材料と方法<ol><li>酵母菌株<ul><li>本研究で使用したすべての菌株はBY4742背景に基づいています。 SILACのコントロール株（ マット、HIS3、LEU2、URA3、LYS2とarg4に：：G418は）：BY4742（マットアルファ、にarg4に欠失株（G418：マット、HIS3、URA3、LEU2、arg4は対象を ）交配することにより行われたHIS3、LEU2、URA3とLYS2）、および減数分裂半数体は四分子解剖によって得られた。したがって、対照株では、リジンおよびアルギニンのための栄養要求株である。餌のひずみは、ベクトルpBS1479を用いてPCRを介した相同組換えによって作られた。したがって、本研究で使用したエピトープタグは、タンデムアフィニティー精製（TAP）タグ（3）です。 </li></ul></li><li> SILAC： <ul><li> SILACの対照株は98％、L -リジン- 4 ,4,5,5 - D4（0.03 g / Lの、ケンブリッジアイソトープ）と98％L -アルギニン- 13 C 6（0.036グラム/を添加した最小のドロップアウトの培地で培養したL、ケンブリッジアイソトープ）。 </li><li>餌の株は、通常の同位体アミノ酸との定期的な富栄養培地で増殖させた。 </li></ul></li><li> TAPの精製の最初の段階で[このプロトコルは、コールドスプリングハーバー研究所コースマニュアル（4）から改作したものです] ** -80 ° Cの冷凍庫でお使い精製、プレチル乳鉢と乳棒の前夜。また、-20プレチルビーカー° C ** 右使用前にこれらのバッファを作る（培養液1 Lの場合）： <ul><li> 25μlのPIC（1 / 2000）、1M DTTを50μlと30ミリリットルNP - 40バッファー</li><li> 200分の1 PIC、1M DTT50μlのとNP - 40バッファー20 mlの</li><li> 2000分の1 PIC、1 M DTTを10μlとTEV - Cの緩衝液10ml </li></ul></li></ol> SILACと準備<ol><li> OD 600 = 1.0から1.3に別々に餌株と対照株の各1L成長する。 </li><li> 500ミリリットルNalgeneの遠心管に文化を注ぐ。 2580X gで負荷し、細胞をペレット化のバランスをとる</li><li> 50ミリリットルの遠心チューブに細胞を転送中に風邪のdH 2 O 50mlのあるデカントメディアとペレットを再懸濁します。細胞をペレット化。 **あなたは、-80 ° C **をペレットを凍結することができる</li><li> **重要**乾燥ペレットの重量で1:1の餌のひずみ文化にコントロール株の文化と一致している。 </li><li> 1 / / 2,000プロテアーゼインヒビターカクテル（PIC）とNP - 40バッファー10mlの中の各細胞ペレットを再懸濁します。 </li><li>直接、片方の管をデカントして、2つの培養細胞を混ぜる。 研削 </li><li>あらかじめ冷却した乳鉢に液体窒素を注ぎ、それが完全に蒸発させることができます。円を描くように乳鉢に細胞の2 mlを加え。細胞を凍結するいくつかの液体N 2を注ぐ。細胞は微粉末になるまで細胞を挽く。すべての細胞が地面になるまでプロセスを繰り返します。 **細胞が融解しないようにしてください。液体N 2に必要な**を追加</li><li>室温で氷冷ビーカーと融解する粉末を転送する。エッジが解凍し始めるときは1 / 200 PICとNP - 40緩衝液20 mlを加える。 </li><li> 40ミリリットルNalgene管に粗ライセートを転送する。 39000 xgfor 30分で回転する。 </li><li>待っている間、セファロース2Bビーズ（GE）の300μlを取る。 NP - 40バッファー（1 / 2、000 PIC）の3倍の300μlでビーズを洗浄する。彼らは1:1のスラリーになるように300μlのバッファーでビーズを再懸濁します。 前洗浄細胞ライセート </li><li>上清を新しいチューブに移し、セファロース2Bのビーズを追加。 30分間の低温室内の回転台にインキュベートする。 </li><li>待っている間、IgGセファロース6ビーズ（GE）の300μlを取る。 NP - 40バッファー（1 / 2、000 PIC）の3倍の300μlでビーズを洗浄する。彼らは1:1のスラリーになるように300 _lバッファーでビーズを再懸濁します。 </li><li>ペレットビーズ。上清を新しいチューブに移す。 **ウェスタンブロッティング後のために細胞溶解液のアリコートを保存する** IgG抗体セファロースビーズに結合する </li><li>遠心分離によってセファロース2Bのビーズを削除します。 IgG抗体ビーズを（以前に午前1時01スラリーに調製）を追加します。より効率的な結合のための2つのチューブに内容を区切ります。 2時間低温室での回転台にインキュベートする。 </li><li>ビーズをスピンダウンします。 **バインドされていない派閥のアリコートを保存する** </li><li> 10ミリリットルNP - 40バッファー（1 / 2、000 PIC）でビーズを洗浄する。 </li><li> 3ミリリットルTEV - Cバッファー（1 / 2、000 PIC）でビーズを洗浄する。 **ビーズのアリコートを保存します。これは、バインディング**の効率性を反映します TEVプロテアーゼ切断により、IgG抗体のビーズから溶出 </li><li> TEV - C 1mlの緩衝液（1 / 2、000 PICと）にAcTEVの5μlを添加する。混合した後、より効率的な混合するための2つの1.5mlのエッペンドルフチュー​​ブに内容を区切ってください。一晩低温室での回転台にインキュベートする。 </li><li>ビーズの回転を停止し、新しい1.5 mlチューブに溶出液を移す。 TEV - Cバッファーの追加の0.5mlでビーズを洗浄</li><li> 1本のチューブにelauteを組み合わせる。あなたは合計で溶出液の1.5 mlを持つ必要があります。** TEV溶出液のアリコートを保存する** トリクロロ酢酸（TCA）precipatationは、（質量分析ベースの分析では、我々は、エタノール/酢酸沈殿法を使用することをお勧めします） </li><li> 100％TCAとTCAを25％にアリコートを調整します。これを行うには、750μlの各2試験管に1.5 mlの溶出液を分離する。チューブのTCA 100％の250μlを追加します。ソリューションは、乳白色にする必要があります。 </li><li>周期的にボルテックスしながら30分間氷上に置きます。 </li><li> 30分間低温室でのテーブルトップ遠心機の最高速度でスピン。 </li><li>最大速度（低温室）で5分間0.05 N HClおよびスピンを含む氷冷アセトンを500μlで1回洗浄します。 </li><li>最大（低温室）で5分間氷冷アセトンとスピンの500μlので1回洗浄します。 </li><li>慎重にアセトンとドライペレットを取り外します。 </li><li>銀染色の場合は、1 × SDSサンプルバッファー50μlでペレットを再懸濁します。 エタノール/酢酸precipatation </li><li>グリコーゲンの20μgを追加する</li><li> 100％EtOHで5倍のサンプルを希釈し、2.5 M在庫切れと50mMのNaCH 3 COO、pHを5.0に調整する</li><li> 2時間のためのソリューションをスタンド</li><li>ペレットは、室温で12,000 × gで10分間の遠心分離によってタンパク質を沈殿させた。 </li></ol> NP - 40バッファー（200 mlの） <table border="0"><tbody><tr><td></td><td>ストック濃度</td><td>追加</td></tr><tr><td> 10mMリン酸ナトリウム緩衝液（pH7.2） </td><td> 0.1 M </td><td> 20ミリリットル</td></tr><tr><td> 150mMのNaCl </td><td> 2 M </td><td> 15ミリリットル</td></tr><tr><td> 1％NP - 40 </td><td> 100パーセント</td><td> 2ミリリットル</td></tr><tr><td> 50mMのNaFを</td><td> 1 M </td><td> 10ミリリットル</td></tr><tr><td> 0.1mMののNa 3 VO 4 </td><td> 10 mMの</td><td> 2ミリリットル</td></tr><tr><td style="text-align:right;" colspan="3"> 200mlにボリューム</td></tr></tbody></table>使用前にADD： <ol><li> 1 M DTTの1 / 1、000 </li><li> PICの1 / 2、000（プロテアーゼ阻害剤のカクテル） </li></ol> TEV - Cバッファー（50 mlの） <table border="0"><tbody><tr><td></td><td>ストック濃度</td><td>追加</td></tr><tr><td> 25mMトリス（pH8.0）に</td><td> 100mMの</td><td> 12.5ミリリットル</td></tr><tr><td> 150mMのNaCl </td><td> 2 M </td><td> 3.75ミリリットル</td></tr><tr><td> 0.1％NP - 40 </td><td> 100パーセント</td><td> _l 50 </td></tr><tr><td> 0.5mMのEDTA </td><td> 500mMの</td><td> __l 50 </td></tr><tr><td style="text-align:right;" colspan="3"> 50mlに容積</td></tr></tbody></table>使用前にADD： <ol><li> 1M DTTの1 / 1、000 </li><li> PICの1 / 2、000 </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>

精製処理中に保存さアリコートは、（1）事前に清澄化細胞ライセート、（2）結合画分、（3）非結合画分、および（4）溶出画分を含める必要があります。我々は、実験の結合と溶出効率を反映するために抗TAP抗体または銀染色を用いてウェスタンブロッティングにより上記アリコートの濃度のタンパク質を分析することをお勧めします。銀染色したゲルとブロットの例を図1に示されていま?...

identification of protein complexes with quantitative proteomics in s. cerevisiae

脂質は細胞膜のビルディングブロックの障壁として及び細胞プロセスの区画で、そして最近では、重要な細胞内シグナル伝達分子として機能です。しかし、タンパク質とは異なり、脂質は主に膜の接触部位（MCSの）で起こると仮定しているが不十分で説明非小胞性の経路によって小さな疎水性分子は、トラフィックです。 MCSのは、小胞体（ER）は提携細胞小器官、例えば、細胞膜（PM）との直接的な物理的接触を行う領域です。 ER - PM MCSのER部分は、その脂質の合成は、これらのサイトにリダイレクトし、MCSのは脂質のトラフィックのために重要であることを意味しているを示唆し、脂質合成酵素に富んでいる。酵母は、1000種類以上の細胞あたりの連絡先、およびすべての真核生物におけるそれらの保存された自然と、ために彼らの豊かなER - PM MCSを研究する理想的なモデルです。 MCSを構成するタンパク質を明らかにする脂質のトラフィックは細胞内でどのように達成されるかを理解する上で重要な、そしてそれらがどのようにシグナル伝達分子として作用です。我々はScs2pと呼ばれるERは、ER - PMのMCSのに局在することを発見し、その形成に重要ですしている。我々はScs2pの分子のパートナーを発掘に重点を置いています。タンパク質複合体の同定は、伝統的に質量分析法によるタンパク質のバンドやペプチドの分析のゲル内消化に続いて、最初にゲル電気泳動によって精製したタンパク質サンプルを解決することに依存しています。これは、しばしばタンパク質の小さなサブセットに調査を制限します。また、タンパク質複合体は、手順の間に変性または非生理的な条件にさらされている。これらの問題を回避するために、我々は公平と定量データを抽出するために大規模な定量的プロテオミクスの手法を実装している。我々は、タグの付いていない対照菌株中のタンパク質にステープル同位体の原子核を組み込むために細胞培養におけるアミノ酸（SILAC）と安定同位体標識法を使用してください。タグ付けされた文化とタグなし、SILAC標識文化の等量を混合し、液体窒素中で粉砕することにより溶解されています。私たちは、その後、タンパク質複合体をプルダウンするアフィニティー精製の手順を実行します。最後に、我々は、高速液体クロマトグラフィー/タンデム質量分析法による分析のための準備ができているタンパク質試料を、沈殿させる。最も重要なことは、対照株のタンパク質は、重い同位体で標識し、餌の株では非標識蛋白質に対する定量することができる質量/電荷シフトを生成します。したがって、汚染物質、または非特異的な結合を容易に除去することができます。このアプローチを使用することによって、我々は、ER - PMのMCSのためにローカライズするいくつかの新規タンパク質を同定した。ここでは、我々のアプローチの詳細な説明を提示する。

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

ここでは、出芽酵母でのタンパク質複合体を識別するための新しい定量的プロテオミクスの手法を説明します。本研究では、我々は、高い特異性でERタンパク質、Scs2pの結合パートナーを同定するためにタンデム質量分析が続くアフィニティー精製と一緒にSILACメソッドを使用している。

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

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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Research

JoVE Journal

Biology

13.1K ビュー.  University of British Columbia - UBC. ここでは、出芽酵母でのタンパク質複合体を識別するための新しい定量的プロテオミクスの手法を説明します。本研究では、我々は、高い特異性でERタンパク質、Scs2pの結合パートナーを同定するためにタンデム質量分析が続くアフィニティー精製と一緒にSILACメソッドを使用している。

ビデオ: 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.

S. cerevisiaeの接着と寒天の侵略のためのアッセイ

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

生物学

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.

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

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.

脂肪酸の定量的なPhosphoproteomicsは刺激を受けてサッカロマイセスセレビシエ</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.

使用年齢に依存するゲノム不安定性を研究して S.出芽酵母時系列寿命モデル

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.

のタンパク質複合体の同定<em&gt;大腸菌</em&gt;タンデム質量分析との組み合わせでシーケンシャルペプチドアフィニティ精製を用いた

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.

のフローサイトメトリーベースの精製<em&gt; S.セレビ</em&gt;受精卵

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.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（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.

のリフォールディング監視タンパク質を結合アッセイ<em&gt;サッカロミセス·セレビシエ（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.

植物タンパク質複合体の翻訳後修飾の同定

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.

SILAC免疫沈降定量的プロテオミクスを用いて哺乳動物細胞におけるタンパク質相互作用パートナーの同定

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