我々はそのCFTR 3次元構造コンソーシアム（承認番号FORD08XX0）を介して、この作業に資金を提供するため嚢胞性線維症財団（CFF）を感謝します。我々はCFTR遺伝子の設計では、特にこの仕事にコンソーシアム内のすべての仲間たちの巨大な貢献を認める。また、博士の貴重な貢献を認める。作業の初期段階ではジェームズBirtley（NCSR Demokritos、ギリシャ）、マーク·ヤング（カーディフ大学、英国）とデビッド·ドリュー（インペリアル·カレッジ、ロンドン、英国）。我々は、特に原稿の重要な読書のために博士伊那Urbatsch（テキサス工科。大学、ラボック）に感謝。

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cystic+fibrosis+transmembrane+conductance+regulator+degradation+depends+on.">Cystic fibrosis transmembrane conductance regulator degradation depends on.</a> the lectins Htm1p/EDEM and the Cdc48 protein complex in. 15, 4125-4135 (2004).</li><li>Yoo, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-conventional+trafficking+of+the+cystic+fibrosis+transmembrane+conductance+regulator+through+the+early+secretory+pathway.">Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.</a> J. Biol. 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Pharmacol. 75, 1430-148 (2009).</li><li>Ramjeesingh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+monomer+is+the+minimum+functional+unit+required+for+channel+and+ATPase+activity+of+the+cystic+fibrosis+transmembrane+conductance+regulator.">A monomer is the minimum functional unit required for channel and ATPase activity of the cystic fibrosis transmembrane conductance regulator.</a> Biochemistry. 40, 10700-10706 (2001).</li><li>Huang, P., Liu, Q., Scarborough, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysophosphatidylglycerol:+a+novel+effective+detergent+for+solubilizing+and+purifying+the+cystic+fibrosis+transmembrane+conductance+regulator.">Lysophosphatidylglycerol: a novel effective detergent for solubilizing and purifying the cystic fibrosis transmembrane conductance regulator.</a> Anal. Biochem. 259, 89-97 (1998).</li></ol>

利害の衝突が宣言されません。

本論文では、嚢胞性線維症の研究を促進すべきである酵母細胞でネズミのCFTR蛋白質の発現方法を提供する。目的は、嚢胞性線維症財団（経由で利用できるようになりますマウスCFTR DNAコンストラクトのリリースでこの論文をリンクすることである<a href="http://www.cff.org/research/CFFT/">http://www.cff.org/research/CFFT/</a> ）。他のオルソログは、後で利用できるようになるはずです。 CFTRを含むベクターを用いた酵母細胞の形質転換は簡単ですが、それはCFTRの高発現コロニーをスクリーニングすることが重要です。変数の発現レベルは、いくつかの要因から発生する可能性がありますが、プラスミド細胞当たりのコピー数は、おそらく変動の重要度を占めています。ここで説明する重要なステップでは、CFTR発現酵母細胞およびCFTR含むミクロソーム膜の生産を許可する必要があります。酵母細胞の形質転換、成長、収穫や溶解が習得されたら、purifタンパク質のicationが可能であるべきであり、図3に我々は、有用なベンチマークとして、このケースでは達成可能でなければなりません純度の例を与えている。それは、タンパク質の精製のために詳細な方法論を提供するために、この原稿では我々の意図ではありません。しかし、そこにそのような細胞溶解とミクロソーム精製などS.cerevisae発現系に固有のいくつかの重要な下流の精製工程があり、これらは本稿で詳細に含まれています。それは離れて我々が使用している二つの方法から、別の酵母細胞の破砕方法は、フランス語圧力セルの使用など、用いることができること、しかし、言及されるべきである。組換えタンパク質は、タンパク質が誘導する（図4）の後に追跡することができTEV-開裂可能なC末端GFPのドメインを持っています。酵母は同じ条件で11、この下に蛍光が役に立つ間を提供することができます本質的な70kDaタンパク質（おそらくデヒドロゲナーゼ12コハク酸）が全細胞抽出物またはミクロソームにおけるCFTRの相対発現レベルの内部キャリブレーション標準（図3）。それはガラクトースで誘導後の細胞収穫のタイミングが重要であることを図4に示すデータから明らかである。 CFTRのドロップの収率は急激に誘導の約16時間後、酵母細胞内の任意の検出可能なCFTRは、誘導の24時間後にやっとそこにあるように。 精製タンパク質の収量は15リットル発酵槽培養液ごとに1-2mgを約CFTR蛋白質である。リカバリは、ミクロソームのステージに合計CFTR-GFP蛋白質までの約70％と推定され、タンパク質の精製約25％回復することができます。 S.のキャラクタリゼーション酵母発現CFTRは進行中である。図に見られる。 4、細胞内の蛋白質の場所は、蛍光顕微鏡で監視することができます。蛍光の多くが予想される10個のセルの周囲に発見されていますが、タンパク質の一部が点状の局在を表示、のいずれか、またはただ、おそらく後期ゴルジ体/エンドソーム経路14またはER 4からの輸送小胞の出芽のコンパートメント下流を通じてリサイクルをCFTRが原因である可能性があり、原形質膜の内側。 PNGaseF、タンパク質をdeglycosylates酵素を用いた治療は、それがグリコシル化されていることを意味し、SDS-PAGE上のCFTRタンパク質のバンドの移行に最小限の変化を示し、または最小限のグリコシル化15を有する。タンパク質のリン酸化状態の実験が進行中である。洗剤の一部にこれまでテストされ、精製されたタンパク質は、以前は2,15公開されたものに類似しているレートでATPase活性を（CFTR-特異的阻害剤16によって阻害されている）が表示されます。 CFTRチャネル活性の測定は、比較的高い臨界ミセル濃度（CMC）17を有する洗剤の最終的な精製工程を含意するでしょう精製タンパク質の再構成が必要になります。酵母ミクロソームのcontaininグラムCFTRは、例えば、一般的に&quot;マイルド&quot;であると考えられているドデシルマルトシド2のような界面活性剤を含むいくつかの一般的に用いられる界面活性剤18とsolublisedすることができます。しかし、最も高いCMC洗剤は、これらの界面活性剤にその交換を示唆し、これまでのところ、solubilsationでは不十分であることが判明している任意の精製スキームの後半の段階で考慮されるべきである。

<table border="1"><tbody><tr><td> 試薬/装置の名前 </td><td> 会社 </td><td> カタログがありません </td></tr><tr><td> PEP4削除10のFGY217 S.cerevisiae株 、 </td><td></td><td></td></tr><tr><td>アミノ酸を含まない酵母窒素塩基</td><td> Formedium </td><td> CYN0410 </td></tr><tr><td>ウラシルなく、サプリメント混合物を完了する</td><td> Formedium </td><td> DCS0169 </td></tr><tr><td>細菌学的寒天</td><td> Sigma-Aldrich社</td><td> A5306 </td></tr><tr><td> D-ガラクトース</td><td>フィッシャー</td><td> BP656-500 </td></tr><tr><td> D-グルコース</td><td>フィッシャー</td><td> D16-500 </td></tr><tr><td>ペプスタチン</td><td> Sigma-Aldrich社</td><td> P4265 </td></tr><tr><td>ロイペプチン</td><td>メルク</td><td> 108975 </td></tr><tr><td>キモスタチン</td><td> Sigma-Aldrich社</td><td> C7268 </td></tr><tr><td> Phenylmethanesulfonylfluoride（PMSF） </td><td> Sigma-Aldrich社</td><td> P7626 </td></tr><tr><td> Epoxysuccinyl-leucylamido-ブタン（E-64） </td><td> Sigma-Aldrich社</td><td> E3132 </td></tr><tr><td> Aminoethylbenzenesulfonylフッ化物（AEBSF） </td><td> Sigma-Aldrich社</td><td> A8456 </td></tr><tr><td>ベンズアミジン塩酸塩</td><td> Sigma-Aldrich社</td><td> 434760 </td></tr><tr><td>ジメチルスルホキシド（DMSO） </td><td> Sigma-Aldrich社</td><td> D8418 </td></tr><tr><td>ジチオスレイトール（DTT） </td><td> Sigma-Aldrich社</td><td> 43815 </td></tr><tr><td>エチレンジアミン四酢酸（EDTA） </td><td>フィッシャー</td><td> BP120500 </td></tr><tr><td>トリス塩基</td><td> Formedium </td><td> TRIS01 </td></tr><tr><td>のTris-HCl </td><td>フィッシャー</td><td> P631 </td></TR&gt; <tr><td> D-ソルビトール</td><td> Sigma-Aldrich社</td><td> S1876 </td></tr><tr><td>グリセロール</td><td>フィッシャー</td><td> 065017 </td></tr><tr><td> NaClを</td><td> Sigma-Aldrich社</td><td> S6191 </td></tr><tr><td> n-ドデシル-β-D-maltopyranoside </td><td>アフィメトリクス</td><td> D310S </td></tr><tr><td>ブロモフェノールブルー</td><td> Sigma-Aldrich社</td><td> 114391 </td></tr><tr><td> PageRulerプラスジウムタンパク質標準</td><td> Fermentas </td><td> SM1811 </td></tr><tr><td> NuSepトリス-Glyの4-20％グラジエントゲル</td><td> NuSep </td><td> NB10-420 </td></tr><tr><td>インスタントブルークマシー染色</td><td> Novexin </td><td> ISB1L </td></tr><tr><td>酸洗浄ガラスビーズ、 </td><td>シグマ</td><td> G8772 </td></tr><tr><td> 50ミリリットル滅菌ファルコンチューブ</td><td>ザルスタット</td><td> 62.547.254 </td></tr><tr><td> 2ミリリットル滅菌スクリュートップバイアル</td><td>ザルスタット</td><td> 72.694.005 </td></tr><tr><td> 250ミリリットル滅菌三角フラスコに困惑</td><td> BDバイオサイエンス</td><td> 355119 </td></tr><tr><td> 2リットル滅菌三角フラスコに困惑</td><td> BDバイオサイエンス</td><td> 355131 </td></tr><tr><td> 2ミリリットルマイクロチューブ</td><td>ザルスタット</td><td> 72.695 </td></tr><tr><td> 0.2μmのシリンジフィルター</td><td>縫工筋</td><td> FC121 </td></tr><tr><td>超遠心管</td><td>ベックマン·コールター</td><td> 355618 </td></tr><tr><td>遠心チューブ</td><td>ベックマン·コールター</td><td> 357000 </td></tr><tr><td> 1リットル遠心ポット</td><td>ベックマン·コールター</td><td> 969329 </td></tr><tr><td>軌道は、温度制御と振とう培養</td><td>ニューブランズウィックサイエンティフィック</td><td></td></tr><tr><td> Deltavision RT復元顕微鏡</td><td>アプライド·ビジョン</td><td></td></tr><tr><td>卓上型遠心機</td><td> HERMLE </td><td> Z300 </td></tr><tr><td>卓上型遠心</td><td>フィッシャー</td><td> 13-100-511 </td></tr><tr><td>ボルテックスミキサー</td><td>スターラボ</td><td></td></tr><tr><td>台風·トリオスキャナ</td><td> GEヘルスケア</td><td> 63-0055-87 </td></tr><tr><td> LAS3000イメージングシステム</td><td>富士</td><td></td></tr><tr><td> 20リットル発酵容器と制御ユニット</td><td> Applikon </td><td></td></tr><tr><td>定数系細胞のかく乱</td><td>定数システム</td><td></td></tr><tr><td> Beadbeater細胞かく乱</td><td> BioSpec </td><td> 1107900 </td></tr><tr><td>ミニbeadbeater-16 </td><td> BioSpec </td><td> 607 </td></tr><tr><td> JA-17ロトR </td><td>ベックマン·コールター</td><td> 369691 </td></tr><tr><td>オプティマL-100超遠心</td><td>ベックマン·コールター</td><td> 392050 </td></tr><tr><td> 50.2Tiローター</td><td>ベックマン·コールター</td><td> <a href="https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=337901" target="_blank">337901</a> </td></tr></tbody></table>

expression and purification of the cystic fibrosis transmembrane conductance regulator protein in saccharomyces cerevisiae

嚢胞性線維症膜コンダクタンス制御因子（CFTR）が変異すると、したがって、このタンパク質にはかなりの関心がhumans.Thereで嚢胞性線維症を生じさせることができるクロライドチャネルであるが、その構造と活性を研究するための努力は困難によって妨げられている1から3蛋白質の十分な量を発現し、精製する。多くの&quot;困難な&quot;真核生物の膜タンパク質と同様に、急成長している生物での発現が望ましいですが、やりがいがあり、酵母S.の酵母は 、これまで少量が得られた組換えタンパク質の急速な劣化が4-9を観察した。酵母における組換えCFTRの処理に関与するタンパク質は6-9に記載されている。本報告では、かなりの量の酵母とその浄化のCFTRの発現のための方法論を記述します。プロトコルは、以前のタンパク質分解の問題は克服することができる方法について説明し、どのように発現レベルのCFTRが大幅に細胞採取の前に特定の期間内に、細胞の成長条件を変更することにより、誘導条件を制御することによって改善することができます。このプロトコル（マウスCFTR発現酵母細胞または酵母プラスミド）に関連付けられているreagantsは、研究を後援している米国の嚢胞性線維症財団を介して配布されます。 CFTRの設計と合成、このレポートで用いコンストラクトを記述した記事が（; Thibodeau、P. ら 、未発表Urbatsch、I.）は別途公開されます。プラスミドを含む酵母（図1） - この記事では、CFTRの構築と酵母細胞の形質転換手法の始まりを説明します。構文は、C末端に融合CFTR緑色蛍光タンパク質（GFP）の配列を持っており、ドリューらによって開発されたシステム。（2008）10に従います 。 GFPは、CFTRの発現と精製は比較的容易に続くことができます。 Joveの可視化機能実装酵母細胞からミクロソームの調製後オール終了すると、我々はミクロソームからのタンパク質の精製のためのいくつかの提案が含まれていますが。読者はそれらに利用可能な蛋白質とローカル機器で実施される最終的な実験に依存し、ミクロソーム精製法に独自の変更を追加したい場合があります。酵母発現CFTR蛋白質は、部分的に本質的なポリヒスチジン精製タグを使用して、金属イオンアフィニティークロマトグラフィーを用いて精製することができる。その後のサイズ排除クロマトグラフィーは、SDS-PAGEとゲルのクマシー染色で判定して、&gt; 90％純粋であると思われるタンパク質が得られます。

Watch this Scientific Journal Video about Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae at JoVE.com

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

で嚢胞性線維症膜コンダクタンス制御因子（CFTR）を表現する試み<em&gt;サッカロマイセスセレビシエ</em&gt;今まで、タンパク質の比較的少量が得られている。嚢胞性線維症財団を介して配布このプロトコルと関連した試薬は、この &quot;難しい&quot;真核生物の膜蛋白質のミリグラム量の準備を許可する必要があります。

で嚢胞性線維症膜コンダクタンス制御因子タンパク質の発現および精製サッカロマイセスセレビシエ

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

expression-purification-cystic-fibrosis-transmembrane-conductance

Research

JoVE Journal

Biology

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

18.4K ビュー.  University of Manchester. で嚢胞性線維症膜コンダクタンス制御因子（CFTR）を表現する試みサッカロマイセスセレビシエ今まで、タンパク質の比較的少量が得られている。嚢胞性線維症財団を介して配布このプロトコルと関連した試薬は、この "難しい"真核生物の膜蛋白質のミリグラム量の準備を許可する必要があります。

ビデオ: Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

の解剖サッカロマイセスセレビシエ ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

生物学

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

クーマシー有機溶媒および酢酸のないG - 250でゲル中のタンパク質の染色

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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 ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

の精製 M. magneticum株AMB - 1 Magnetosome関連タンパク質MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

マイクロアレイ解析のためのサッカロマイセスセレビシエ</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

酵母でMitophagyを監視するための蛍光顕微鏡アッセイサッカロマイセスセレビシエ

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

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 ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

で表現する嚢胞性線維症膜コンダクタンス制御因子タンパク質の精製<em&gt;サッカロマイセス·セレビシエ</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to identify and quantify the cofactors in their detectable redox state.
The technique is complementary to direct electrochemistry (voltammetry) approaches, as it does not offer information on electron transfer rates, but does establish the identity and redox state of the cofactors in the protein under study. The technique is widely applicable to any protein containing an electron paramagnetic resonance (EPR) detectable cofactor.
A typical titration requires 2 ml protein with a cofactor concentration in the range of 1-100 &#181;M. The protein is titrated with a chemical reductant (sodium dithionite) or oxidant (potassium ferricyanide) in order to poise the sample at a certain potential. A platinum wire and a Ag/AgCl reference electrode are connected to a voltmeter to measure the potential of the protein solution. A set of 13 different redox mediators is used to equilibrate between the redox cofactors of the protein and the electrodes. Samples are drawn at different potentials and the Electron Paramagnetic Resonance spectra, characteristic for the different redox cofactors in the protein, are measured. The plot of the signal intensity versus the sample potential is analyzed using the Nernst equation in order to determine the midpoint potential of the cofactor.

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

The goal of this protocol is to use electron paramagnetic resonance (EPR) monitored redox titrations to identify different cofactors of Saccharomyces cerevisiae Nar1. Redox titrations offer a very robust way to obtain midpoint potentials of different redox active cofactors in enzymes and proteins.

EPRは、補因子の酸化還元滴定を監視対象<em&gt;サッカロマイセス·セレビシエ</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...