato 1,5 ml em 2 tubos de 750 mL cada. Adicionar 250 mL de TCA 100% para o tubo. Sua solução deve girar leitoso. </li><li> Colocar no gelo por 30 minutos com vortex periódica. </li><li> Giram em velocidade máxima na centrífuga de mesa na sala fria por 30 min. </li><li> Lave uma vez com 500 mL de acetona gelada contendo 0,05 N HCl e girar por 5 min em velocidade máxima (sala fria). </li><li> Lave uma vez com 500 mL de acetona gelada e girar por 5 min no max (sala fria). </li><li> Remova cuidadosamente acetona e pellet seco. </li><li> Para a coloração de prata, ressuspender o sedimento em 50 ul de 1X tampão de amostra SDS. EtOH precipatation / acetato </li><li> Adicionar 20 mg de glicogênio </li><li> Diluir as amostras de 5X com EtOH 100% e ajuste para 50 mM nach 3 COO com um estoque 2,5 M, pH 5,0 </li><li> Suportar a solução para 2hr </li><li> Pellet proteína precipitada por centrifugação por 10 min a 12.000 X g à temperatura ambiente. </li></ol> NP-40 Buffer (para 200 ml) <table border="0"><tbody><tr><td></td><td> Concentração de ações </td><td> Adicionar </td></tr><tr><td> 10 mM tampão fosfato de sódio (pH 7,2) </td><td> 0,1 M </td><td> 20 ml </td></tr><tr><td> 150 mM NaCl </td><td> 2 M </td><td> 15 ml </td></tr><tr><td> 1% NP-40 </td><td> 100% </td><td> 2 ml </td></tr><tr><td> 50 mM NaF </td><td> 1 M </td><td> 10 ml </td></tr><tr><td> 0,1 mM Na 3 VO 4 </td><td> 10 mM </td><td> 2 ml </td></tr><tr><td style="text-align:right;" colspan="3"> Volume para 200 ml </td></tr></tbody></table> ADD antes de usar: <ol><li> 01/01, de 1 000 M DTT </li><li> 02/01, 000 do PIC (Protease Cocktail Inibidores) </li></ol> TEV-C Buffer (para 50 ml) <table border="0"><tbody><tr><td></td><td> Concentração de ações </td><td> Adicionar </td></tr><tr><td> 25 mM Tris (pH 8,0) </td><td> 100 mM </td><td> 12,5 ml </td></tr><tr><td> 150 mM NaCl </td><td> 2 M </td><td> 3,75 ml </td></tr><tr><td> 0,1% NP-40 </td><td> 100% </td><td> 50 _l </td></tr><tr><td> 0,5 mM EDTA </td><td> 500 mM </td><td> 50 __l </td></tr><tr><td style="text-align:right;" colspan="3"> Volume para 50 ml </td></tr></tbody></table> ADD antes de usar: <ol><li> 01/01, 000 de 1M TDT </li><li> 02/01, 000 do PIC </li></ol>

<ol>
	<li>Loewen, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inheritance+of+cortical+ER+in+yeast+is+required+for+normal+septin+organization.">Inheritance of cortical ER in yeast is required for normal septin organization.</a> Jour. Cell Biol. 179, 467-483 .</li><li>Ong, S., Foster, L. J., Hoog, C. L., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric-based+approaches+in+quantitative+proteomics.">Mass spectrometric-based approaches in quantitative proteomics.</a> Methods. 29, 124-130 .</li><li>Puig, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tandem+Affinity+Purification+(TAP)+method:+a+general+procedure+of+protein+complex+purification.">The Tandem Affinity Purification (TAP) method: a general procedure of protein complex purification.</a> Methods. 24, 218-229 .</li><li>Amberg, D. C., Burke, D. J., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+Yeast+Genetics.">Methods in Yeast Genetics.</a> , 123-125 .</li></ol>

As alíquotas salvo durante o processo de purificação deve incluir (1) lisado celular pré-limpo, (2) fração ligada, (3) fracção livre, e (4) fração eluída. Recomendamos analisar o conteúdo de proteína das alíquotas acima por Western blotting utilizando anticorpo anti-TAP ou coloração pela prata para refletir a eficiência de ligação e eluição do experimento. Exemplos de um gel de prata manchada e uma mancha são mostrados na Figura 1. Desde SILAC nos fornece medições imp...

identification of protein complexes with quantitative proteomics in s. cerevisiae

Lipídios são os blocos de construção das membranas celulares que funcionam como barreiras e na compartimentalização de processos celulares e, recentemente, como importantes moléculas sinalizadoras intracelulares. No entanto, ao contrário de proteínas, lipídios são pequenas moléculas hidrofóbicas que o tráfego principalmente pelo mal descritos rotas não vesiculares, que são a hipótese de ocorrer em locais de contato da membrana (MCS). MCS são regiões onde o retículo endoplasmático (ER) faz o contato físico direto com uma organela parceria, por exemplo, membrana plasmática (PM). A porção de ER ER-PM MCS é enriquecida em lipídios, síntese de enzimas, sugerindo que a síntese lipídica é direcionado para esses sites e dando a entender que SMCs são importantes para o tráfego de lipídios. A levedura é um modelo ideal para estudar ER-PM SMCs por causa de sua abundância, com mais de 1.000 contatos por celular, e sua natureza preservada em todos os eucariontes. Descobrindo as proteínas que constituem MCS é fundamental para a compreensão de como o tráfego de lipídios é realizada nas células, e como eles agem como moléculas de sinalização. Descobrimos que uma ER chamado Scs2p localizar a ER-PM MCS e é importante para sua formação. Estamos focados em descobrir os parceiros moleculares da Scs2p. Identificação de complexos de proteínas tradicionalmente se baseia em primeiro resolver amostras de proteínas purificadas por eletroforese em gel, seguido por in-gel a digestão de bandas de proteínas e análise de peptídeos por espectrometria de massa. Isso muitas vezes limita o estudo a um pequeno subconjunto de proteínas. Além disso, complexos de proteínas são expostas a desnaturação ou condições não fisiológicas durante o procedimento. Para contornar esses problemas, temos implementado um grande escala técnica proteômica quantitativa para extrair dados imparciais e quantificados. Usamos rotulagem isótopo estável com aminoácidos em cultura de células (SILAC) para incorporar os núcleos de isótopos grampo em proteínas em uma estirpe de controlo untagged. Volumes iguais de cultura marcados e não marcados cultura, SILAC-rotulados são misturados e lisadas por moagem em nitrogênio líquido. Em seguida, realizar um processo de purificação de afinidade para puxar para baixo complexos de proteína. Finalmente, precipitar a amostra de proteína, que está pronto para análise por cromatografia líquida / espectrometria de massa tandem. Mais importante ainda, proteínas em uma estirpe de controlo são rotulados pela isótopo pesado e produzirá uma mudança em massa / carga que pode ser quantificado contra as proteínas não marcado na cepa isca. Portanto, contaminantes, ou ligação inespecífica pode ser facilmente eliminada. Usando essa abordagem, identificamos várias novas proteínas que localizar a ER-PM MCS. Aqui nós apresentamos uma descrição detalhada da nossa abordagem.

Watch this Scientific Journal Video about Identification of protein complexes with quantitative proteomics in S. cerevisiae at JoVE.com

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Aqui nós descrevemos uma nova técnica proteômica quantitativa para a identificação de complexos de proteína em Saccharomyces cerevisiae. Neste estudo, nós utilizamos o método SILAC juntamente com purificação de afinidade seguido por espectrometria de massa para identificar com alta especificidade dos parceiros de ligação de uma proteína ER, Scs2p.

Identificação de complexos de proteínas com proteômica quantitativa em S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

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13.1K visualizações.  University of British Columbia - UBC. Aqui nós descrevemos uma nova técnica proteômica quantitativa para a identificação de complexos de proteína em Saccharomyces cerevisiae. Neste estudo, nós utilizamos o método SILAC juntamente com purificação de afinidade seguido por espectrometria de massa para identificar com alta especificidade dos parceiros de ligação de uma proteína ER, Scs2p.

Vídeo: Identification of protein complexes with quantitative proteomics in S. cerevisiae

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

Ensaio para a Adesão e Invasão Agar em S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

Biologia

A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning.  Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Um método de alto rendimento para estudar a morfologia global organela em S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

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.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

Phosphoproteomics quantitativa em ácidos graxos estimulada Saccharomyces cerevisiae</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

Estudando Idade-dependente Instabilidade Genômica usando o S. cerevisiae Modelo Lifespan cronológica

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Identificação de complexos de proteínas em<em&gt; Escherichia coli</em&gt; Utilizando a purificação por afinidade sequencial Peptide em combinação com espectrometria de massa tandem

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

Purificação Citometria de Fluxo com base em<em&gt; S. cerevisiae</em&gt; Zigotos

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualização e análise de moléculas de mRNA Usando Fluorescência<em&gt; In Situ</em&gt; A hibridação em<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Ensaios para Monitoramento juntamente redobramento de proteínas em<em&gt; Saccharomyces cerevisiae</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Identificação de modificações pós-translacionais de Complexos de proteína vegetal

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

Identificação de parceiros de interação de proteínas em células de mamíferos utilizando SILAC-imunoprecipitação quantitativos Proteômica

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...