We thank Dr. Oliver Kerscher for support and advice and members of the Basrai laboratory for their support and comments on the paper. This work was supported by the National Institutes of Health Intramural Research Program.

酵母細胞の1成長<ol><li>小さ ​​なフラスコにYPD（ 表1）30mlに酵母細胞を接種します。振とうしながら30℃で一晩インキュベートします。 </li><li> YPD 50mlに600 nmの（OD 600 = 0.2）で0.2の光学濃度に細胞を希釈し、振盪しながら30℃でインキュベートします。 </li><li> 600 nmの（OD 600 = 1.0）で1.0の光学密度に文化を育てます。 </li><li> 2,000×gで5分間遠心操作し細胞と上清を捨てます。 </li><li>細胞を洗浄するために2,000×gで5分間、40ミリリットル滅菌水と遠心機で細胞ペレットを再懸濁します。上清を捨て、-20℃で細胞ペレットを保存します。 </li></ol>タンパク質の2抽出<ol><li>またはNi-NTAスーパーフロービーズを用いたプルダウンアッセイの氷冷グアニジン緩衝液（表1）0.5mlに再懸濁細胞を、免疫沈降した（ 表1）をバッファ。すべての回で氷の上にチューブを保管してください。 </li><li> 2メートルへの転送Lスクリューキャップチューブ。 </li><li>ガラスビーズの同じ容量（ 材料表）を追加します。 </li><li> 2-3分間氷上に置く、その後、室温で2分間のミニビーズビーター（材料表）で細胞をビーズ破りました。この3回繰り返します。 </li><li> 30〜60分間、4℃で高速の渦。細胞が溶解されていることを確認するために顕微鏡下で可視化して細胞を確認してください。 注：溶解した細胞は、暗い幽霊が表示され、境界または規定された形状を欠くであろう。最適細胞の少なくとも80％が溶解されるべきです。 </li><li> 15ミリリットルコニカルチューブ（スクリューキャップが緩んでなければならない）コレクションに押しピンと場所を使用して、チューブの底に穴を穿刺。 </li><li>溶解物を収集するために1分間1000×gで遠心分離します。 </li><li>マイクロ遠心チューブにライセートを移します。 </li><li> 4°Cで30分間15,000×gで遠心分離は、抽出されたタンパク質を回収します。 </li><li>タンパク質ASSAを用いて抽出されたタンパク質の濃度を測定しますYキット（ 材料の表）と同量のタンパク質を含有するすべての抽出物を正規化します。適切な緩衝液で1ミリリットルの全容量を持参してください。 注：総タンパク質の約5 mgを50 OD 600細胞から得られます。 </li><li>全細胞抽出物（WCE）として（ステップ2.10から抽出した全タンパク質は、5mgである場合に250μgタンパク質）50μLを保存します。 2×Laemmliサンプルバッファー（ 表1）の50μlを添加して、3〜5分間ヒートブロックで100℃でインキュベートします。負荷ウエスタンブロット分析（第5節：ウェスタンブロット分析）のためのSDS-PAGEゲルの各サンプルを10μl。 </li></ol> HF-SMT3コンジュゲートの3精製<ol><li> 1分間低速遠心（800-1,500 XG）による実験（サンプルあたりのビーズを100μl）のために必要なのNi-NTAスーパービーズ（ 材料表）を取得します。上清を取り除きます。 </li><li>反転トップオーバー：洗浄ビーズを1mlのPBS（材料表）で5倍底ビーズを再懸濁されるまで、低速遠心分離によりビーズを収集し、上清を除去します。 </li><li>グアニジン緩衝液（ 表1）1mlのビーズを中断し、処理対象のサンプルに対応する管の数にそれらをアリコート。低速遠心分離によりビーズを集め、上清を除去します。トップオーバー底を反転させてよく、ビーズを混ぜます。 注：トップオーバー底を反転させてよく、ビーズを混ぜます。 </li><li>のNi-NTAスーパービーズ100μlに：各サンプルについて、（タンパク質の抽出ステップ2.11から）残りのWCEの950μlを添加します。 </li><li>少なくとも4時間、または4℃で一晩、ロッキングプラットフォーム上でインキュベートします。 </li><li> 4°Cで1分間800-1,500×gで遠心分離します。 </li><li>上清（SUP）として50μLを保存します。 2×Laemmliサンプル緩衝液50μlを添加し、3〜5分間ヒートブロックで100℃でインキュベートします。ウェスタンブロット分析のためにSDS-PAGEゲルにおける負荷10μlの各サンプルを（セクション5：ウェスタンブロット肛門ysis）。 </li><li>ロッキングプラットフォーム上で5〜10分間、グアニジン緩衝液1mlで一回ビーズを洗浄します。 </li><li>洗浄ビーズをロッキングプラットフォーム上で5〜10分間のバッファ（ 表1）を破る1mlの5倍。 </li><li> 2×Laemmliサンプルバッファー90μlの再懸濁ビーズ。 </li><li> 1 Mイミダゾールの10μlを添加します。 注：イミダゾールは、イミダゾールとビーズとの間の相互作用を競合に起因したNi-NTAビーズからのHisタグ化タンパク質を解離するのに役立ちます。 </li><li> 3-5分間ヒートブロックで100℃でインキュベートします。 </li><li>ボルテックスし、30秒間13,000×gで遠心します。上清を新しいチューブに移します。 </li><li>ウェスタンブロット分析のためにSDS-PAGEゲル（：ウェスタンブロット分析セクション5）の各サンプルのロード10-20μL。 注：WCEの5 mgを使用した場合、10〜20μlを、入力の0.5〜1.0ミリグラムに相当します。 </li></ol> Myc標識動原体タンパク質の4免疫沈降<ol><li>抗c-Mycのアガロースアフ​​ィニティーゲルANを取得tibody（ 材料表）低速遠心分離（800-1,500 XG）による実験（サンプルあたりの樹脂の25μl）をするために必要な。上清を取り除きます。 </li><li>緩衝液A 1mlで洗浄樹脂5X：反転トップオーバーボトム樹脂を再懸濁されるまで、低速遠心分離によって樹脂を集め、上清を除去します。 </li><li>緩衝液A 1mlの樹脂をサスペンドし、処理対象のサンプルに対応する管の数にそれらをアリコート。低速遠心分離によって樹脂を集め、上清を除去します。 注：トップオーバー底を反転させて十分に樹脂を混ぜます。 </li><li>樹脂の25μlに：（タンパク質の抽出ステップ2.11から）残りのWCEの950μlを添加します。 </li><li>一晩4℃でロッキングプラットフォーム上でインキュベートします。 </li><li> 4°Cで1分間800-1,500×gで遠心分離します。 </li><li>上清（SUP）として50μLを保存します。 2×Laemmliサンプル緩衝液50μlを添加し、3〜5分間ヒートブロックで100℃でインキュベートします。ローウェスタンブロット分析のためにSDS-PAGEゲル（：ウェスタンブロット分析セクション5）の各サンプルの10μlの広告。 </li><li>緩衝液A 1mlで洗浄樹脂5X：反転トップオーバーボトム樹脂を再懸濁されるまで、低速遠心分離によって樹脂を集め、上清を除去します。 </li><li> SUMEBサンプル緩衝液（ 表1）100μlの中に樹脂を再懸濁します。 注：SUMEBサンプル緩衝液を8 M尿素および1％SDS（強い変性条件）を含みます。 </li><li> 3-5分間ヒートブロックで100℃でインキュベートします。 </li><li>ボルテックスし、30秒間13,000×gで遠心します。上清を新しいチューブに移します。 </li><li>ウェスタンブロット分析のためにSDS-PAGEゲル（：ウェスタンブロット分析セクション5）の各サンプルのロード10-20μL。 注：WCEの5 mgを使用した場合、10〜20μlを、入力の0.5〜1.0ミリグラムに相当します。 </li></ol> 5.ウェスタンブロット分析<ol><li> 4~12％のBis-Trisゲル（材料の表）とPERFにタンパク質抽出物をロードORM電気泳動は、90分間120 Vで（ランニング緩衝液： 材料の表）。 </li><li> 90分（： 材料の表転送バッファ）、30 Vで転写装置を用いてニトロセルロース膜（ 材料表）にゲルからタンパク質を転送します。 </li><li>室温で1時間の5％ミルク/ 1×TBST（ 表1）で膜をブロックします。 </li><li> 4℃で一晩、5％ミルク/ 1×TBST中で一次抗体（ 材料表）で膜をインキュベートします。千（抗Flagおよび抗Ubの抗体）または1：5,000（抗Myc抗体）一次抗体は、1の希釈を使用しています。 </li><li> 1×TBSTで10分間ずつの膜3回洗浄します。 </li><li>室温で1時間、5％ミルク/ 1×TBSTで二次抗体（ 材料表）で膜をインキュベートします。二次抗体の希釈5,000：1を使用してください。 </li><li> 1×TBSTで10分間ずつの膜3回洗浄します。 </li><li> ECL workiで膜をインキュベートngの溶液（ 材料表）5分間。 </li><li>青感性X線フィルムに膜（ 材料表）を公開し、自動現像機（ 材料表）を使用して開発しています。 </li></ol>

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The authors have nothing to disclose.

そのようなHA、Mycの、フラグ、GSTなどのエピトープタグは、広くタンパク質の生化学的分析のために使用されます。 HF-SMT3などNdc10やNdc80としてMyc標識動原体タンパク質、を有する株の構築は、SUMO化およびユビキチン化などのPTMの検出を容易にしています。 HF-SMT3アッセイをプルダウンNdc10とNdc80（ 図1）、SUMO化動原体タンパク質の検出を可能にします。修飾タンパク質は、HF-SMT3な?...

それぞれ、標的タンパク質へ;（S.セレビシエ 1 SMT3 SUMO）ユビキチンとSUMO化は、ユビキチンと小さなユビキチン様修飾剤の結合を可能にします。動原体蛋白質のPTMは、忠実な染色体の分離を確実にするために、異なる細胞周期の段階でそれらの細胞レベルおよびタンパク質 - タンパク質相互作用に影響を与えます。例えば、Cse4 / CENP-Aと外側動原体タンパク質DSN1の細胞レベルは、ゲノムの安定性2-5を確保するためのユビキチンを介したタンパク質分解によって調節されています。不正な動原体微小管の添付ファイルの不安定化は、直接微小管6-8と対話DAM1とNdc80複合体をリン酸化するIPL1 /オーロラBキナーゼを必要とします。 70以上の動原体タンパク質の同定にもかかわらず、PTMを、 例えば 、ユビキチンおよびSUMOとのこれらのタンパク質の修飾を調査する非常に少数の研究があります。主な制限は、PTMを維持する能力であります精製およびそのようなSUMO化、リン酸化、メチル化、および他のようなPTMを検出するためのカスタム抗体の不足時にS。 SUMO化動原体タンパク質の特性はNdc10、Cep3は、BIR1、およびNdc80カスタム抗体9を利用しました。さらに、Ndc10は、ユビキチン化10の基質として関与しています。ヒトHec1（S. cerevisiaeにおけるNdc80）も、APC / C-hCdh1 E3リガーゼ11によって規制、ユビキチン化の基質です。したがって、Ndc10とNdc80はSにSUMO化とユビキチン化の両方を検出するためのプロトコルの最適化のための良好な候補でありますセレビシエ 。 SUMO化の識別を容易にするために、我々は、HF-SMT3とMyc標識Ndc10またはNdc80を発現する株を構築しました。エピトープタグ（HF：のHis6フラッグ）の使用は、頻繁に候補タンパク質に対するポリクローナル血清で観察された交差反応性に背景を最小限に抑えることができます。私たちは、親和性のプロトコルを考案しましたHF-SMT3複合体を精製した後、精製SMT3準備にsumolyated Ndc10とNdc80の存在を検出するために、市販の抗フラッグ及び抗Myc抗体を用いました。ユビキチン化のために、我々はmycタグ動原体タンパク質のユビキチン化を保持する修飾された免疫沈降プロトコルを考案し、Ndc10とNdc80のユビキチン化を検出するために、市販の抗Ubの抗体を用いたウエスタンブロット分析を行いました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Glass beads</td>
			<td>BioSpec Products</td>
			<td align="right">11079105</td>
			<td>0.5 mm dia.</td>
		</tr>
		<tr>
			<td>Mini beadbeater</td>
			<td>BioSpec Products</td>
			<td>#693</td>
			<td>8 cell disrupter</td>
		</tr>
		<tr>
			<td>Ni-NTA superflow</td>
			<td>Qiagen</td>
			<td align="right">30430</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail</td>
			<td>Sigma-Aldrich</td>
			<td>P8215</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Anti-c-Myc agarose affinity gel antbody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>A7470</td>
			<td>1 ml</td>
		</tr>
		<tr>
			<td>Monoclonal anti-Flag M2 antibody produced in mouse</td>
			<td>Sigma-Aldrich</td>
			<td>F1804</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>c-Myc antibody (A-14)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-789</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Purified mouse antibody monoclonal 9E10</td>
			<td>Covance</td>
			<td>MMS-150P</td>
			<td>Primary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>Ubiquitin (P4G7) monoclonal antibody</td>
			<td>Covance</td>
			<td>MMS-258R</td>
			<td>Primary antibody, dilution 1:1000</td>
		</tr>
		<tr>
			<td>ECL Rabbit IgG, HRP-linked whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA934V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>ECL Mouse IgG, HRP-Linked Whole Ab</td>
			<td>GE Healthcare Life Sciences</td>
			<td>NA931V</td>
			<td>Secondary antibody, dilution 1:5000</td>
		</tr>
		<tr>
			<td>DC protein assay</td>
			<td>Bio-Rad</td>
			<td>500-0116</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Novex</td>
			<td>LC2001</td>
			<td>0.45 mm pore size</td>
		</tr>
		<tr>
			<td>NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td>Novex</td>
			<td>NP0321BOX</td>
			<td>1.0 mm, 10 well</td>
		</tr>
		<tr>
			<td>NuPAGE MES SDS Running Buffer</td>
			<td>Novex</td>
			<td>NP0002</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>NuPAGE Transfer Buffer</td>
			<td>Novex</td>
			<td>NP0006-1</td>
			<td>20x</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td align="right">34078</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS pH7.4</td>
			<td>GIBCO</td>
			<td>70011-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blue sensitive X-Ray film</td>
			<td>Dbio</td>
			<td>DBOF30003</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic developer</td>
			<td>Kodak</td>
			<td>M35AX-OMAT</td>
			<td></td>
		</tr>
		<tr>
			<td>Nocodazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1404</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>MG-132</td>
			<td>Selleck Chemicals</td>
			<td>S2619</td>
			<td>25 mg</td>
		</tr>
	</tbody>
</table>

protein purification technique that allows detection of sumoylation and ubiquitination of budding yeast kinetochore proteins ndc10 and ndc80

このようなリン酸化、メチル化、アセチル化、ユビキチン化、およびSUMO化などの翻訳後修飾（PTMを）は、多くのタンパク質の細胞機能を調節します。動原体DNAに関連付ける動原体タンパク質のPTMを、ゲノムの安定性を維持するために忠実な染色体分離を媒介します。このような質量分析およびウェスタンブロット分析のような生化学的アプローチは、最も一般的のPTMの同定のために使用されます。ここで、タンパク質精製法は、サッカロミセス·セレビシエに、Ndc10とNdc80、動原体タンパク質のSUMO化及びユビキチン化の両方の検出を可能にすることが記載されています。ポリヒスチジンFlagタグ付きSMT3（HF-SMT3）およびMyc標識Ndc10またはNdc80を発現株を構築し、私たちの研究のために使用しました。 SUMO化の検出のために、我々は、ニッケルビーズを用いて、HisタグSUMO化タンパク質をアフィニティー精製するためのプロトコルを考案し、SUMO化Ndc10 Aを検出するために抗Myc抗体を用いたウエスタンブロット分析を使用しましたND Ndc80。ユビキチン化の検出のために、我々はMyc標識タンパク質の免疫沈降のためのプロトコルを考案し、Ndc10とNdc80がユビキチン化されていることを示すために抗Ubの抗体を用いたウェスタンブロット分析を使用していました。我々の結果は、彼のフラッグで目的のエピトープタグ化タンパク質はSMT3株は、複数のPTMの検出を容易にタグ付けされたことを示しています。今後の研究では、この技術の利用は、特定のPTMに依存しているタンパク質相互作用を同定し、特徴付けるために許可する必要があります。

以前9,12に記載されているように動原体タンパク質Ndc80とNdc10のSUMO化を検出するために、HF-SMT3とMyc標識動原体タンパク質（Ndc80またはNdc10）を有する株は、（ 表2）を構築しました。 HF-SMT3結合体は、親和性のNi-NTAビーズを用いて精製しました。 HF-SMT3（ 図1A及び図1Bに示すように 、左パネル）を含まない対照株には存在しなかったSUMO基...

Watch this Scientific Journal Video about Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80 at JoVE.com

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

この原稿は、出芽酵母サッカロミセス·セレビシエ（Saccharomyces cerevisiae）において、動原体タンパク質、Ndc10とNdc80のSUMO化とユビキチン化の検出について説明します。

出芽酵母動原体タンパク質Ndc10とNdc80のSUMO化とユビキチン化の検出を可能にするタンパク質の精製技術

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

protein-purification-technique-that-allows-detection-sumoylation

Research

JoVE Journal

Biology

12.0K ビュー.  National Cancer Institute, National Institute of Health. この原稿は、出芽酵母サッカロミセス·セレビシエ（Saccharomyces cerevisiae）において、動原体タンパク質、Ndc10とNdc80のSUMO化とユビキチン化の検出について説明します。 

ビデオ: Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

出芽酵母の複製寿命の測定

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

生物学

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

タンパク質のユビキチン化の検出

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

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 ...

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

酵母細胞からミトコンドリアの精製

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104の精製、タンパク質Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母のリボソームのクロマトグラフィー精製

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

Acquiring Fluorescence Time-lapse Movies of Budding Yeast and Analyzing Single-cell Dynamics using GRAFTS

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies depicting the temporal dependence of gene expression provide insight into the dynamics of its regulation; however, there are many technical challenges to obtaining and analyzing fluorescence movies of single cells. We describe here a simple protocol using a commercially available microfluidic culture device to generate such data, and a MATLAB-based, graphical user interface (GUI) -based software package to quantify the fluorescence images. The software segments and tracks cells, enables the user to visually curate errors in the data, and automatically assigns lineage and division times. The GUI further analyzes the time series to produce whole cell traces as well as their first and second time derivatives. While the software was designed for S. cerevisiae, its modularity and versatility should allow it to serve as a platform for studying other cell types with few modifications.

We present a simple protocol to obtain fluorescence microscopy movies of growing yeast cells, and a GUI-based software package to extract single-cell time series data. The analysis includes automated lineage and division time assignment integrated with visual inspection and manual curation of tracked data.

出芽酵母の蛍光タイムラプス作品を取得し、移植片を使用したシングルセルダイナミクスの解析

Fluorescence time-lapse microscopy has become a powerful tool in the study of many biological processes at the single-cell level. In particular, movies ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

機能の研究、相互作用、および翻訳後修飾のため出芽酵母タンパク質の抽出と精製

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜タンパク質の緑色蛍光タンパク質ベースの発現スクリーニングで<em&gt;エシェリヒア·コリ</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...