この作業は、FAPESP 認可 (15/06477-9) によってサポートされました。

1. 酵母総抽出物の調製
<ol><li>1 L YNB-glu培地(酵母窒素基底に2%m/vグルコースを補充)のTAPタグ9 に融合したスプライシング因子の1つを発現する酵母細胞を、適切なアミノ酸または核塩基で増殖させ、 この場合、アデニン(20μg/mL)、ロイシン(30μg/mL)、トリプトファン(30μg/mL)、OD600= 1.0まで。</li>
	<li>1L培養から細胞を1L培養から採取し、4°Cで10分間17,000xgの3本の500mL遠心分離器ボトルで遠心し、10mL冷たい滅菌水で2回洗浄します。 </li>
	<li>採取した酵母細胞をコールドバッファーAの細胞体積の1/10に再懸濁させる(10 mmolL-1 HEPES pH = 7.9,1.5 mmolL-1 MgCl2,50mmolL-1 KCl,5%v/vグリセロール,0.5 mmol L-1 DTT,EDTAフリープロテアーゼ阻害剤カクテル)。</li>
	<li>液体窒素中の細胞懸濁液の小さな滴を凍結します。小さな滴は、再懸濁された細胞溶液をピペット化し、50 μLを液体窒素に直接滴下することによって得ることができる。 注意:液体窒素は皮膚や目と接触して傷害を引き起こす可能性があります。適切な安全装置を使用して取り扱います。 注: プロトコルはここで一時停止することができます。細胞懸濁液の凍結滴は-80°Cで保存することができる。</li>
	<li>ボールミル装置で20 Hz/sで6サイクル3分間粉砕して、酵母細胞のライセートを調製します。 注:各サイクルの後、凍結した細胞を含む容器を液体窒素に浸して、溶融を避けてください。プロトコルはここで一時停止することができます。凍結した抽出物は、-80°Cで保存することができる。 酵母スプライシング抽出物は、ホモジナイザーを用いて、スフェロプラスト10をリセートしたり、乳鉢および害虫11,12を使用して調製することもできる。</li>
	<li>それを含むチューブを室温で水に入れ、時折揺れ動かして抽出物を溶かします。</li>
	<li>遠心分離機抽出物は4°Cで1時間45,000xgで抽出する。 </li>
	<li>BCA法13によりクリアされた上澄みのタンパク質含有量を定量化する。</li>
	<li>抽出物のアリコートを準備し、液体窒素で速く凍結し、-80°Cで保存します。 注: プロトコルはここで一時停止することができます。</li>
</ol>2. グリセロール勾配製剤
<ol><li>バッファーAに2つのグリセロール溶液を調製し、1つは10%v/vを含み、もう1つは30%v/vグリセロールを含みます。</li>
	<li>30%v/vグリセロール溶液中に0.1%v/vに架橋剤グルタルアルデヒドを加え、均質化するために混合する。 注意:適切な安全装置を使用して、ヒュームフード内のグルタルアルデヒドを取り扱います。</li>
	<li>12 mL 遠心管の底部に冷10%v/vグリセロール溶液(14 x 89 mm)を加えます。</li>
	<li>10%v/vグリセロール溶液のすぐ下に、チューブの底にグラジマスターデバイスを備えた長い針に付着した注射器を備えたグルタルアルデヒドを添加した冷たい30%v/vグリセロール溶液の6 mLを加えます。 注:または、10%v/vグリセロール溶液は、30%v/vグリセロール/グルタルアルデヒド溶液の上部に慎重にパイプすることができます。</li>
	<li>グラデーションマスターデバイスを使用して、連続した密度勾配を生成します。 注:勾配マスターデバイスでは、チューブは適切なラックに配置され、パラメータ(時間/角度/速度)を決定するためのメーカーの推奨事項に従って、短時間回転されます。この作業では、2:25分/81.5°/11rpmを使用しました。</li>
	<li>細胞抽出物を添加する直前に、7%v/vグリセロール/バッファーAクッションを200 μL慎重に加えます。 注:クッションのグリセロール濃度は、線形勾配を作成するために使用されるあまり濃縮されたグリセロール溶液よりも低くなければなりません。</li>
</ol>3. 遠心分離を抽出する
<ol><li>各12 mLリニアグリセロール勾配10%~30%のグルタルアルデヒドの上部に、合計タンパク質の約2mgをクッション付きでロードします。</li>
	<li>チューブをあらかじめ冷却されたスイングバケットローターに入れ。 メモ:超遠心分離の前に混合を避けるために、チューブを穏やかに処理します。</li>
	<li>遠心分離機 194,000 x g で 16 時間 4 °C.</li>
	<li>アリコート 24 500 μL フラクションで、適合した Econo システムを使用するか、慎重にピペット処理を行います。 注:適合Econoシステムは、蠕動ポンプ、UV検出器、チューブ穿光装置に接続された分数コレクターで構成されています。沈み込みプロファイルは、280 nmでの吸光度の測定によって監視することができます。</li>
	<li>ペリスタリポンプを通して40%v/vグリセロール溶液を使用して、グリセロールをチューブから分画コレクターに10%~30%の勾配で押し込みます。 注: プロトコルはここで一時停止することができます。分数は使用するまで-80°Cで保管してください。</li>
	<li>ドットブロットまたはスロットブロットデバイス上のニトロセルロース膜に直接各画分の50 μLをロードします。2次抗体としてCBPに対する抗体(1:6,000、TAPタグのCBP部分を検出する)と抗ウサギIgG(1:15,000)を併用してタンパク質を検出した。</li>
</ol>

<ol>
	<li>Kastner, B., Will, C. L., Stark, H., L&#252;hrmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+insights+into+nuclear+pre-mRNA+splicing+in+higher+eukaryotes.">Structural insights into nuclear pre-mRNA splicing in higher eukaryotes.</a> Cold Spring Harbor Perspectives in Biology. 11 (11), 032417 (2019).</li><li>Yan, C., Wan, R., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+pre-mRNA+splicing+through+structural+biology+of+the+spliceosome.">Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome.</a> Cold Spring Harbor Perspectives in Biology. 11 (1), 032409 (2019).</li><li>Cordin, O., Hahn, D., Beggs, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+regulation+of+spliceosomal+RNA+helicases.">Structure, function and regulation of spliceosomal RNA helicases.</a> Current Opinion in Cell Biology. 24, 431-438 (2012).</li><li>Kastner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GraFix:+sample+preparation+for+single-particle+electron+cryomicroscopy.">GraFix: sample preparation for single-particle electron cryomicroscopy.</a> Nature Methods. 5 (1), 53-55 (2008).</li><li>Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafix:+stabilization+of+fragile+macromolecular+complexes+for+single+particle+cryo-EM.">Grafix: stabilization of fragile macromolecular complexes for single particle cryo-EM.</a> Methods in Enzymology. 481, 109-126 (2010).</li><li>Yan, C., Wan, R., Bai, R., Huang, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+yeast+activated+spliceosome+at+3.5+&#197;+resolution.">Structure of a yeast activated spliceosome at 3.5 &#197; resolution.</a> Science. 353 (6302), 895-904 (2016).</li><li>Ohi, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomics+analysis+revelas+stable+multiprotein+complexes+in+both+fission+and+budding+yeasts+containing+Myb-related+Cdc5p/Cef1p,+novel+pre-mRNA+splicing+factors,+and+snRNAs.">Proteomics analysis revelas stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.</a> Molecular and Cellular Biology. 22 (7), 2011-2024 (2002).</li><li>Lourenco, R. F., Leme, A. F. P., Oliveira, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+yeast+mutant+RNA+exosome+complexes.">Proteomic analysis of yeast mutant RNA exosome complexes.</a> Journal of Proteome Research. 12 (12), 5912-5922 (2013).</li><li>Rigaut, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generic+protein+purification+method+for+protein+complex+characterizatioin+and+proteome+exploration.">A generic protein purification method for protein complex characterizatioin and proteome exploration.</a> Nature Biotechnology. 17, 1030-1032 (1999).</li><li>Cheng, S. C., Newman, A., Lin, R. J., McFarland, G. D., Abelson, 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=Preparation+and+fractionation+of+yeast+splicing+extract.">Preparation and fractionation of yeast splicing extract.</a> Methods in Enzymology. 181, 89-96 (1990).</li><li>Umen, J. G., Guthrie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+for+a+U5+snRNP+protein+in+3'+splice+site+selection.">A novel role for a U5 snRNP protein in 3' splice site selection.</a> Genes &amp; Development. 9, 855-868 (1995).</li><li>Dunn, E. A., Rader, S. D., Hertel, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+yeast+whole+cell+splicing+extract.">Preparation of yeast whole cell splicing extract.</a> Spliceosomal Pre-mRNA Splicing: Methods and Protocols. Methods in Molecular Biology. 1126, 123-135 (2014).</li><li>Hill, H. D., Straka, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+determination+using+bicinchoninic+acid+in+the+presence+of+sulfhydryl+reagents.">Protein determination using bicinchoninic acid in the presence of sulfhydryl reagents.</a> Analalytical Biochemistry. 170 (1), 203-208 (1988).</li><li>Cepeda, L. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+assembly+factor+Nop53+controls+association+of+the+RNA+exosome+with+pre-60S+particles+in+yeast.">The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.</a> The Journal of Biological Chemistry. 294 (50), 19365-19380 (2019).</li><li>Lin, R. J., Newman, A. J., Cheng, S. C., Abelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+mRNA+splicing+in+vitro.">Yeast mRNA splicing in vitro.</a> The Journal of Biological Chemistry. 260 (27), 14780-14792 (1985).</li></ol>

著者は利益相反を持っていません。

タンパク質とリボ核酸-タンパク質相互作用は、架橋剤を使用して安定化することができます。得られた複合体は、グリセロール勾配に対する超遠心化に耐える安定であることが重要である。さらに、バッファー条件では、相互作用を許可する必要がありますが、非特異的なバインドを回避するのに十分な厳格な必要があります。ここに示す実験では、インビトロスプライシング反応<sup class="...

スプライシングは、多数の因子の結合と解放を必要とする非常に動的なプロセスです。これらのスプライシング因子は、RNA結合タンパク質、ATPAses、ヘリカーゼ、タンパク質キナーゼおよびホスファターゼ、ユビキチンリガーゼ、とりわけ1、2、3を含む。また、分子の再調整を可能にするために、これらの要因のいくつかは、スプライセソームサブコンプレックスに非常に一時的に結合し、これらのRNP中間複合体の単離および同定を非常に困難にする。
ここでは、Grafix法4,5を使用して、酵母スプライシング因子Cwc24とBアクトコンプレックス6との相互作用を安定させ、そのサブ複合体に付随して結合する他の因子を同定し、ユビキチンリガーゼPrp19がCwc24の結合または放出に何らかの役割を果たしているかどうかを判断しました。場所。グリセロール勾配に沿って架橋剤の濃度を増加させる高分子を暴露する利点は、複合体間架橋4、5、したがって、凝集体の形成を回避することです。
この方法は、タンパク質共免疫沈降およびプルダウンアッセイの補完として使用されたが、これは、大きな複合体の単離を可能にするにもかかわらず、大きな動的複合体7,8内の一過性相互作用を維持するために信頼できない可能性がある。グリセロール勾配における固定化試薬の使用は、このような因子の結合を安定化させ、特定のタンパク質とスプライシングサブコンプレックスとの相互作用を確認することを可能にする。選択した架橋剤は化学的に不可逆的であったため、回収された画分に存在するタンパク質を、勾配遠心分離後のドットブロットで分析した。

<table>
	<tbody>
		<tr>
			<td>Anti-Calmodulin Binding Protein Epitope</td>
			<td>Millipore</td>
			<td>07-482</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL anti-Rabbit IgG</td>
			<td>GE Healthcare</td>
			<td>NA934</td>
			<td></td>
		</tr>
		<tr>
			<td>EconoSystem</td>
			<td>Bio-Rad</td>
			<td>1-800-424-6723</td>
			<td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td>
		</tr>
		<tr>
			<td>EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Recovery System</td>
			<td>Beckman Coulter</td>
			<td>270-331580</td>
			<td>Tube-perforating device that was connected to the parts of the EconoSystem</td>
		</tr>
		<tr>
			<td>Gradient Master Model 107ip</td>
			<td>Biocomp</td>
			<td>107-201M</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer Mill MM 200</td>
			<td>Retsch</td>
			<td>207460001</td>
			<td>Ball Mill device</td>
		</tr>
		<tr>
			<td>Rotor F12-6x500Lex</td>
			<td>Thermo Scientific</td>
			<td>096-062375</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall RC 6 Plus Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>36-101-0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Swinging Bucket Rotor P40ST</td>
			<td>Hitachi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge CP 80 NX</td>
			<td>Hitachi</td>
			<td>901069</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td>
			<td>Beckman Coulter</td>
			<td>344059</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

プレmRNAスプライシングは、組立、RNA処理、および複合成分の放出中に、多くの分子再構成を伴う非常に動的なプロセスです。グリセリン勾配遠心分離は、機能および構造研究のためのタンパク質またはRNP(RiboNucleoProtein)複合体の分離のために使用されています。ここでは、単一粒子の凍結電子顕微鏡用の高分子複合体を精製・安定化するために最初に開発されたGrafix(勾配固定)の利用について述べ、一過性のスプライセソー複合体と結合するスプライシング因子間の相互作用を同定する。この方法は、複合体を安定化させる固定化試薬の濃度の増加にサンプルの遠心分離に基づいています。グリセロール勾配にロードされた酵母総抽出物の遠心分離後、回収された分率は、個々のスプライシング因子の存在の決定およびスプライセソームサブ複合体の同定のためにドットブロットによって分析される。

Cwc24-TAPの沈殿プロファイルを解析し、グラフィックス法がスプライシングサブコンプレックスへの結合を安定化するのに有効であったかどうかを判断するために、Cwc24-TAPを発現する細胞の全酵母抽出物を、クロスリンク剤としてのグルタルアルデヒドの存在下または存在下にグリセロール勾配に遠心分離して分離した。24個の500 μL画分のサンプルを、次に、TAPタグのCBP部分に対する抗体を用?...

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

ここでは、架橋器の存在下でのグリセロール勾配遠心分離であるグラフィックス(勾配固定)の利用について、一過性にスプライセソーム複合体に結合するスプライシング因子間の相互作用を同定する。

サッカロミセス・セレビシエ・スプライセソソーム・サブコンプレックスの過渡性インターアクター検出のためのグラフィックスの利用

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

utilization-grafix-for-detection-transient-interactors-saccharomyces

Research

JoVE Journal

Biochemistry

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

3.5K ビュー.  University of Sao Paulo. ここでは、架橋器の存在下でのグリセロール勾配遠心分離であるグラフィックス(勾配固定)の利用について、一過性にスプライセソーム複合体に結合するスプライシング因子間の相互作用を同定する。

ビデオ: Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral RNAs. When bound to viral double-stranded RNAs (dsRNAs), PKR undergoes dimerization and subsequent autophosphorylation. Phosphorylated PKR (pPKR) becomes active and induces phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2&#945;) to suppress global translation. Increasing evidence suggests that PKR can be activated under physiological conditions such as during the cell cycle or under various stress conditions without infection. However, our understanding of the RNA activators of PKR is limited due to the lack of a standardized experimental method to capture and analyze PKR-interacting dsRNAs. Here, we present an experimental protocol to specifically enrich and analyze PKR bound RNAs during the cell cycle using HeLa cells. We utilize the efficient crosslinking activity of formaldehyde to fix PKR-RNA complexes and isolate them via immunoprecipitation. PKR co-immunoprecipitated RNAs can then be further processed to generate a high-throughput sequencing library. One major class of PKR-interacting cellular dsRNAs is mitochondrial RNAs (mtRNAs), which can exist as intermolecular dsRNAs through complementary interaction between the heavy-strand and the light-strand RNAs. To study the strandedness of these duplex mtRNAs, we also present a protocol for strand-specific qRT-PCR. Our protocol is optimized for the analysis of PKR-bound RNAs, but it can be easily modified to study cellular dsRNAs or RNA-interactors of other dsRNA binding proteins.

We present experimental approaches for studying RNA-interactors of double-stranded RNA binding protein kinase RNA-activated (PKR) during the mammalian cell cycle using HeLa cells. This method utilizes formaldehyde to crosslink RNA-PKR complexes and immunoprecipitation to enrich PKR-bound RNAs. These RNAs can be further analyzed through high-throughput sequencing or qRT-PCR.

哺乳類の細胞周期中に RNA 活性化蛋白質キナーゼの RNA インターアクターを勉強

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

生化学

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

茶由来の新たに確立された細胞懸濁液培養における6種の全身殺虫剤の代謝に関する研究(カメリアシネンシスL.葉

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

液体クロマトグラフィーとタンデム質量分析法を用いたサッカロミセス・セレビシエ細胞リピドームの定量的解析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A mitochondrial genome of baker&#8217;s yeast encodes eight proteins: three subunits of the cytochrome c oxidase (Cox1p, Cox2p, and Cox3p), three subunits of the ATP synthase (Atp6p, Atp8p, and Atp9p), a subunit of the ubiquinol-cytochrome c oxidoreductase enzyme, cytochrome b (Cytb), and mitochondrial ribosomal protein Var1p. The purpose of the method described here is to specifically label these proteins with 35S methionine, separate them by electrophoresis and visualize the signals as discrete bands on the screen. The procedure involves several steps. First, yeast cells are cultured in a galactose-containing medium until they reach the late logarithmic growth stage. Next, cycloheximide treatment blocks cytoplasmic translation and allows 35S methionine incorporation only in mitochondrial translation products. Then, all proteins are extracted from yeast cells and separated by polyacrylamide gel electrophoresis. Finally, the gel is dried and incubated with the storage phosphor screen. The screen is scanned on a phosphorimager revealing the bands. The method can be applied to compare the biosynthesis rate of a single polypeptide in the mitochondria of a mutant yeast strain versus the wild type, which is useful for studying mitochondrial gene expression defects. This protocol gives valuable information about the translation rate of all yeast mitochondrial mRNAs. However, it requires several controls and additional experiments to make proper conclusions.

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Baker&#8217;s yeast mitochondrial genome encodes eight polypeptides. The goal of the current protocol is to label all of them and subsequently visualize them as separate bands.

ベーカー酵母サッカロミセス・セレビシエにおけるミトコンドリアゲノム発現産物の表示と可視化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

液体クロマトグラフィーとタンデム質量分析法を用いたサッ カロミセス・セレビシエ の定量的メタボロミクス

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de novo&#160;from acetyl-CoA and are primarily present in eukaryotes in the form of triglycerides (TGs) and sterol-esters (SEs). The enzymes responsible for the synthesis of NLs are highly conserved from Saccharomyces cerevisiae (yeast) to humans, making yeast a useful model organism to dissect the function and regulation of NL metabolism enzymes. While much is known about how acetyl-CoA is converted into a diverse set of NL species, mechanisms for regulating NL metabolism enzymes, and how mis-regulation can contribute to cellular pathologies, are still being discovered. Numerous methods for the isolation and characterization of NL species have been developed and used over decades of research; however, a quantitative and simple protocol for the comprehensive characterization of major NL species has not been discussed. Here, a simple and adaptable method to quantify the de novo&#160;synthesis of major NL species in yeast is presented. We apply 14C-acetic acid metabolic labeling coupled with thin layer chromatography to separate and quantify a diverse range of physiologically important NLs. Additionally, this method can be easily applied to study in vivo reaction rates of NL enzymes or degradation of NL species over time.

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Here, a protocol is presented for the metabolic labeling of yeast with 14C-acetic acid, which is coupled with thin layer chromatography for the separation of neutral lipids.

代謝標識と薄層クロマトグラフィーによる サッカロミセス・セレビシエ における中性脂質合成の解析

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

We present a protocol for lamella preparation of plunge frozen biological specimens by cryo-focused ion beam micromachining for high-resolution structural studies of macromolecules in situ with cryo-electron tomography. The presented protocol provides guidelines for the preparation of high-quality lamellae with high reproducibility for structural characterization of macromolecules inside the&#160;Saccharomyces cerevisiae.

クライオ電子断層撮影のための サッカロミセス・セレビシエ の調製とクライオFIBマイクロマシニング

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

出芽酵母サッカロミセスセレビシエにおけるサブソキアドタンパク質の局在化の評価

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

内因性 ショウジョウバエ 一過性受容器電位チャネルの精製

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

S. cerevisiae細胞から精製された双方向キネシン-5 Cin8の一分子およびクラスターの運動性

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...