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</em...

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 border="0" cellpadding="0" cellspacing="0" width="607">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Desired yeast strains, plasmids, standard medium and buffer components</td>
			<td style="width:105px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable borosilicate glass tubes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">14-961-32</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Temperature-regulated incubator (e.g. Heratherm Incubator Model IMH180)</td>
			<td style="width:105px;">Dot Scientific</td>
			<td style="width:119px;">51028068</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="357">
			<td height="357" style="height:357px;width:216px;">New Brunswick Interchangeable Drum for 18 mm tubes (tube roller)</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:119px;">M1053-0450</td>
			<td style="width:167px;">A tube roller is recommended to maintain overnight&#160; 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 height="42">
			<td height="42" style="height:42px;width:216px;">New Brunswick TC-7 Roller Drum 120V 50/60 H</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:119px;">M1053-4004</td>
			<td>For use with tube roller</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">SmartSpec Plus Spectrophotometer</td>
			<td style="width:105px;">Bio-Rad</td>
			<td>170-2525</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Centrifuge 5430</td>
			<td style="width:105px;">Eppendorf</td>
			<td>5427 000.216&#160;</td>
			<td style="width:167px;">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 height="105">
			<td height="105" style="height:105px;width:216px;">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 style="width:105px;">Eppendorf</td>
			<td>F-35-6-30</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">15-ml screen printed screw cap tube 17 x 20 mm conical, polypropylene</td>
			<td style="width:105px;">Sarstedt</td>
			<td style="width:119px;">62.554.205</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.5-ml flex-tube, PCR clean, natural microcentrifuge tubes</td>
			<td style="width:105px;">Eppendorf</td>
			<td style="width:119px;">22364120</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="357">
			<td height="357" style="height:357px;width:216px;">Analog Dri-Bath Heaters</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">1172011AQ</td>
			<td style="width:167px;">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 &#176;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 height="105">
			<td height="105" style="height:105px;width:216px;">Heating block for 12 x 15-ml conical tubes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td>11-473-70</td>
			<td style="width:167px;">For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Heating block for 20 x 1.5-ml conical tubes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td>11-718-9Q</td>
			<td style="width:167px;">For use with Dri-Bath Heater to boil lysates for protein denaturation.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">SDS-PAGE running and transfer apparatuses, power supplies, and imaging equipment or darkrooms for SDS-PAGE and transfer to membrane</td>
			<td style="width:105px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Western blot imaging system (e.g. Li-Cor Odyssey CLx scanner and Image Studio Software)</td>
			<td style="width:105px;">Li-Cor</td>
			<td>9140-01</td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EMD Millipore Immobilon PVDF Transfer Membranes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">IPFL00010</td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Primary antibodies (e.g. Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8))</td>
			<td style="width:105px;">Life Technologies</td>
			<td style="width:119px;">459250</td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Secondary antibodies (e.g. Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H+L))</td>
			<td>Life Technologies</td>
			<td>A-21065</td>
			<td style="width:167px;">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?...

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

Research

JoVE Journal

Biology

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

28.7K 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

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

囊性纤维化跨膜传导调节蛋白表达和纯化酿酒酵母

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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 ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

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.