Name: growth-based determination and biochemical confirmation of genetic requirements for protein degradation in saccharomyces cerevisiae
Uploaded: 2015-02-16T15:00:00.000+00:00
Duration: 10 min 57 s
Description: Watch this Scientific Journal Video about Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae at JoVE.com

我们感谢鲁宾斯坦实验室的现任和前任成员提供支持和热情的科研环境。我们感谢瑞安T.吉布森在协议优化的援助。我们感谢马克Hochstrasser（耶鲁大学）和迪特·沃尔夫（斯图加特大学）的酵母菌株和质粒。我们感谢我们的匿名审稿人对他们在改善这个手稿的清晰度和实用的帮助。这项工作是由希格玛西的州立巴尔大学章SGW研究奖的支持，卫生授予国家研究院​​（R15 GM111713）为EMR，一个州立巴尔大学向往研究奖，以EMR，资金从波尔州立大学教务办公室和生物学系。

1.酵母生长试验，以确定候选人突变体有缺陷的蛋白质降解<ol><li>变换的野生型和突变体的酵母细胞用质粒编码融合不稳定蛋白质，在帧中，记者代谢酶。 </li><li>接种在5ml合成的定义（SD）的基本培养基中是选择性的细胞窝藏质粒分子的转化体。孵育过夜，在30℃，旋转。 </li><li>测量在每个过夜培养物在600nm（OD 600）的光密度。 注:按照过夜温育后，细胞在培养中可在任一对数或稳定生长期，但应已达到一个最小的OD 600为0.2。非常缓慢生长的酵母菌株，可能需要的孵育时间长于一晚，或接种的更大数目的细胞，如根据经验确定。 </li><li>制备转化的酵母细胞的6倍连续稀释在无菌96孔板，与细胞稀释至开始ñOD 600为0.2。将每个酵母转化体中，在96孔板不同行待测定。 <ol><li>对于每一个转化体，计算稀释细胞至0.2的OD 600在200微升最终体积所需过夜培养物的体积。过夜培养这个体积添加到相应井柱1.添加无菌水适量使体积为200微升。 </li><li>用于酵母中的每一行，在列2，3和4中添加125微升无菌水到孔中。 注:独立包装的无菌96孔板可与无菌盖包装。盖子可以用作藏为分布在这个步骤中的无菌水。这允许无菌水同时传输到所有孔中一个给定列用多通道移液器。 </li><li>通过上下吹打用多通道移液器混合的第一列（酵母稀释至OD 600为0.2）的内容。 </li><li> TRANSF呃25微升酵母从第1列至第2列，采用了多通道移液器。通过上下吹打混匀。转移25微升从第2列到第3列，以及25微升从第3列到第4列（混合以及在每一个步骤）。 </li></ol></li><li>混合用多通道移液器各样品。从最稀进行到至少稀酵母，吸管4微升各样品的柱到含有适当的选择培养基两个板。使用一个板中维持质粒的选择（这个板块作为酵母斑点和生长控制）。使用第二板介质，其选择用于融合到报道酶不稳定蛋白的质粒保持和表达。因为酵母沉降快速，通过上下抽吸混合细胞定期。 注:干燥机板会更容易吸收液体比新制备的板，因此推荐用于这些实验。湿板可以通过在室温下孵育在LO被干燥瓦特湿度下1 - 2天或更短的孵育在层流罩。板可干不均匀，如果空气层流平行于板凳。使用模板可以更容易地发现酵母细胞在固定距离。两个样品模板在图1中提供的，这些可以被印刷，切出，并固定在培养皿盖的内侧。 </li><li>允许板晾干在板凳上面。 </li><li>孵育板在30℃下进行2 - 6天。 </li><li>拍摄培养后各板。 </li></ol><img alt="图1" src="/files/ftp_upload/52428/52428fig1large.jpg" width="650" /> 图1.模板为察觉酵母细胞到100毫米的琼脂平板上。这些模板可以用来促进在与多通道移液器规则距离斑点酵母。模板可以被打印，切出，并固定在培养皿盖的内侧。将培养皿中生长中内盖与模板贴。模板都标有一个缺口，以追踪定位。因此建议在生长测定中使用板可以同样标有一个缺口以跟踪方向。模板察觉四（A）或五个（B）提供了酵母细胞的连续稀释， <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52428/150126 Fig 1.pdf" target="_blank">请点击这里查看这个数字与100毫米的模板的打印版本。</a> 酵母生长试验2.生化确认<ol><li>酵母细胞和后碱性蛋白抽提增长（从31修改） <ol><li>变换的野生型和突变体的酵母细胞用质粒编码不稳定的蛋白质。 </li><li>在5毫升的SD培养基是选择性的细胞窝藏质粒分子的接种转化体。孵育过夜，在30℃，旋转。 </li><li>测量每个隔夜立方米的OD 600lture。 注:培养过夜后，细胞可在任一对数或稳定生长期，但应已达到的OD 600，其将允许稀释至0.2在10毫升新鲜的选择性培养基（步骤2.1.4）的外径600。非常缓慢生长的酵母菌株，可能需要的孵育时间长于一晚，或接种的更大数目的细胞，如根据经验确定。 </li><li>稀释的酵母细胞，以0.2的10ml新鲜的选择性培养基的OD 600。 </li><li>继续孵育细胞在30℃，旋转或摇动，直到培养物达到OD 600 0.8和1.2之间（ 即在中期对数生长）。 注意:如果感兴趣的不稳定的蛋白质是一个可调节的启动子的控制下，诱导蛋白表达和细胞收获的最佳时机可以根据以往的研究或经验观察不一。 </li><li>收集2.5 OD 600单位文化在一个15毫升的锥形人管离心5000 xg离心5分钟，在室温下进行。吹打或抽吸去除上清液。 注:600单元被以1.0的OD 600定义为酵母的存在量在1ml培养的一个外径。 V = 2.5的OD 600单位/测量的OD 600:培养（以毫升）收获2.5的OD 600单位（Ⅴ）所需的体积可以使用以下等式来确定</li><li>重悬的细胞在1ml蒸馏水中。转移的悬浮细胞至微量离心管。 </li><li>通过离心沉淀细胞在6500×g离心30秒，在室温下进行。吹打或抽吸去除上清液。 </li><li>重悬的细胞在100μl蒸馏水通过上下抽吸或涡旋，并加入100微升的0.2M NaOH中。通过上下吹打混匀。孵育样品5分钟，在室温下进行。 </li><li>丸细胞（其中大部分尚未释放的蛋白质和仍然存活）离心18000×克5分钟。吹打或抽吸去除上清液。 </li><li>重悬沉淀在50 - 100微升1×Laemmli样品缓冲液，将细胞裂解，通过上下吹打或涡旋。 注意:使用Tris-甘氨酸电泳缓冲液系统和免疫印迹在pH用十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）兼容除去碱性上清液以下离心并将细胞在Laemmli样品缓冲液的后续再悬浮的提取物的蛋白质。 </li><li>要充分使蛋白质变性，孵育裂解物在95℃下5分钟。 注意:当在95℃下温育聚集倾向的蛋白（ 例如，蛋白的几个跨膜片段）可能成为不溶性的。因此，裂解物应在较低温度下孵育（ 例如 37℃ - 70℃）10 - 30分钟，因为经验确定，这样的蛋白质的分析。 </li><li>凉爽裂解物通过放置在冰上5分钟。 </li><li>离心裂解物在18000×g离心，在室温下1分钟以沉淀不溶物质。取上清液（溶解提取的蛋白质）通过SDS-PAGE之前，随后的免疫印迹分析（2.2节）分开。可替代地，存储裂解物在-20℃。 </li></ol></li><li>代表西方的印迹协议<ol><li>加载裂解物的经验确定的体积的SDS-PAGE凝胶。 </li><li>运行凝胶在200V直到染料前沿到达凝胶的底部。 </li><li>在4℃下90分钟 - 从凝胶到聚偏二氟乙烯（PVDF）膜通过湿转印转移蛋白在20 V 60。 </li><li>块膜孵育在5％脱脂乳的Tris缓冲盐水（TBS）中，摇摆，进行1小时，在室温下过夜或在4℃下。 </li><li>倒出封闭溶液。 </li><li>孵育膜用1％脱脂乳的TBS特异于感兴趣蛋白质一级抗体（或表位标签体）用0.1％吐温20（TBS / T）1小时，在室温德mperature，摇摆。 </li><li>滗抗体溶液，并用TBS / T洗膜3×5分钟，在室温下，摇摆。 </li><li>孵育膜用适当的荧光团共轭的二级抗体在1％脱脂乳的TBS / T为1小时，在室温下，摇摆。 注意:由于荧光团是光敏感，荧光团共轭抗体的稀释度应在黑暗中来制备。另外，在荧光团共轭抗体的存在膜的孵化应发生在遮光容器中。这可以通过包装孵育塔板在铝箔来完成。 </li><li>滗抗体溶液，并用TBS / T洗膜3×5分钟，在室温下，摇摆。 </li><li>收购膜采用LI-COR奥德赛CLX和图像Studio软件（或类似的影像设备和软件）的图像，根据制造商的建议。 </li><li>成像膜后，孵育该膜与特异于负载的第一抗体在室温下1小时，以1％脱脂牛奶的TBS / T控制时蛋白质，摇摆。 </li><li>滗抗体溶液，并用TBS / T洗膜3×5分钟，在室温下，摇摆。 </li><li>孵育膜用适当的荧光团共轭的二级抗体在1％脱脂乳的TBS / T为1小时，在室温下，摇摆。 </li><li>滗抗体溶液，并用TBS / T洗膜3×5分钟，在室温下，摇摆。 </li><li>收购膜采用LI-COR奥德赛CLX和图像Studio软件（或类似的影像设备和软件）的图像，根据制造商的建议。 </li></ol></li></ol>

作者什么都没有透露。

这里介绍的方法允许快速测定和蛋白质降解酵母细胞中的遗传要求生物化学确认。这些实验中突出酵母的效用和权力作为一种模式真核生物（协议的处理，存储和操纵酵母细胞（ 如酵母41-44生物学的几个优秀的审查和汇编），可用于调查新的生物体）。的技术可以很容易地被应用到调查的各种类的蛋白质的降解和丰富。例如，其他人已采用这种策略来表征不稳定胞质，细胞核和ER腔...

选择性蛋白质降解是对真核生命至关重要，并且改变的蛋白质降解有助于若干医疗条件，其中包括几种类型的癌症，神经变性疾病，心血管疾病，和囊性纤维化1-5。泛素-蛋白酶体系统（UPS），其催化选择性的蛋白质降解，是在这些条件6-10一个新兴的治疗靶点。泛素连接酶共价连接的76个氨基酸的泛素蛋白质11的聚合物。已标有泛素链蛋白识别并〜2.5 megadalton 26S蛋白酶体蛋白水解12。已经在模型真核生物酿酒酵母 （芽殖酵母）发起的研究已经基本在真核细胞中的蛋白质降解机理的阐明。 UPS的首次展示的生理基板是酵母转录抑制MATα213，UPS的14，和许多高度保守的成分被首次发现或特征酵母（ 如 15-26）。在这种多功能和基因听话的模式生物中发现的有可能继续提供重要见解泛素介导的降解机制保守。 识别大多数UPS基板和降解需要多种蛋白质的一致行动。因此，在表征一个给定的不稳定蛋白的调节的降解的一个重要目标是确定蛋白水解基因的需求。经典方法（ 例如脉冲追踪和放线菌酮追踪实验27），用于监测蛋白降解在哺乳动物或酵母细胞是费力和耗时的。虽然这些类型的方法中，用于检测蛋白质的降解提供高度敏感的装置，它们不适合用于蛋白降解或大规模screeni快速分析纳克的突变，防止蛋白质的降解。这里，酵母生长为基础的检测的快速鉴定为不稳定的蛋白质的降解的遗传要求提出。 在酵母生长系的分析方法，蛋白降解，感兴趣（或降解信号）不稳定的蛋白质融合，在帧中，对所需要的特定环境下酵母生长的蛋白质。其结果是一种人工基质，可作为一种强大的工具，以确定所关注的不稳定蛋白的蛋白质降解的遗传要求。便利地，最常用的实验室酵母菌株怀有突变的基因编码涉及的特定氨基酸或含氮碱（ 例如 20,28-30）的生物合成的代谢酶的小组。这些酶可用于在不存在在其合成中的酶参与外源提供的代谢物的细胞增殖至关重要。这样因而代谢酶可充当生长基记者不稳定蛋白质的降解与它们稠合。对蛋白降解的基因的需求可以容易地阐明，因为突变阻止蛋白水解将允许细胞窝藏降解记者选择条件下生长。 生长优势是一个间接指示特定的突变增加目的蛋白质的丰度。然而，直接生化分析，以确认突变允许的增长，通过增加蛋白质的水平，而不是通过间接的或人为的原因。在蛋白丰度突变的作用可能是通过稳态蛋白水平的细胞和不怀有特定突变蛋白质印迹分析来确定。一种酵母蛋白的快速高效提取（酵母细胞用氢氧化钠和样品缓冲的顺序孵化）在适当的形式方法通过免疫印迹分析也提出了31。总之，这些实验有助于快速识别蛋白质降解的候选人监管机构。

<table><tbody><tr><td>Desired yeast strains, plasmids, standard medium and buffer components</td><td></td><td></td><td>Yeast strains with desired mutations may be generated in the investigator&#039;s laboratory. Wild-type yeast and a variety of mutants are also commercially available (&lt;em&gt;e.g.&lt;/em&gt; from GE Healthcare). Plasmids encoding fusion proteins may be generated in the investigator&#039;s laboratory.</td></tr><tr><td>3-amino-1&lt;em&gt;H&lt;/em&gt;-1,2,4-triazole</td><td>Fisher Scientific</td><td>AC264571000</td><td>Competitive inhibitor of His3 enzyme. May be included in medium to increase stringency of growth assay using His3 reporter constructs.</td></tr><tr><td>Endoglycosidase H (recombinant form from &lt;em&gt;Streptomyces plicatus&lt;/em&gt;)</td><td>Roche</td><td>11088726001</td><td>May be used to assess N-glycosylation of proteins; compatible with SDS and beta-mercaptoethanol concentrations found in 1x Laemmli sample buffer.</td></tr><tr><td>Disposable borosilicate glass tubes</td><td>Fisher Scientific</td><td>14-961-32</td><td>Available from a variety of manufacturers</td></tr><tr><td>Temperature-regulated incubator (&lt;em&gt;e.g.&lt;/em&gt; Heratherm Incubator Model IMH180)</td><td>Dot Scientific</td><td>51028068</td><td>Available from a variety of manufacturers</td></tr><tr><td>New Brunswick Interchangeable Drum for 18 mm tubes (tube roller)</td><td>New Brunswick</td><td>M1053-0450</td><td>Tube roller is recommended to maintain overnight yeast starter cultures of yeast cells in suspension. A platform shaker or tube roller may be used to maintain larger cultures in suspension.</td></tr><tr><td>New Brunswick TC-7 Roller Drum 120V 50/60 H</td><td>New Brunswick</td><td>M1053-4004</td><td>For use with tube roller</td></tr><tr><td>SmartSpec Plus Spectrophotometer</td><td>Bio-Rad</td><td>170-2525</td><td>Available from a variety of manufacturers</td></tr><tr><td>Sterile 96-well flat bottom microtest plates with lid individually wrapped</td><td>Sarstedt</td><td>82.1581.001</td><td>Available from a variety of manufacturers</td></tr><tr><td>Pipetman Neo P8x20N, 2-20 &amp;mu;l</td><td>Gilson</td><td>F14401</td><td>Available from a variety of manufacturers&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&lt;br /&gt;&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Pipetman Neo P8x200N, 20-200 &amp;mu;l</td><td>Gilson</td><td>F14403</td><td>Single-channel and multichannel pipettors are used at various stages of the protocol. While multichannel pipettors reduce the pipetting burden at several steps, single-channel pipettors may be used throughout the entire protocol. Available from a variety of manufacturers.</td></tr><tr><td>Centrifuge 5430</td><td>Eppendorf</td><td>5427 000.216</td><td>Rotor that is sold with unit holds 1.5 and 2.0 ml microcentrifuge tubes. Rotor may be swapped for one that holds 15 ml and 50 ml conical tubes.</td></tr><tr><td>Plate imaging system (&lt;em&gt;e.g.&lt;/em&gt; Gel Doc XR+ System)</td><td>Bio-Rad</td><td>170-8195</td><td>A variety of systems may be used to image plates, including sophisticated imaging systems, computer scanners, and camera phones.</td></tr><tr><td>Fixed-Angle Rotor F-35-6-30 with Lid and Adapters for Centrifuge Model 5430/R, 15/50 ml Conical Tubes, 6-Place</td><td>Eppendorf</td><td>F-35-6-30</td><td></td></tr><tr><td>15 ml screen printed screw cap tube 17 x 20 mm conical, polypropylene</td><td>Sarstedt</td><td>62.554.205</td><td>Available from a variety of manufacturers</td></tr><tr><td>1.5 ml flex-tube, PCR clean, Natural microcentrifuge tubes</td><td>Eppendorf</td><td>22364120</td><td>Available from a variety of manufacturers</td></tr><tr><td>Analog Dri-Bath Heater</td><td>Fisher Scientific</td><td>1172011AQ</td><td>Boiling water bath with hot plate may also be used to denature proteins</td></tr><tr><td>SDS-PAGE running and transfer apparatuses, power supplies, and imaging equipment or darkrooms for SDS-PAGE and transfer to membrane</td><td></td><td></td><td>Will vary by lab and application</td></tr><tr><td>Western blot imaging system (&lt;em&gt;e.g.&lt;/em&gt; Li-Cor Odyssey CLx scanner and Image Studio Software)</td><td>Li-Cor</td><td>9140-01</td><td>Will vary by lab and application</td></tr><tr><td>EMD Millipore Immobilon PVDF Transfer Membranes</td><td>Fisher Scientific</td><td>IPFL00010</td><td>Will vary by lab and application</td></tr><tr><td>Primary antibodies (&lt;em&gt;e.g.&lt;/em&gt; Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8))</td><td>Life Technologies</td><td>459250</td><td>Will vary by lab and application</td></tr><tr><td>Secondary antibodies (&lt;em&gt;e.g.&lt;/em&gt; Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H+L))</td><td>Life Technologies</td><td>A-21065</td><td>Will vary by lab and application</td></tr></tbody></table>

growth-based determination and biochemical confirmation of genetic requirements for protein degradation in saccharomyces cerevisiae

调节蛋白的降解是几乎每一个细胞功能的关键。大部分所了解的分子机制和真核细胞的蛋白质降解基因的要求，最初成立于酿酒酵母 。蛋白质降解的经典分析都依赖于生化脉冲追踪和放线菌酮追方法。尽管这些技术提供了敏感装置，用于观察蛋白降解，它们是费力，费时，并且低通量。这些方法不适合于快速或大规模筛选突变防止蛋白质降解。这里，酵母生长为基础的检测为浅显鉴定为蛋白降解基因的要求进行说明。在该测定中，需要对特定的选择条件下生长记者酶融合到一种不稳定的蛋白。细胞缺乏内源性报告酶，但表达该融合蛋白可以在塞莱生长只有当融合蛋白被稳定莫如条件下（ 即 ，当蛋白质的降解受到损害）。在这里所描述的生长测定，野生型和突变体的酵母细胞携带的质粒编码的融合蛋白的系列稀释液点样到选择性和非选择性培养基。选择条件下的生长是由给定的突变降解减值一致。增加蛋白质丰度应生化证实。一种酵母蛋白在适合电泳和免疫印迹形式的快速提取方法也证实。甲生长基于读出的蛋白质稳定性，并以简单的协议，用于蛋白提取用于生化分析，有利于快速鉴定对蛋白降解的基因的需求。这些技术可以适用于监视各种短命的蛋白质的降解。在呈现的例子中，HIS3酶，这是需要组氨酸的生物合成，融合到Deg1 -Sec62。Deg1 -Sec62是针对退化后异常从事内质网位子。细胞窝藏Deg1 -Sec62-HIS3能够选择性条件下生长时，蛋白质被稳定化。

为了说明这种方法，在HIS3酶已被稠合到模型内质网（ER）相关退化的羧基末端（ERAD）基板，Deg1 -Sec62（ 图2A）来创建Deg1 -Sec62-HIS3（ 图3） 。Deg1 -Sec62代表在与位子持续，异常协会有针对性一类新的ERAD基板的创始成员之一，该通道主要负责跨ER膜32-34动蛋白。此类不稳定蛋白已经暂时被称为ERAD-T（对于易位子相关）衬底。以往的研究表明，当异?...

Watch this Scientific Journal Video about Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae at JoVE.com

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 ...

growth-based-determination-biochemical-confirmation-genetic

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

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

9.7K 次观看.  Ball State University. 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.

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

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 ...

科研

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 ...

生物化学

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

调整降解以实现特定和高效的蛋白质消耗

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

免疫与感染

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

记者的增长分析的蛋白质降解的系统分析

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

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 ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;:突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

蛋白质降解的放线菌酮大通分析<em&gt;酿酒酵母</em

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

成长型测定和遗传需求生化确认的蛋白质降解

摘要

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此视频中的章节

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

萌芽酵母酿酒酵母中软骨蛋白定位的评估

调整降解以实现特定和高效的蛋白质消耗

耦合检测监控蛋白复性

记者的增长分析的蛋白质降解的系统分析

化学遗传相互作用的快速鉴定

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

定向进化法

蛋白质降解的放线菌酮大通分析

基于蛋白质的继承在酿酒酵母中的高通量筛选