我们感谢所​​有成员的Kerscher实验室的鼎力支持。我们感谢:马克Hochstrasser酵母菌株MHY3765的。这项工作是由NSF资助1051970（OK）和霍华德·休斯医学研究所授予本科旅游和梦露学者项目资助（ES）的支持。

纯化6xHis-tagged蛋白表达在芽殖酵母细胞在天然条件下 1。酵母细胞的生长和诱导蛋白表达 （修改自2） 可选:使用对数生长的酵母培养物表达感兴趣的蛋白质，而不是下面描述的半乳糖诱导的培养。 <ol><li>转化细胞的半乳糖+酵母菌株的质粒编码半乳糖诱导的6xHis标签的蛋白给出。例如，试剂列表。 </li><li>合适的选择性培养基（ 如 SD-尿嘧啶）在5毫升含2％蔗糖，接种转化体。孵育在30°CO / N，旋转。 </li><li>使稀释的O / N培养物在600nm处的光密度测量0.3（OD 600 = 0.3），33毫升的选择性培养基中，用2％蔗糖。增长在30°C，晃动（Δ150转）。 </li><li>当培养已达到OD 600 = 0.8-1.5，通过加入17毫升的3倍的YEP 6％的半乳糖（配方在表1中），以使最终浓度为1x YEP用2％的半乳糖诱导表达所需蛋白。总培养体积是现在50毫升。孵育，振摇，在30℃下5-6小时。 注意:培养体积可以变化。在步骤1.3，稀释成所需的最终体积的三分之二的。在步骤1.4中，加入三分之一的最终体积的3倍YEP / 6％的半乳糖。 </li><li>测量OD 600诱导培养物的离心Δ150-200 OD 600单位的细胞在4℃下以5,000 rpm 5分钟</li><li>重悬细胞沉淀用1 ml冰冷的1X PBS 1X蛋白酶抑制剂和转移到2毫升的螺丝帽筒。 </li><li>离心细胞在4℃下以15,000 rpm的1分钟倒出上清。 </li><li>在液氮中速冻细胞沉淀，随后立即细胞裂解或储存于-80℃下，直到进一步使用。 </li></ol><ol><li>前一步骤中的冷冻细胞沉淀，加入200μl的酸洗过的玻璃珠，加入500μl冰冷的裂解缓冲液（配方在表1中，或使用细胞裂解缓冲液中给出）。 </li><li>简言之吸取向上和向下。它不要求完全重悬细胞沉淀。冰在任何时候都保持管。 注:枪头年底前可能会被切掉上下吹打。 </li><li>在4℃，地方管（S）与细胞成珠磨机，平衡，锁，并按照生产商的指示机器运行。 </li><li>运行含珠打浆机管（次），持续20秒，在5.5米/秒，然后放置在泥泞的冰，持续1分钟。总重复6X。 </li><li>清除所提取的蛋白质通过在15,000 rpm下离心15分钟，在4℃下可选:离心通过一个SpinX过滤去除小颗粒。 </li><li>准备样品的全细胞Extract（WCE）的感兴趣的蛋白质的Western印迹，以确认存在: <ol><li>添加WCE对应的2 OD 600单位的单元格（ 例如，5微升清零的提取物，如果200 OD 600单位的细胞收获），于800微升20％三氯乙酸（TCA）。旋涡混合。 </li><li>在4℃下以15,000 rpm离心2.5分钟倒出上清，但小心保留沉淀。 </li><li>添加800微升2％三氯乙酸颗粒与涡管，然后离心2.5分钟，以15,000 rpm于4℃。倒出上清，但小心保留沉淀。 </li><li>添加100微升TCA样品缓冲液（配方见表1），涡旋解散颗粒。注:如果样品缓冲液变成酸性（除了沉淀后变成黄色），加等分Δ10微升Tris碱[1M]直到样品再次是蓝色。 </li><li>孵育2-5分钟，在100℃加热块。 </li><li>涡AG泉充分溶解，如果仍然存在残余颗粒。用这种方法制备的颗粒饲料是出了名的难以充分溶解。这可能需要Δ10分钟涡旋颗粒完全溶解。 </li><li>商店样品在-80°C，直到进一步的使用。 </li></ol></li><li>急速冷冻，直到进一步利用剩余清除WCE在-80°C的液态氮和存储等分。 </li></ol> 3。批量纯化蛋白酵母细胞匀浆注:此纯化方法进行了优化，CO 2 +金属亲和树脂纯化6xHis-tagged蛋白。 <ol><li> 树脂平衡 <ol><li>清除WCEΔ20-40 OD 600个单位的细胞制备的样品，加50-100μl的亲和树脂离心管。提取从细胞中不表达所需蛋白或一种非特异性的树脂，如直链淀粉，蛋白质可被用作控制S非特异性结合。 </li><li>用1ml的洗涤缓冲液洗涤树脂5倍:翻转顶部比底部，直到树脂再悬浮，然后在每分钟5000转旋转1分钟，在4℃下吸出上清液。 注意:如果在同一天进行提取和纯化，树脂的平衡可以在提取前进行。 </li></ol></li><li> 亲和纯化的蛋白结合 <ol><li>加入100-200微升的澄清的裂解液（Δ20-40 OD 600单位），以50〜100微升的洗涤过 ​​的有孔玻璃珠，使总体积为1毫升用裂解缓冲液。 </li><li>章动或摆动，在4℃孵育2-5小时。 </li><li>自旋为1分钟，在5,000 rpm在4℃下</li><li>如果需要的话，除未结合的蛋白，用于随后的分析的样品的剩余的上清液。三氯乙酸沉淀详细在WCE（步骤2.6）以上。 </li><li>洗树脂与结合蛋白5倍用1ml的洗涤缓冲液在5,000 rpm，1分钟，然后通过旋4℃下在洗涤过程中保持样品冷。 </li></ol></li><li> 洗脱结合蛋白 <ol><li>树脂加入150μl的洗脱缓冲液，章动，在4℃下5分钟，自旋为1分钟，在5,000 rpm在4℃下保存一个新的管中的上层清液。可选:重复2倍和游泳池洗脱。 </li><li>准备洗脱样品的Western blot分析:将25μl洗脱的蛋白质，加入25μl的2倍的锂十二烷基硫酸钠（LDS）样品缓冲液中与2μl的β-巯基乙醇（BME），并在100℃的加热块中孵育2分钟。 注意:也可以使用标准的2X Laemmli样品缓冲液。 </li><li>速冻多余洗脱蛋白在液氮。 可选:带材的剩余树脂的蛋白质与等体积的2×LDS样品缓冲液中于65℃下进行5分钟，取出LDS洗脱的蛋白质，以一个新的管，然后加入2微升的BME的洗脱蛋白，而不是到树脂。此命令是为了防止拆除任何离子或抗体共轭到珠。 </li><li>样品储存在-80°C，直到进一步利用。 </li></ol></li><li> 可视化蛋白质印迹和探针与相应的抗体 。 <ol><li>装入10-20μl的各样品给出的SDS-PAGE凝胶中的蛋白质入水口和3-10微升（Δ1-3 OD 600单位）。常规用于此协议包括4-12％的Bis-Tris和8％Tris-甘氨酸凝胶。 </li><li>在200伏50分钟运行凝胶。 </li><li>半干转移蛋白质从凝胶转移到PVDF膜上，在19伏特，约20-30分钟（在表1中的配方）。 </li><li> 1小时RT或O / N挡膜在4％milk/1x的Tris缓冲生理盐水吐温（TBST）在4°C（配方见表1）。 </li><li>在4℃下孵育膜与初级抗体的表位标记的感兴趣的蛋白在4％milk/1x，TBST为1-3小时，在室温下或O / N </li><li>各5分钟，用1×TBST洗膜3倍。 </li><li>适当的孵育膜次级辣根过氧化物酶（HRP）标记的抗体为1-3小时，在RT。 </li><li>为各15分钟，在一个大的体积为1x TBST洗膜3倍。 </li><li> ，ECL底和包裹在保鲜膜覆盖膜。 </li><li>公开膜膜，并开发可视化蛋白质。 </li></ol></li></ol>

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该协议的重点编制完整的，原生的，和翻译后修饰的蛋白质从芽殖酵母细胞为下游应用。然后再尝试该协议，以确定是否可以很容易地在变性条件下12制备的蛋白提取物中检测到感兴趣的蛋白质，这是至关重要的。如果不可用多克隆抗体，它可能是有利的，使感兴趣的蛋白质的表位标记的融合蛋白的Western印迹上可检测到。在本协议中，我们标记SIZ1与医管局的6xHis-V5，或myc表位的标?...

酵母遗传学的令人敬畏的力量是有口皆碑的，但是从芽殖酵母， 酿酒酵母，天然蛋白的制备和分析往往是充满了问题。后者是由于相当大的机械强度和弹性的酵母细胞壁1。已经描述了不同的手段的酶 ​​，化学，机械，和基于压力的破坏的酵母细胞获得全细胞蛋白提取物2-6。这些技术有很大的不同，在它们的功效产生代表细胞，天然蛋白质提取物，可用于随后的分析或纯化步骤。例如，可以去除酵母细胞壁溶解酶（ 如酶解酶）和由此产生的spheroblasts会受到剪切，洗涤剂，或渗透裂解，释放蛋白。这种方法已经成功地采用了许多亚细胞分馏的起点，但它需要漫长的孵化与一些蛋白7的稳定性是不兼容的。 专有的化学提取的酵母细胞的蛋白质的的酵母裂解试剂（如清洁剂）是市售的，但在蛋白提取这些试剂的功效在随后的蛋白质的生化特性和其效果并不总是很清楚8。高压均质机，通常被称为法国印刷机，有效突破首先对他们进行高压，然后挤出来通过一个小口压力细胞的酵母细胞。该技术生产出高品质的提取物，但设备是非常昂贵的，可能不适合小批量的细胞或多个样品9。因此，机械破碎酵母细胞在珠磨机往往是原生酵母蛋白制剂10给出的方法。这项技术涉及的酵母细胞壁的机械破碎，用酸娃流下的玻璃珠，它可以与各种振动筛，旋涡仪或珠磨机进行。值得注意的是，此方法可用于同时处理多个更小的样品（1毫升或更小的细胞）。许多不同的有孔玻璃珠或珠磨机干扰矩阵现在市售扰乱几乎任何种类的细胞类型在2ml管。考虑到其他的技术和设备，珠磨机具有额外的好处，酵母细胞中的中断发生得非常快，从而有助于保持翻译后修饰，如SUMO化的，尤其是当使用蛋白酶和/或蛋白酶抑制剂与合适的缓冲液提取物的温度控制。 该协议的重点在温和的条件下，其最终目标保持蛋白质的功能，相互作用和翻译后修饰的快速，有效，可靠的内源性和过度表达的蛋白质提取。生长培养基，细胞裂解液组合物，珠磨机设置优化，保持蛋白质相互作用和SUMO化的和泛素化等翻译后修饰。

<table><tbody><tr><td>Omni Bead Ruptor 24</td><td>Omni International</td><td>19-010</td><td></td></tr><tr><td>Yeast ORF strain in BG1805</td><td>ThermoScientific</td><td>YSC3869</td><td>GAL+ yeast strain that can be used for induction and extraction</td></tr><tr><td>pYES2.1 TOPO vector</td><td>Life Technologies</td><td>K4150-01</td><td>GAL+ yeast strain that can be used for induction and extraction; contains a V5/6xHIS tag</td></tr><tr><td>Halt Protease Inhibitor Single-Use Cocktail (100x)</td><td>Thermo Scientific</td><td>1860932</td><td></td></tr><tr><td>2.0 ml Skirted Tube with Tethered Screw Cap</td><td>BioExpress</td><td>C-3369-3</td><td>Make sure that the tube properly fits in the bead ruptor</td></tr><tr><td>Glass beads, acid-washed, 425-600 &amp;micro;m (30-40 US sieve)</td><td>Sigma-Aldrich</td><td>G8772</td><td></td></tr><tr><td>Corning Costar Spin-X centrifuge tube filters</td><td>Sigma-Aldrich</td><td>CLS8161</td><td>Use for additional filtering of clarified lysate</td></tr><tr><td>TALON Metal Affinity Resin</td><td>Clontech</td><td>635502</td><td>For the purification of 6xHIS tagged proteins</td></tr><tr><td>NuPAGE LDS Sample Buffer (4x)</td><td>Life Technologies</td><td>NP0007</td><td>To prepare and/or elute samples prior to SDS-PAGE and Western Blotting</td></tr><tr><td>NuPAGE 4-12% Bis-Tris Gel</td><td>Life Technologies</td><td>NP0321BOX</td><td>Precast gels often used for SDS-PAGE prior to Western Blotting</td></tr><tr><td>Simply Blue</td><td>Life Technologies</td><td>#LC6060</td><td>Protein gel stain</td></tr><tr><td>Mammalian Lysis Buffer</td><td>Promega</td><td>G9381</td><td>Alternative commercial lysis buffer</td></tr><tr><td>Anti-V5 agarose</td><td>Sigma</td><td>A7345</td><td>Method of immunoprecipitation</td></tr><tr><td>ECL</td><td>Millipore</td><td>WBKL S0 050</td><td></td></tr><tr><td>PVDF membrane</td><td>Millipore</td><td>IPVH00010</td><td></td></tr><tr><td>BIS-TRIS gels</td><td>Life Technologies</td><td>NP0321BOX</td><td></td></tr><tr><td>anti-myc antibody</td><td>Covance</td><td>mms-150R</td><td></td></tr><tr><td>secondary antibody</td><td>Abcam</td><td>ab97040</td><td>Goat pAb to mouse IgG (HRP)</td></tr></tbody></table>

budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications

珠跳动的同质化是一个快速和有效的方式来释放DNA，RNA，蛋白质和代谢产物，从芽殖酵母细胞，这是出了名的难以破坏。在这里，我们描述了使用珠磨机，均化器优化，保持的功能，相互作用的蛋白质的翻译后修饰的蛋白质的提取到缓冲区。对数生长期的细胞表达的蛋白质还给出的液体生长介质中生长。生长培养基可以补充试剂诱导蛋白表达诱导型启动子（ 如半乳糖），细胞周期阶段的同步（ 例如，诺考达唑），或抑制蛋白酶体的功能（ 例如，蛋白酶体抑制剂MG132）。细胞再沉淀，再悬浮在适当的缓冲液含有蛋白酶和/或磷酸酶抑制剂，被立即处理，或在液氮中冷冻，以供稍后使用。同质化是通过6个周期的20秒BEA的d的跳动（5.5米/秒），然后在冰上保温一分钟。通过离心分离所得到的匀浆被清除，小颗粒可通过过滤除去。由此产生的清除的全细胞提取物（WCE）沉淀，用20％三氯乙酸的总蛋白，通过SDS-PAGE，然后通过Western印迹法直接分析。提取物也适合用于亲和纯化的特定蛋白质的翻译后修饰，或共同净化蛋白的分析，检测。大多数蛋白质的纯化协议的情况下，可能需要一些酶和蛋白质的独特条件或缓冲液的组合物的纯化和其他规定的条件下可能是不稳定的或不溶性的。在后者的情况下，提出的协议可提供一个有用的起点，凭经验确定最佳的蛋白提取和纯化珠跳动策略。我们显示了提取和纯化的表位标记的SUMO E3连接酶，SIZ1，一个细胞周期调节蛋白变得SUMO化和磷酸化，以及SUMO针对性的泛素连接酶亚基，Slx5。

我们的代表性的结果揭示了用于各种下游应用（总结在表2）的蛋白质的制备重现性，所描述的珠跳动和蛋白质提取协议是有用的。然后通过考马斯亮蓝染色的WCEs的SDS-PAGE表明，蛋白质（〜12&gt; 250 kDa）的广泛的可再现性地从酵母细胞中（ 图1A）萃取。分子量在一定范围内的离散频段的优质蛋白质提取物的指标。提取物的质量可以针对特定抗原标记的蛋白质免疫印迹证实。...

Watch this Scientific Journal Video about Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications at JoVE.com

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

高品质的酵母细胞提取物的制备是一个必要的分析单个蛋白质或整个蛋白质组的第一步。在这里，我们描述了一个快速，高效，可靠的同质化协议已被优化，以保持蛋白质的功能，相互作用和翻译后修饰的芽殖酵母细胞。

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

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

budding-yeast-protein-extraction-purification-for-study-function

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

24.8K 次观看.  The College of William & Mary. 高品质的酵母细胞提取物的制备是一个必要的分析单个蛋白质或整个蛋白质组的第一步。在这里，我们描述了一个快速，高效，可靠的同质化协议已被优化，以保持蛋白质的功能，相互作用和翻译后修饰的芽殖酵母细胞。 

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

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

科研

生物化学

Expression, Solubilization, and Purification of Eukaryotic Borate Transporters

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also known as Band 3), the most abundant membrane protein in the red blood cells. The SLC4 family is homologous with borate transporters, which have been characterized in plants and fungi. It remains a significant technical challenge to express and purify membrane transport proteins to homogeneity in quantities suitable for&#160;structural or functional studies. Here we describe detailed procedures for the overexpression of borate transporters in Saccharomyces cerevisiae, isolation of yeast membranes, solubilization of protein by detergent, and purification of borate transporter homologs from S. cerevisiae, Arabidopsis thaliana, and Oryza sativa. We also detail a glutaraldehyde cross-linking experiment to assay multimerization of homomeric transporters. Our generalized procedures can be applied to all three proteins and have been optimized for efficacy. Many of the strategies developed here can be utilized for the study of other challenging membrane proteins.

Here we present a protocol to express, solubilize, and purify several eukaryotic borate transporters with homology to the SLC4 transporter family using yeast. We also describe a chemical cross-linking assay to assess the purified homomeric proteins for multimeric assembly. These protocols can be adapted for other challenging membrane proteins.

真核硼酸背液转运体的表达、增溶及纯化

The Solute Carrier 4 (SLC4) family of proteins is called the bicarbonate transporters and includes the archetypal protein Anion Exchanger 1 (AE1, also ...

Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

萌芽酵母 SNX-BAR 异体体的表达、纯化和脂质结合

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and ...

遗传学

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

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

生物学

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.

萌芽酵母酿酒酵母中软骨蛋白定位的评估

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

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.

在芽殖酵母中的蛋白浓度的功能形态学表型的发展分析

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

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

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

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

蛋白质纯化技术，可以检测SUMO化修饰和芽殖酵母蛋白动粒和Ndc10泛素Ndc80的

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

An Introduction to Saccharomyces cerevisiae

Saccharomyces cerevisiae (commonly known as baker&rsquo;s yeast) is a single-celled eukaryote that is frequently used in scientific research. S. cerevisiae is an attractive model organism due to the fact that its genome has been sequenced, its genetics are easily manipulated, and it is very easy to maintain in the lab. Because many yeast proteins are similar in sequence and function to those found in other organisms, studies performed in yeast can help us to determine how a particular gene or protein functions in higher eukaryotes (including humans).
This video provides an introduction to the biology of this model organism, how it was discovered, and why labs all over the world have selected it as their model of choice. Previous studies performed in S. cerevisiae that have contributed to our understanding of important cellular processes such as the cell cycle, aging, and cell death are also discussed. Finally, the video describes some of the many ways in which yeast cells are put to work in modern scientific research, including protein purification and the study of DNA repair mechanisms and other cellular processes related to Alzheimer&rsquo;s and Parkinson&rsquo;s diseases.

芽殖酵母蛋白质的提取和纯化功能研究，交互和翻译后修饰

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