Name: cycloheximide chase analysis of protein degradation in saccharomyces cerevisiae
Uploaded: 2016-04-18T15:00:00.000+00:00
Duration: 9 min 5 s
Description: Watch this Scientific Journal Video about Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae at JoVE.com

The authors thank current and former members of the Rubenstein lab for providing a supportive and enthusiastic research environment. The authors thank Mark Hochstrasser (Yale University) for sharing reagents and expertise. E.M.R. thanks Stefan Kreft (University of Konstanz) and Jennifer Bruns (University of Pittsburgh) for sharing invaluable expertise in kinetic analysis of proteins. This work was supported by a National Institutes of Health grant (R15 GM111713) to E.M.R., a Ball State University ASPiRE research award to E.M.R, a research award from the Ball State University chapter of Sigma Xi to S.M.E., and funds from the Ball State University Provost's Office and Department of Biology.

酵母细胞的生长1和收获<ol><li>如果不分析内源酵母蛋白的降解动力学，变换所需的酵母菌株（多个）与编码感兴趣的蛋白质的质粒。用于酵母转化的可靠方法先前已被描述37。 </li><li>在5ml适当的培养基中接种酵母（ 例如，选择性的合成定义（SD），用于质粒维持转化的细胞或对未转化的细胞的非选择性酵母提取物-蛋白胨-葡萄糖（YPD）培养基的培养基）。在30℃孵育过夜，旋转。 注:30℃为典型的野生型实验室酵母菌株38的最佳生长温度。但是，由于一些突变酵母菌株不于30℃最 ​​佳生长和某些蛋白质经受依赖于温度的降解，用于酵母细胞的生长和放线菌酮追逐的温度应根据经验确定39。 </li><li>测量日在每个过夜培养物在600nm（OD 600）在线光密度。 注:细胞可以是在对数或稳定生长期但应最低限度地达到的密度，这将允许稀释到OD 600 =新鲜培养基0.2在15毫升。 </li><li>稀释过夜培养至0.2在15毫升新鲜培养基的OD 600值。 </li><li>在30℃下孵育酵母，振摇直到细胞达到中期对数生长期（ 即 ，的OD在0.8和1.2之间600）。 </li><li>在酵母细胞的生长，执行了放线菌酮追逐程序准备以下几点: <ol><li>设置一个热块，可容纳的15毫升锥形管，以30℃在放线菌酮的存在下温育细胞。加水至热块的孔中，以允许有效的热分配到培养物。加水到各孔中，使得一个15毫升的锥形管将导致水位上升到，但不会溢出，井的唇。 </li><li>设置的第二加热块，可容纳1.5毫升以下细胞裂解离心管至95℃使蛋白质变性。 </li><li>预暖新鲜生长培养基（每人培养时间1.1点ml至待测定）至30℃。 </li><li>加入50μl的20倍停止混合到预标记的微量离心管（每培养每个时间管指向待测定）。试管置于冰上。 注意:叠氮化钠，在20倍的停止混合的成分，是通过口服或皮肤接触有毒。准备时，存储，处理和叠氮化钠按照制造商的建议。意外暴露的情况下，请咨询厂家提供材料安全数据表。 </li></ol></li><li>当细胞达到中期对数生长，收集2.5的OD 600单位每个时间点每种培养物待测定（ 例如 ，7.5的OD 600单位来分析蛋白质丰度在三个时间点）。离心收集的细胞在15ml锥形管中在3,000 XG在室温下进行2分钟。 注意:一个外径600单位是在一个外径为1.0 600等于酵母本在1ml培养的量。 V =点¯xOD 600单位/测量OD 600:培养（以毫升）收获点¯xOD 600单位（Ⅴ）所需的体积可以通过使用以下等式来确定。例如，收获7.5 OD 600单位的酵母细胞培养在一个外径为1.0 600，收集7.5 OD 600单位/ 1.0 = 7.5 ml酵母文化。 </li><li>重悬每个细胞沉淀在1ml 30℃每2.5的OD 600单位的细胞（预热）的新鲜生长培养基（ 如 3毫升7.5的OD 600单位）。 </li></ol> 2.放线菌酮大通<ol><li>孵育在30℃加热块5分钟平衡酵母细胞悬浮液。 </li><li>准备一个计时器，从0:00计数。 </li><li>要开始追酮，按"开始"计时器。很快，不过仔细，请执行下列步骤: <ol><li>添加放线菌酮，以250微克/毫升到第一酵母细胞悬浮液的最终浓度（ 例如，添加37.5微升20毫克/毫升放线菌酮的库存至3ml细胞悬浮液），并涡旋简要地混合。 注意:放线菌酮是一种皮肤的刺激性，可经口摄入中毒。当准备，储存和处理酮按照制造商的建议。意外暴露的情况下，请咨询厂家提供材料安全数据表。 </li><li>添加放线菌酮和涡旋后，立即与加入酮转移950微升（〜2.4的OD 600单位）的酵母细胞悬浮于含有50微升冰冷的20倍停止混合标记的预离心管中。直到所有样品都被收集涡离心管中，并置于冰上。 </li><li>返回酵母细胞悬浮液至30℃。 </li></ol></li><li>重复步骤2.3.1至2.3.3每在一定的时间间隔（剩余的酵母细胞悬浮液例如，每隔30秒，使得放线菌酮加入到样品＃1 0:00，样品＃2 0点30分，试样＃3在1:00 等 ）。 </li><li>在每个随后的时间点，涡酵母细胞悬液和转让950微升含50微升预冷20X停止混合标记的离心管。涡流并在冰上的地方收集到的细胞。返回的15毫升锥形管中至30℃加热块。 <ol><li>例如，对于环己酰亚胺加入后30分钟收集细胞，涡流（在时间过程的开始假设酮加成之间30秒的间隔，以酵母细胞悬浮液）中并在30:00从样品＃1中取出950微升细胞悬浮液。在30:30重复试样＃2，依此类推。 注:为了防止整个追逐的过程中在15ml锥形管大约每5分钟酵母，涡细胞悬液沉降。此外，酵母细胞悬液S可以在一个连续搅拌的水浴中的放线菌酮追实验的持续时间被保持。 </li></ol></li><li>当所有的样品已经被收集起来，沉淀收集的细胞通过离心在6500 xg离心在室温下持续30秒。吹打或抽吸去除上清液。细胞现在已经准备好碱裂解。可替换地，管理单元冷冻在液氮中并储存沉淀的细胞在-80℃下。 </li></ol> 3.后碱性蛋白提取（从16,40修改） <ol><li>加入100微升的蒸馏水，以每个细胞沉淀。吹打上下或涡旋重悬。 </li><li>加入100微升的0.2M NaOH中向每个样品。吹打向上和向下或震荡混合。 </li><li>在室温下孵育悬浮的细胞5分钟。在此阶段，酵母细胞没有被裂解，蛋白质没有被释放40。 </li><li>离心沉淀细胞18,000 XG在室温脾气ATURE 30秒。吹打或吸入去除上清液。 </li><li>裂解细胞，添加100μl的Laemmli样品缓冲液中向每个细胞沉淀。吹打上下或涡旋重悬。 注意:使用一个Tris-甘氨酸电泳缓冲液系统中与十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS-PAGE）兼容的形式NaOH和Laemmli样品缓冲液释放蛋白的细胞的连续培养。 </li><li>孵育在95℃下5分钟以充分使蛋白质变性。 注意:孵育在95℃下未必适用于容易发生聚合的蛋白质的分析（ 例如 ，与几个跨膜片段的蛋白）。当在升高的温度41温育这些蛋白质可以成为不溶的。因此，对于这样的蛋白质的分析，裂解物应在较低的温度下孵育（ 例如 ，37℃ - 70℃）10 - 30分钟，因为经验确定。 </li><li>在RO在XG 18000裂解液离心嗡温度1分钟以沉淀不溶物质。上清液（溶解提取的蛋白质）准备好通过SDS-PAGE及随后的免疫印迹分析的分离。可替代地，存储裂解物在-20℃。 </li></ol>代表性的SDS-PAGE和Western印迹程序（从16改编） <ol><li>负载蛋白质分子量标准和裂解物的经验确定的体积上的SDS-PAGE凝胶。 注:选择基于所关注的蛋白质的分子量丙烯酰胺和双丙烯酰胺的在SDS-PAGE凝胶的百分比。在一般情况下，较低百分比凝胶更适合解决较高分子量蛋白质。 </li><li>在200V运行凝胶直到染料前沿到达凝胶的底部边缘。 </li><li>在4℃下90分钟 - 从凝胶到聚偏二氟乙烯（PVDF）膜通过湿转印在20 V转移蛋白60。 </li><li>通过在Tris缓冲盐水孵育（TB方框膜S） - 含5％脱脂乳，摇摆，在室温下1小时或在4℃过夜。 注:为了防止在培养过夜的膜的存在的微生物生长，建议包括在封闭溶液叠氮化钠以0.02％的终浓度。 </li><li> 1小时孵育与特异于的在TBS含0.1％Tween-20（TBS / T）和1％脱脂乳，摇摆兴趣蛋白质的第一抗体的膜，在室温下。 </li><li>在室温下用TBS / T洗膜3×5分钟，摇摆。 </li><li>在室温下孵育，用1％脱脂牛奶的TBS / T适当荧光标记的二抗膜1小时，摇动。 注:荧光团对光敏感。因此，缀合至荧光基团的抗体的稀释度应在黑暗中进行制备，并且在偶联于荧光团和随后的洗涤步骤的抗体的存在应该发生在避光（ 例如，铝箔包裹）共膜孵化ntainers。 </li><li>在室温下用TBS / T洗膜3×5分钟，摇摆。 </li><li>使用LI-COR的奥德赛CLX和影像工作室软件（或类似的成像设备和软件），按照制造商的建议形象膜。 </li><li>获取膜图像后，孵育在室温下特异于在TBS / T上样对照蛋白质的初级抗体用1％脱脂乳的膜1小时，摇动。 </li><li>在室温下用TBS / T洗膜3×5分钟，摇摆。 </li><li>在室温下孵育，用1％脱脂牛奶的TBS / T适当荧光标记的二抗膜1小时，摇动。 </li><li>在室温下用TBS / T洗膜3×5分钟，摇摆。 </li><li>图像膜如在步骤4.9。 </li></ol>

The authors have nothing to disclose.

在本文中，提出了用于分析蛋白质的降解动力学的方法。这种技术可以容易地应用于一系列由各种机制降解的蛋白质。要注意的是放线菌酮追踪实验给​​定的蛋白质的稳态池的降解动力学报告是很重要的。其它技术可以用于分析蛋白质的特定人群的降解动力学。例如，新生多肽的降解的命运可以通过脉冲追踪分析55进行跟踪。在这样的实验中，新生的蛋白质是短暂脉冲标记（ 例如 ，用放射性氨基酸）。在丰标记新生蛋白质（其可以或可以不反映在稳态蛋白质丰度的变化）的变化可以随时间被跟踪。 酮追是适于在相对 ​​较短的时间过程分析蛋白稳定性（ 即，最多两个小时）。在较长时间Courses（ 即两个数小时至数天），放线菌酮，翻译的一个全球性的抑制剂，它是细胞毒性，可能是由于遍在蛋白56的枯竭。此外，在较长的时间进程的蛋白质稳定性的分析更容易由感兴趣的蛋白质（的降解全局受损的蛋白质合成的间接影响受到损害例如，涉及的蛋白质的降解一个短暂的蛋白质的降解利益）。因此更适合于学习的长寿命蛋白的降解的其它技术，例如脉冲追踪代谢标记实验，是和可被执行以证实在放线菌酮追踪实验获得的结果。 加入细胞的放线菌酮和收集的定时是在此过程中的一个重要的考虑因素。精度是非常短暂的蛋白质的分析尤为重要，因为在时间的小偏差酮阿迪经过重刑细胞采集可以有明显的降解动力学产生实质性的影响。另外，由于不执行这些时间敏感的步骤的明确计划，实验可能很快退化陷入混乱和挫折。出于这个原因，建议酮在计划，定期添加到培养物。 30秒的间隔是建议在这个协议中，但时间可以调节，以匹配研究者的舒适程度和灵巧。新手调查人员可能会发现它有用启动实验前的样品采集时间调度写，因为它发生划掉每个计划集合。 在步骤1.7和这里所描述的协议的1.8，酵母细胞培养物随后酮追逐过程期间集中在减少为了易于操纵的生长培养基的体积。然而，酵母细胞的离心迅速诱导某些细胞应激反应（ 例如 ，信号通路即葡萄糖剥夺）57,58被激活。因此，调查人员告诫不要离心长时间。当分析蛋白质的稳定性，可能是​​离心敏感，调查不妨直接添加到酮对数中期培养，恕不另行离心。在这样的情况下，放线菌酮应该被添加到相同的最终浓度（250微克/毫升），和酵母细胞样品可以通过不要求初始离心步骤（ 例如 ，快速过滤）57的方法进行收获。另外，在步骤2.3 - 2.5，在每个时间点收集的样品转移至含有叠氮化钠的溶液中，并置于冰上。这些条件继续抑制蛋白质降解为追逐的持续时间。应当指出的是叠氮降低的三磷酸腺苷59细胞浓度，从而特异性抑制ATP依赖的蛋白酶的活性。虽然降低温度应该在LSO减少在该非ATP依赖的蛋白水解过程功能，调查研究通过这种机制降解蛋白质可替换地选择每个样品收获到管理单元冷冻细胞沉淀在液氮中的速率。 对细胞裂解及包含在这个手稿印迹具有代表性的协议样本协议已经被改编自以前的报告16,40。在这里所描述的碱性后裂解法，氢氧化钠预处理提高在随后的加成Laemmli样品缓冲液的酵母蛋白质提取。由这种增强的发生机制的特点是很差40。然而，多糖如在酵母细胞壁中发现它们溶于碱的条件60。因此它是诱人的推测，孵育在NaOH的存在下部分地或完全球状体的酵母细胞（ 即，除去细胞壁组分），使它们更sensitivE要SDS目前在Laemmli样品缓冲液洗涤剂。因为蛋白质是高度变性条件下释放，蛋白酶抑制剂不必包括在Laemmli样品缓冲液。细胞裂解的具体方法可以被修改以适合一个给定的实验。例如，该碱性后裂解方法可能不适合用作要受到不良通过Western印迹检测到的低丰度蛋白质或蛋白质。在这种情况下，包括一个三氯乙酸沉淀步骤以浓缩的蛋白质样品可以是最合适的61的方法。此外，关于抗体选择几个重要的考虑因素，装载控制选择，检测的替代手段（ 例如，使用胶片或数字成像系统，化学发光免疫印迹法）已经讨论了最近16。可以进行蛋白质丰度以下酮治疗的定量。许多成像系统与软件包，促进销售的这种分析。 酮追可经实施以分析各种酵母蛋白质的降解，并且可适于研究在其它真核细胞中的蛋白质稳定性。如在该协议描述的，放线菌酮处理之后，细胞裂解和蛋白丰度通过蛋白质印迹分析检测。根据不同的应用，但是，蛋白质丰度以下菌酮处理可以通过一系列的技术评估，以适合研究目标。例如，如果蛋白质大小不是用于分析的相关因子，细胞裂解物可以经受斑点印迹或酶联免疫吸附测定（ELISA）分析，这对蛋白质丰度报告，但不是表观分子量62。对在细胞表面上的蛋白质（或内部荧光标记内部蛋白质），流式细胞术，可以使用酮除了51后，量化在不同时间的样品的蛋白丰度。 FLUOrescence显微镜下治疗酮将提供两种蛋白丰度和本地化18的信息。基板，生物，以及下游应用服从酮追逐的范围使该技术研究蛋白质降解的高度通用和信息的手段。

蛋白质在几乎每一个细胞的过程中执行的关键功能。许多生理过程要求特定的蛋白（或蛋白）的一段确定的时间或在特定情况下周期的存在。因此，生物监测和调节蛋白丰度，以满足移动需求1。例如，细胞周期蛋白（控制细胞分裂的蛋白质），存在于细胞周期的特定阶段，和管制细胞周期蛋白水平的损失已经伴随恶性肿瘤的形成2相关联。除了 ​​调节蛋白水平，以满足蜂窝需求，电池使用降解的质量控制机制，以消除错误折叠，未组装，或以其它方式异常蛋白质分子3。蛋白丰度的控制既包括高分子合成（转录和翻译）和降解（RNA降解和蛋白水解）的监管。受损或过度的蛋白质降解有助于多个病症，包括癌症，囊性纤维化病，神经变性病症和心血管疾病4-8。因此，蛋白水解机制代表了一系列疾病9-12有前途的治疗靶点。 在单个时间点的蛋白质分析（ 例如 ，通过免疫印迹13，流式细胞仪14，或荧光显微镜15）提供稳定状态的蛋白丰度的快照不透露合成或降解的相对贡献。同样地，基于生长的报告分析反映在延长的时间周期稳态蛋白质水平而不合成和降解15-20的影响之间进行鉴别。有可能通过前比较丰度和抑制降解机制（ 例如特定组件，由药理学失活prote后推断降解过程稳态蛋白质水平的贡献asome 21或敲除推测的基因以需要为降解13）。抑制降解途径后，处于稳定状态的蛋白水平的变化提供了蛋白水解的蛋白质丰度13的控制的贡献的有力证据。然而，这样的分析仍然没有提供关于蛋白质周转的动力学信息。放线菌酮追逐其次是免疫印迹法，允许研究人员在一段时间22-24可视化蛋白质降解克服这些弱点。另外，不是必需的，因为以下酮追蛋白质检测通常通过蛋白质印迹，放射性同位素和冗长免疫沉淀步骤执行用于放线菌酮追逐，与许多通常使用的脉冲追踪技术，其也进行可视化的蛋白质降解25。 酮最初被鉴定为具有抗真菌合适的化合物由革兰氏阳性菌灰色链霉菌 26,27产生联系。它是一种细胞渗透性分子，通过阻碍核糖体易位28-31特异性抑制真核细胞内（但不细胞器）的翻译。在一个酮追踪实验，放线菌酮加入到细胞中，并且细胞的等分试样，立即在以下另外的化合物22的特定时间点收集。细胞裂解，并且蛋白质丰度在每个时间点进行分析，通常通过免疫印迹。降低在蛋白丰度加入环己酰亚胺可以自信归因于蛋白降解以下。不稳定的蛋白质将纷纷随时间而减少，而相对稳定的蛋白质会表现出丰富的变化不大。 选择性的蛋白质降解机理在整个真核生物的高度保守的。大部分已知的关于蛋白质降解是第一次听说该模型的单细胞真核生物， 酿酒酵母 （芽殖酵母）25,32-36。用酵母的研究很可能会继续提供新的和重要的见解的蛋白质降解。这里介绍一种在酵母细胞随后蛋白丰度的印迹分析酮追逐方法。

<table><tbody><tr><td>Desired yeast strains, plasmids, standard medium and buffer components</td><td></td><td></td><td></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>A tube roller is recommended to maintain overnight&amp;nbsp; starter cultures of yeast cells in suspension. Alternatively, if a tube roller is unavailable, a platform shaker in a temperature-controlled incubator may be used for overnight starter cultures. 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 120 V 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>Centrifuge 5430</td><td>Eppendorf</td><td>5427 000.216&amp;nbsp;</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 and 50 ml conical tubes</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 Heaters</td><td>Fisher Scientific</td><td>1172011AQ</td><td>It is recomended that two heaters are available (one for incubating cells during cycloheximide treatment and one for boiling lysates to denature proteins). Alternatively, 30 &amp;deg;C water bath may be used for incubation of cells in the presence of cycloheximide. Boiling water bath with hot plate may altertnatively be used to denature proteins.</td></tr><tr><td>Heating block for 12 x 15 ml conical tubes</td><td>Fisher Scientific</td><td>11-473-70</td><td>For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.</td></tr><tr><td>Heating block for 20 x 1.5 ml conical tubes</td><td>Fisher Scientific</td><td>11-718-9Q</td><td>For use with Dri-Bath Heater to boil lysates for protein denaturation.</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>

cycloheximide chase analysis of protein degradation in saccharomyces cerevisiae

蛋白丰度的调节是几乎所有的细胞过程的关键。蛋白丰度反映蛋白质合成和蛋白质降解率的结合。许多测定法对蛋白质丰度的报告（ 例如 ，单时间点印迹，流式细胞术，荧光显微镜，或基于生长的报告分析）不允许的翻译和蛋白水解的蛋白的水平的相对效果歧视。本文介绍了采用放线菌酮追逐其次是免疫印迹法来具体分析模型中的单细胞真核生物的蛋白质降解， 酿酒酵母 （芽殖酵母）。在此过程中，酵母细胞中的翻译抑制剂放线菌酮的存在下温育。细胞的等分试样后，并在下面的附加酮的特定时间点立即收集。细胞溶解，并且裂解物通过聚丙烯酰胺凝胶电泳FO分离R在每次时间点的蛋白丰度的免疫印迹分析。的放线菌酮追逐过程允许多种细胞蛋白质的稳态人口的降解动力学的可视化。该过程可被用来研究对蛋白质降解的遗传需求和环境的影响。

为了说明酮追方法，Deg1 -Sec62的稳定性（ 图1），一个模型酵母的内质网（ER）相关的降解（ERAD）基板，分析42-44。在ERAD，质量控制泛素连接酶的酶共价结合的小分子蛋白泛素以定位于内质网膜异常蛋白链。这样polyubiquitylated蛋白质随后从ER除去由蛋白酶体，一个大的，胞质蛋白酶45降解。所述Deg1 -Sec62蛋白是针对降解后持续和异常接合位子，促进蛋白质转运进入内质网腔或膜43的信道。同样地，哺乳动物载脂蛋白B，低密度脂蛋白的蛋白质成分，持续啮合ER位子并随后通过一个相关的机构时，其唇经历降解ID绑定的合作伙伴是缺席46-48。因此，在酵母细胞Deg1 -Sec62降解的分析可能会产生洞察持久或异常接合位子蛋白质的降解，暂时称为ERAD-T（对于易位子关联的）衬底43。 先前的研究已经表明，与泛素结合酶Ubc7对ER-驻地泛素连接酶Hrd1函数来催化Deg1 -Sec62 16,42-44的劣化。该跨膜蛋白Cue1锚Ubc7到内质网膜并激活酶49-51。然而，在Deg1 -Sec62的降解为Cue1要求没有直接的影响。为了测试Cue1，像Hrd1和Ubc7，需要Deg1 -Sec62高效降解假设，野生型和突变体的酵母细胞中表达Deg1 -Sec62经受酮追逐和韦斯燕鸥印迹分析（ 图2A）。该Deg1 -Sec62蛋白质迁移在SDS-PAGE为多个物种。这与先前的研究表明蛋白43,44,52的广泛的翻译后修饰是一致的。在野生型酵母细胞中，Deg1 -Sec62是容易降解。正如前面所观察到的，无论是Hrd1或Ubc7损失大致稳定的Deg1 -Sec62蛋白42-44。如预测的，Deg1 -Sec62在没有Cue1的（以类似的程度为在不存在Ubc7的）稳定时，确认为在ERAD-T中Ubc7相互作用蛋白的作用。 剩余在每个时间点Deg1 -Sec62蛋白质的比例进行定量，并在图2B中画出。我们注意到，在野生型细胞Deg1 -Sec62消失的表观速率可以是新生Deg1 -Sec62的降解速率的低估（ 即，所述蛋白质的半衰期，这可通过脉冲追踪分析43最直接地确定）。因为野生型酵母积极降解之前酮加入Deg1 -Sec62蛋白，Deg1 -Sec62稳态丰度在这些细胞中显着减少（相比于降解受损细胞）。这可以通过在初始时间点为图2A中的每个应变Deg1 -Sec62的丰度进行比较来理解。另外，底物蛋白质的任何降解抗性的亚群（ 例如，如果该蛋白在从其中防止降解细胞内池积累）可能出现在野生型细胞在实验的时间过程相对稳定。 <img alt="图1" src="/files/ftp_upload/53975/53975fig1.jpg" /> 图1.模型 Deg1 降解 </em&gt; -Sec62以下位子的异常参与 （A）中 Deg1 -Sec62 Deg1 -Sec62由以下元素的，在顺序的示意性描绘:Deg1（从酵母转录阻遏MATα2氨基末端67个氨基酸），一个标志（F）的表位，2跨膜蛋白Sec62，并继正常共翻译插入其两个跨膜段到内质网（ER）膜中的金黄色葡萄球菌蛋白A（PRA）。（B）的两个副本，持久性关联的Deg1 -Sec62的位子触发异常，Deg1依赖，翻译后的位子参与。胞质氨基端的一部分异常进入，并有可能保持在-的位子。（C）继位子接合时，Hrd1泛素连接酶泛素Deg1 -Sec62。 Hrd1被泛素结合酶ù协助BC7，这是在由Cue1 ER膜锚。 UB，泛素蛋白单体（D）泛素化Deg1 -Sec62从内质网膜提取和蛋白酶体降解，解除梗阻位子。 <a href="https://www.jove.com/files/ftp_upload/53975/53975fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53975/53975fig2.jpg" /> 图2.放线菌酮大通其次是免疫印迹表明Cue1是需要有效 Deg1 -Sec62降解。指示的基因型（A）的酵母细胞用酵母着丝粒质粒编码Deg1 -Sec62转化（pRS416-P MET25 - Deg1 -flag-Sec62-2xProtA; 是AmpR / URA3 43），并进行放线菌酮追分析。蛋白从每个时间点（相当于0.125的OD 600单位）■分离在8％SDS-PAGE凝胶并通过用兔抗小鼠次级抗体，其直接结合Deg1的C末端蛋白A表位的免疫印迹检测- Sec62融合蛋白。作为上样对照，膜随后用特定于Pgk1基因抗体温育。该实验中已被复制三次;代表性的结果被描绘中的数据（B）定量（A），使用成像软件（见材料表 ）进行。的区域包围Deg1 -Sec62蛋白质的总荧光强度对每个样品确定。对于各样品的背景荧光强度从Deg1 -Sec62蛋白质邻近像素的平均荧光强度推断。此外推的背景荧光强度从总荧光强度中减去，以产生经调整fluorescENCE强度每个样品。为Pgk1基因，上样对照蛋白，其丰度在测定的条件不变化进行类似quantifications总量，背景，和调整后的荧光强度。比较样品中的丰度Deg1 -Sec62蛋白质，对每个样品测定Deg1 -Sec62到Pgk1基因的调节信号强度的比率。所述Deg1 -Sec62 / Pgk1基因比被定义为100％，为第一个时间点（ 即 ，放线菌酮加入后立即）根据分析每个菌株。 Deg1 -Sec62的其余在其后的时间点（ 即 Deg1 -Sec62 /比率Pgk1基因）计算为在第一时间点Deg1 -Sec62 /比率Pgk1基因的百分比的金额。 <a href="https://www.jove.com/files/ftp_upload/53975/53975fig2large.jpg" target="_blank">请点击此处查看大图这个数字。</a> <table border="1" fo:keep-together.within-page="1" fo:kEEP-与-next.within页="总是"&gt; <tbody><tr><td rowspan="3"> 合成的确定（SD）最小酵母培养基 </td><td rowspan="3"> 2％葡萄糖，0.67％酵母氮​​碱无氨基酸，0.002％腺嘌呤，0.004％尿嘧啶，0.002％精氨酸，0.001％组氨酸，0.006％异亮氨酸，0.006％亮氨酸，0.004％的赖氨酸，0.001％的蛋氨酸，0.006％苯丙氨酸，0.005苏氨酸％，0.004％色氨酸。对于固体（板）中，包括2％琼脂。 </td><td> 1.选择性介质被省略合适的氨基酸（S）或含氮碱制备。 </td></tr><tr><td>为方便起见，如下这些成分可以保持为浓原液。氨基酸可以保持为包含所有所需的氨基酸100×储液。酵母氮碱可以以20倍的原液（13.4％）被保持。葡萄糖可以以40％的原液被维持。腺嘌呤和尿嘧啶可被维持为1％储备溶液在0.1M NaOH中。 </td></tr><tr><td> 3。高压灭菌网上平台。 </td></tr><tr><td rowspan="3"> 酵母提取物，蛋白胨葡萄糖（YPD）丰富的酵母中 </td><td rowspan="3"> 1％酵母提取物，2％蛋白胨，2％葡萄糖（可选:0.002％腺嘌呤）。对于固体（板）中，包括2％琼脂。 </td><td> 1.为防止腺嘌呤的生长缓慢和特定实验室酵母菌株的红色着色特性在没有（或低丰度），腺嘌呤可加入YPD培养基。 </td></tr><tr><td>为方便起见，如下一些成分可以保持为浓原液。葡萄糖可以以40％的原液被维持。腺嘌呤可以维持在0.1M NaOH中1％的储液。 </td></tr><tr><td> 3.高压灭菌网上平台。 </td></tr><tr><td> 20X停止混合 </td><td> 200mM的叠氮化钠，5毫克/毫升的牛血清白蛋白</td><td>叠氮化钠可通过口服或皮肤接触有毒。按照制造商制备建议时，存储和处理叠氮化钠。意外暴露的情况下，请咨询厂家提供材料安全数据表。 </td></tr><tr><td rowspan="2"> 20毫克/毫升放线菌酮 </td><td rowspan="2"></td><td> 1.准备乙醇。储存于-20℃。 </td></tr><tr><td> 2.放线菌酮是一种皮肤的刺激性，可经口摄入中毒。当准备，储存和处理酮按照制造商的建议。意外暴露的情况下，请咨询厂家提供材料安全数据表。 </td></tr><tr><td> 0.2M的氢氧化钠 </td><td></td><td>制备在水中。氢氧化钠玻璃反应。因此，对于长期储存，0.2M氢氧化钠应在塑料容器保持。 </td></tr><tr><td rowspan="2"> Laemmli样品缓冲液 </td><td rowspan="2"> 2％SDS，10％甘油，5％＃946;巯基乙醇，60毫米的Tris盐酸pH值为6.8，0.008％溴酚蓝</td><td> 1. Laemmli样品缓冲液通常是通过稀释较为集中（ 例如，5倍）的股票准备。 </td></tr><tr><td> 2.染料溴酚蓝可加入到期望的强度。 A"捏"（很少量从刮刀边缘抽头）通常就足够了。 </td></tr><tr><td> 拉姆力电泳缓冲液（5倍） </td><td> 125毫摩尔Tris，960毫甘氨酸，0.5％SDS中</td><td>在卫生署2 O 5:制备1升1X拉姆力的电泳缓冲液，稀释1 </td></tr><tr><td> 三醋酸-SDS传输缓冲区（5倍） </td><td>的125mM Tris乙酸盐（pH值为8.8），960毫甘氨酸，0.05％SDS的</td><td>为了准备20升1X Tris乙酸盐，SDS的转移缓冲液，结合4升5倍的股票，4升甲醇，12升的dh 2 O的</td></tr><tr><td> 10倍的Tris缓冲盐水（TBS） </td><td> 500毫米的Tris，1.5 M氯化钠;将pH调节至7.5</td><td>为了准备1升1X TBS，淡化1:10在卫生署2 O. 1X TBS可以补充与洗涤剂吐温-20和脱脂奶粉，作为合适的。 </td></tr></tbody></table> 表1.解决方案中使用这一研究进展。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 菌株名称 </td><td> 别号 </td><td> 相关基因型 </td><td> 参考 </td></tr><tr><td rowspan="7"> VJY42 </td><td rowspan="7"> MHY501 </td><td> MATα </td><td rowspan="7"> Chen等人 ，1993年</td></tr><tr><td> HIS3-Δ200 </td></tr><tr><td> leu2-3,112 </td></tr><tr><td> ura3-52 </td></tr><tr><td> lys2-801 </td></tr><tr><td> trp1-1 </td></tr><tr><td> GAL2 </td></tr><tr><td rowspan="8"> VJY47 </td><td rowspan="8"> YWO55; MHY507 </td><td> MATα </td><td rowspan="8">容曼等人，1993;鲁宾斯坦等人 ，2011 </td></tr><tr><td> HIS3-Δ200 </td></tr><tr><td> leu2-3,112 </td></tr><tr><td> ura3-52 </td></tr><tr><td> lys2-801 </td></tr><tr><td> trp1-1 </td></tr><tr><td> GAL2 </td></tr><tr><td> ubc7Δ:: LEU2 </td></tr><tr><td rowspan="8"> VJY56 </td><td rowspan="8"> YTX105; MHY1364 </td><td> MATα </td><td rowspan="8"> Biederer 等人，1997; Ravid 等人 ，2006年</td></tr><tr><td> HIS3-Δ200 </td></tr><tr><td> leu2-3,112 </td></tr><tr><td> ura3-52 </td></tr><tr><td> lys2-801 </td></tr><tr><td> trp1-1 </td></tr><tr><td> GAL2 </td></tr><tr><td> cue1Δ:: HIS3 </td></tr><tr><td rowspan="8"> VJY172 </td><td rowspan="8"> MHY6199 </td><td> MATα </td><td rowspan="8">这项研究</td></tr><tr><td> HIS3-Δ200 </td></tr><tr><td> leu2-3,112 </td></tr><tr><td> ura3-52 </td></tr><tr><td> lys2-801 </td></tr><tr><td> trp1-1 </td></tr><tr><td> GAL2 </td></tr><tr><td> hrd1Δ:: kanMX4 </td></tr></tbody></table> 表2.酵母菌株用于该研究中，所有菌株与同类53 MHY500。 VJY42，VJY47和VJY56以前49,53,54描述。详细株的产生和确认战略VJY172（本研究编制）可根据要求提供。

Watch this Scientific Journal Video about Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae at JoVE.com

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 ...

cycloheximide-chase-analysis-protein-degradation-saccharomyces

Nanyang Technological University

Research

JoVE Journal

Biology

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

28.8K 次观看.  Ball State University. 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).

视频: Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give rise to aneuploidy occur during meiosis in oocytes, but why oocyte meiosis is error-prone is still not fully understood. During cell division, cells prevent errors in chromosome segregation by activating the spindle assembly checkpoint (SAC). This control mechanism relies on detecting kinetochore (KT)-microtubule (MT) attachments and sensing tension generated by spindle fibers. When KTs are unattached, the SAC is activated and prevents cell-cycle progression. The SAC is activated first by MPS1 kinase, which triggers the recruitment and formation of the mitotic checkpoint complex (MCC), composed of MAD1, MAD2, BUB3, and BUBR1. Then, the MCC diffuses into the cytoplasm and sequesters CDC20, an anaphase-promoting complex/cyclosome (APC/C) activator. Once KTs become attached to microtubules and chromosomes are aligned at the metaphase plate, the SAC is silenced, CDC20 is released, and the APC/C is activated, triggering the degradation of Cyclin B and Securin, thereby allowing anaphase onset. Compared to somatic cells, the SAC in oocytes is not as effective because cells can undergo anaphase despite having unattached KTs. Understanding why the SAC is more permissive and if this permissiveness is one of the causes of chromosome segregation errors in oocytes still needs further investigation. The present protocol describes the three techniques to comprehensively evaluate SAC integrity in mouse oocytes. These techniques include using nocodazole to depolymerize MTs to evaluate the SAC response, tracking SAC silencing by following the kinetics of Securin destruction, and evaluating the recruitment of MAD2 to KTs by immunofluorescence. Together these techniques probe mechanisms needed to produce healthy eggs by providing a complete evaluation of SAC integrity.

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

评估小鼠卵母细胞中纺锤体组件检查点完整性

Aneuploidy is the leading genetic abnormality causing early miscarriage and pregnancy failure in humans. Most errors in chromosome segregation that give ...

科研

发育生物学

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

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

生物学

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 ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

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 ...

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome segregation. As DNA synthesis defects underlie chromosome rearrangements, monitoring DNA replication has become essential to understand the basis of genome instability. Saccharomyces cerevisiae is a classical model to study cell cycle regulation, but key DNA replication parameters, such as the fraction of cells in the S phase or the S-phase duration, are still difficult to determine. This protocol uses short and non-toxic pulses of 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, in engineered TK-hENT1 yeast cells, followed by its detection by Click reaction to allow the visualization and quantification of DNA replication with high spatial and temporal resolution at both the single-cell and population levels by microscopy and flow cytometry. This method may identify previously overlooked defects in the S phase and cell cycle progression of yeast mutants, thereby allowing the characterization of new players essential for ensuring genome stability.

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

We describe two complementary protocols to accurately determine S-phase duration in S. cerevisiae using EdU, a thymidine analog, which is incorporated in vivo and detected using Click chemistry by microscopy and flow cytometry. It allows for the easy characterization of the duration of DNA replication and overlooked replication defects in mutants.

使用酿酒酵母中的5-乙炔基-2'-脱氧尿苷掺入测定S相持续时间

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome ...

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often not seen by imaging fixed cells. Budding yeast has proved to be an important model organism to study meiotic chromosome segregation because many meiotic genes are highly conserved. Time-lapse microscopy of meiosis in budding yeast allows the monitoring of different meiotic mutants to show how the mutation disrupts meiotic processes. However, many proteins function at multiple points in meiosis. The use of loss-of-function or meiotic null mutants can therefore disrupt an early process, blocking or disturbing the later process and making it difficult to determine the phenotypes associated with each individual role. To circumvent this challenge, this protocol describes how the proteins can be conditionally depleted from the nucleus at specific stages of meiosis while monitoring meiotic events using time-lapse microscopy. Specifically, this protocol describes how the cells are synchronized in prophase I, how the anchor away technique is used to deplete proteins from the nucleus at specific meiotic stages, and how time-lapse imaging is used to monitor meiotic chromosome segregation. As an example of the usefulness of the technique, the kinetochore protein Ctf19 was depleted from the nucleus at different time points during meiosis, and the number of chromatin masses was analyzed at the end of meiosis II. Overall, this protocol can be adapted to deplete different nuclear proteins from the nucleus while monitoring the meiotic divisions.

Use of Time-Lapse Microscopy and Stage-Specific Nuclear Depletion of Proteins to Study Meiosis in S. cerevisiae

Time-lapse microscopy is a valuable tool for studying meiosis in budding yeast. This protocol describes a method that combines cell-cycle synchronization, time-lapse microscopy, and conditional depletion of a target protein to demonstrate how to study the function of a specific protein during meiotic chromosome segregation.

使用延时显微镜和蛋白质的阶段特异性核耗竭来研究酿酒酵母的减数分裂

Time-lapse fluorescence microscopy has revolutionized the understanding of meiotic cell-cycle events by providing temporal and spatial data that is often ...

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.

Saccharomyces cerevisiae Yeast as a Model Organism

酿酒酵母简介

Saccharomyces cerevisiae (commonly known as baker&rsquo;s yeast) is a single-celled eukaryote that is frequently used in scientific research.  S. ...

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模式生物I:酵母，果蝇和秀丽线虫

Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation

Source: Guo, L. et al., <a href="https://app.jove.com/v/54266">Assays for the Degradation of Misfolded Proteins in Cells.</a> J. Vis. Exp. (2016).
This video describes a degradation assay for misfolded proteins using cycloheximide treatment along with detergent fractionation. This method aids in studying the dynamics of misfolded proteins and uncovering the in-depth mechanisms of protein turnover.

易错折叠的蛋白质降解测定:一种使用放线菌酮处理和去污剂分级分离监测错误折叠蛋白质降解的技术

Source: Guo, L. et al., Assays for the Degradation of Misfolded Proteins in Cells. J. Vis. Exp. (2016).
This video describes a degradation assay for ...

蛋白质降解的放线菌酮大通分析

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