Matériel et méthodes <ol><li> Les souches de levures <ul><li> Toutes les souches utilisées dans cette étude sont basées sur le fond BY4742. La souche de contrôle SILAC (Mat A, his3, leu2, ura3, lys2 et arg4:: G418) a été faite par l&#39;accouplement arg4 souche suppression (Mat A, his3, ura3, leu2, et arg4:: G418) pour BY4742 (Mat alpha, his3, leu2, ura3 et lys2), et les haploïdes méiotique ont été obtenus par dissection tétrade. Par conséquent, la souche de contrôle est un auxotrophe pour la lysine et l&#39;arginine. La souche d&#39;appât a été faite par PCR médiée par recombinaison homologue en utilisant le vecteur pBS1479. Par conséquent, la balise épitope utilisé dans cette étude est la purification par affinité en tandem (TAP) tag (3). </li></ul></li><li> SILAC: <ul><li> La souche de contrôle SILAC été cultivées dans des milieux d&#39;abandon minimum complété avec 98% de L-lysine-4 ,4,5,5-D4 (0,03 g / L, Cambridge Isotope) et 98% de L-arginine-13 C 6 (0,036 g / L, Cambridge Isotope). </li><li> La souche d&#39;appât a été cultivé en milieu riche régulier avec normale isotopique des acides aminés. </li></ul></li><li> Première étape de la purification TAP [Ce protocole est adapté de la Cold Spring Harbor Laboratory Manual sûr (4)] ** La nuit avant votre purification, de pré-refroidir mortier et un pilon en -80 ° C au congélateur. En outre, de pré-refroidir un bécher de -20 ° C ** Faire ces tampons juste avant utilisation (pour 1 L de culture): <ul><li> 30 ml de tampon NP-40 avec 25 ul PIC (1 / 2000), 50 pl de 1M TNT </li><li> 20 ml de NP-40 tampon avec 1 / 200 PIC, 50 pi de 1M TNT </li><li> 10 ml de VET-C tampon avec 1 / 2000 PIC, 10 pi de 1 M de DTT </li></ul></li></ol> SILAC et la préparation <ol><li> Cultivez 1L chacun de la souche appât et le contrôle souche séparément à DO600 = 1,0 à 1,3. </li><li> Verser la culture dans des tubes à centrifuger Nalgene 500ml. Équilibrer la charge et les cellules de bouletage à 2580X g. </li><li> Décanter les médias et resuspendre culot avec 50 ml de froid O DH 2 tout en transférant les cellules pour des tubes à centrifuger 50ml. Cellules Pellet. ** Vous pouvez congeler la pastille à -80 ° C ** </li><li> ** IMPORTANT ** match de la culture souche témoin à la culture souche appât dans 1:1 en granulés poids sec. </li><li> Resuspendre chaque cellule dans 10ml boulette de NP-40 tampon avec cocktail Inhibiteur de la protéase 1 / / 2000 (PIC). </li><li> Mélanger les deux cultures cellulaires par décantation directement un tube à l&#39;autre. Meulage </li><li> Verser le liquide de N 2 dans le mortier pré-réfrigérée et lui permettre de s&#39;évaporer complètement. Ajouter 2 ml de cellules au mortier dans le mouvement circulaire. Verser un peu de liquide de N 2 à geler les cellules. Broyer les cellules jusqu&#39;à ce que les cellules deviennent une poudre fine. Répétez le processus jusqu&#39;à ce que toutes les cellules sont broyées. ** Ne pas permettre aux cellules de dégel. Ajouter le liquide de N 2 si nécessaire ** </li><li> Transférer la poudre à un bécher glacée et décongeler à température ambiante. Lorsque les bords commencent à dégeler, ajouter 20 ml de NP-40 tampon avec 1 / 200 PIC. </li><li> Transfert lysat brut à 40 ml tubes Nalgene. Centrifuger à 39000 xgfor 30 min. </li><li> En attendant, prenez 300 ul de billes de Sépharose 2B (GE). Laver les billes dans 300 ul de tampon NP-40 (1 / 2, 000 PIC) 3 fois. Resuspendre les perles dans 300 pi de tampon de sorte qu&#39;ils sont en suspension à 01h01. Lysat cellulaire pré-compensation </li><li> Transférer le surnageant dans un nouveau tube et ajouter les perles 2B Sépharose. Incuber sur une plateforme rotative dans une chambre froide pendant 30 min. </li><li> En attendant, prenez 300 pl d&#39;IgG Sépharose 6 perles (GE). Laver les billes dans 300 ul de tampon NP-40 (1 / 2, 000 PIC) 3 fois. Resuspendre les billes en mémoire tampon 300 _l de sorte qu&#39;ils sont en suspension à 01h01. </li><li> Pellet les perles. Transférer le surnageant dans un nouveau tube. ** Enregistrer une aliquote du lysat cellulaire pour Western Blot tard ** La liaison aux IgG billes de sépharose </li><li> Retirer les billes de Sépharose 2B par centrifugation. Ajouter les perles d&#39;IgG (préalablement préparé en 1:1 lisier). Séparez le contenu dans deux tubes de liaison plus efficace. Incuber sur une plateforme rotative dans une chambre froide pendant 2 heures. </li><li> Isoler les perles. ** Enregistrer une aliquote de la faction non liée ** </li><li> Lavez perles avec 10 ml de tampon NP-40 (1 / 2, 000 PIC). </li><li> Lavez perles avec 3 ml VET-tampon C (1 / 2, 000 PIC). ** Enregistrer une aliquote des billes. Ceci reflète l&#39;efficacité de l&#39;** contraignante Élution à partir de perles d&#39;IgG par clivage de la protéase TEV </li><li> Ajouter 5 ul d&#39;AcTEV à 1ml de la VET-tampon C (avec 1 / 2, 000 PIC). Après mélange, séparer le contenu en deux tubes Eppendorf de 1,5 ml pour un mélange plus efficace. Incuber sur une plateforme rotative dans une chambre froide pendant la nuit. </li><li> Isoler les perles et le transfert de l&#39;éluat dans un nouveau tube de 1,5 ml. Laver les billes avec additionnels de 0,5 ml de tampon C-VET </li><li> Combinez les elaute dans un tube. Vous devriez avoir 1,5 ml d&#39;éluat au total.** Enregistrer une aliquote de l&#39;éluat VET ** Acide trichloracétique (TCA) précipitations sur (Pour une analyse par spectrométrie de masse à base, nous recommandons d&#39;utiliser EtOH / précipitation d&#39;acétate) </li><li> Ajustez aliquotes de 25% à 100% de TCA TCA. Pour ce faire, séparer l&#39;éluat 1,5 ml dans 2 tubes de 750 ul chacun. Ajouter 250 pi de 100% de TCA dans le tube. Votre solution doit tourner laiteux. </li><li> Placez-le sur la glace pendant 30 min avec vortex périodiques. </li><li> Centrifuger à vitesse maximale dans la centrifugeuse de table dans une chambre froide pendant 30 min. </li><li> Laver une fois avec 500 pi d&#39;acétone glacée contenant 0,05 N HCl et de spin pendant 5 min à vitesse maximale (chambre froide). </li><li> Laver une fois avec 500 pi d&#39;acétone glacée et de spin pendant 5 min à max (chambre froide). </li><li> Retirer délicatement l&#39;acétone et culot sec. </li><li> Pour coloration à l&#39;argent, resuspendre le culot dans 50 ul de tampon 1X échantillon de SDS. Précipitations sur EtOH / acétate </li><li> Ajouter 20 g de glycogène </li><li> Diluer les échantillons 5X avec EtOH 100% et ajuster à 50 mM NACH 3 COO avec un stock de 2,5 M, pH 5,0 </li><li> Placez la solution pour 2h </li><li> Pellet précipité de protéines par centrifugation pendant 10 min à 12000 xg à température ambiante. </li></ol> NP-40 tampon (pour 200 ml) <table border="0"><tbody><tr><td></td><td> Concentration de stock </td><td> Ajouter </td></tr><tr><td> 10 mM de tampon phosphate de sodium (pH 7,2) </td><td> 0,1 M </td><td> 20 ml </td></tr><tr><td> 150 mM de 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 à 200 ml </td></tr></tbody></table> Ajouter avant utilisation: <ol><li> 1 / 1, 000 de 1 M de DTT </li><li> 1 / 2, 000 du PIC (inhibiteurs de protéase Cocktail) </li></ol> VET-C Tampon (pour 50 ml) <table border="0"><tbody><tr><td></td><td> Concentration de stock </td><td> Ajouter </td></tr><tr><td> Tris 25 mM (pH 8,0) </td><td> 100 mM </td><td> 12,5 ml </td></tr><tr><td> 150 mM de NaCl </td><td> 2 M </td><td> 3,75 ml </td></tr><tr><td> 0,1% de NP-40 </td><td> 100% </td><td> 50 _l </td></tr><tr><td> EDTA 0,5 mM </td><td> 500 mM </td><td> 50 __l </td></tr><tr><td style="text-align:right;" colspan="3"> Volume à 50 ml </td></tr></tbody></table> Ajouter avant utilisation: <ol><li> 1 / 1, 000 de 1 million de TNT </li><li> 1 / 2, 000 du 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>

Les aliquotes enregistrées au cours de la procédure de purification doit inclure (1) lysat de cellules pré-autorisé, (2) fraction liée, fraction libre (3), et (4) fraction éluée. Nous vous recommandons d&#39;analyser les teneurs en protéines des aliquotes ci-dessus par Western blot utilisant des anticorps anti-anticorps ou TAP coloration à l&#39;argent afin de refléter l&#39;efficacité de fixation et d&#39;élution de l&#39;expérience. Exemples d&#39;un gel coloré à l&#39;argent et une tache sont présent?...

identification of protein complexes with quantitative proteomics in s. cerevisiae

Les lipides sont les constituants des membranes cellulaires qui fonctionnent comme des barrières et dans le cloisonnement des processus cellulaires, et récemment, aussi importantes molécules de signalisation intracellulaire. Cependant, contrairement aux protéines, les lipides sont des petites molécules hydrophobes que le trafic principalement par les routes mal décrits nonvesicular, qui sont supposé se produire à des sites de contact à membrane (MCS). MCS sont des régions où le réticulum endoplasmique (RE) fait un contact physique direct avec un partenariat organite, par exemple, la membrane plasmique (MP). La portion des ER ER-PM MCS est enrichi en lipides synthétiser des enzymes, ce qui suggère que la synthèse des lipides est dirigé vers ces sites et ce qui implique que MCS sont importantes pour le trafic des lipides. La levure est un modèle idéal pour étudier ER-PM PMRs raison de leur abondance, avec plus de 1000 contacts par cellule, et leur nature préservée chez tous les eucaryotes. Découvrir des protéines qui constituent MCS est essentielle pour comprendre comment le trafic des lipides est accompli dans les cellules, et comment ils agissent comme des molécules de signalisation. Nous avons trouvé que l&#39;ER appelé Scs2p localiser à ER-PM MSSC et est important pour leur formation. Nous nous concentrons sur la découverte des partenaires moléculaires de la Scs2p. Identification des complexes protéiques repose traditionnellement sur la première résolution échantillons protéine purifiée par électrophorèse sur gel, suivie par la digestion dans le gel des bandes de protéines et d&#39;analyse des peptides par spectrométrie de masse. Cette limite souvent l&#39;étude d&#39;un petit nombre de protéines. En outre, les complexes protéiques sont exposés à la dénaturation ou non des conditions physiologiques lors de la procédure. Pour contourner ces problèmes, nous avons mis en œuvre à grande échelle en protéomique technique quantitative d&#39;extraire des données impartiales et quantifiés. Nous utilisons l&#39;étiquetage des isotopes stables avec les acides aminés en culture cellulaire (SILAC) à intégrer des noyaux d&#39;isotopes de base en protéines dans une souche témoin non balisé. Des volumes égaux de la culture marqués et non marqués, la culture SILAC marqués sont mélangés et lysées par broyage dans l&#39;azote liquide. Nous avons ensuite effectuer une procédure de purification par affinité pour abattre des complexes protéiques. Enfin, nous précipiter l&#39;échantillon de protéine, qui est prêt pour l&#39;analyse par chromatographie liquide à haute performance / spectrométrie de masse en tandem. Surtout, les protéines de la souche de contrôle sont marquées par l&#39;isotope lourd et va produire un changement de masse / charge qui peut être quantifiée contre les protéines non marqué dans la souche d&#39;appât. Par conséquent, les contaminants, ou une liaison non spécifique peut être facilement éliminé. En utilisant cette approche, nous avons identifié de nouvelles protéines de plusieurs qui localisent à l&#39;ER-PM MCS. Nous présentons ici une description détaillée de notre approche.

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

Ici, nous décrivons une nouvelle technique quantitative en protéomique pour identifier les complexes protéiques chez Saccharomyces cerevisiae. Dans cette étude, nous avons utilisé la méthode SILAC avec purification par affinité suivie par spectrométrie de masse en tandem afin d&#39;identifier avec une grande spécificité les partenaires de liaison d&#39;une protéine ER, Scs2p.

Identification des complexes protéiques avec la protéomique quantitative chez S. cerevisiae

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

identification-protein-complexes-with-quantitative-proteomics-s

Research

JoVE Journal

Biology

13.1K vues.  University of British Columbia - UBC. Ici, nous décrivons une nouvelle technique quantitative en protéomique pour identifier les complexes protéiques chez Saccharomyces cerevisiae. Dans cette étude, nous avons utilisé la méthode SILAC avec purification par affinité suivie par spectrométrie de masse en tandem afin d'identifier avec une grande spécificité les partenaires de liaison d'une protéine ER, Scs2p.

Vidéo: 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.

Dosage de l&#39;adhésion et l&#39;invasion Agar chez S. cerevisiae

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

Recherche

Biologie

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.

Une méthode à haut débit pour étudier la morphologie globalement organite chez 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.

Phosphoprotéomique quantitatives en acide gras Stimulé 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.

Etudier Instabilité génomique fonction de l&#39;âge en utilisant les S. cerevisiae Modèle chronologique Lifespan

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.

Identification des complexes protéiques dans<em&gt; Escherichia coli</em&gt; En utilisant séquentielle Purification par affinité peptide en combinaison avec la spectrométrie de masse en 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.

Cytométrie en flux à base de purification de<em&gt; S. cerevisiae</em&gt; Zygotes

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.

Visualisation et d&#39;analyse de molécules d&#39;ARNm par fluorescence<em&gt; In Situ</em&gt; Hybridation dans<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.

Les analyses couplées pour la surveillance protéines repliement<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.

Identification des modifications post-traductionnelles des protéines végétales Complexes

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.

Identification des protéines d&#39;interaction partenaires dans les cellules de mammifères à l&#39;aide SILAC-immunoprécipitation quantitatives en protéomique

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