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本文内容

  • 摘要
  • 摘要
  • 引言
  • 研究方案
  • 结果
  • 讨论
  • 披露声明
  • 致谢
  • 材料
  • 参考文献
  • 转载和许可

摘要

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).

摘要

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。

引言

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。

研究方案

酵母细胞的生长1和收获

  1. 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。
  2. 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。
  3. 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。
  4. 稀释过夜培养至0.2在15毫升新鲜培养基的OD 600值。
  5. 在30℃下孵育酵母,振摇直到细胞达到中期对数生长期( ,的OD在0.8和1.2之间600)。
  6. 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:
    1. 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。
    2. 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。
    3. 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℃。
    4. 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。
  7. 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。
  8. 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( 3毫升7.5的OD 600单位)。

2.放线菌酮大通

  1. 孵育在30℃加热块5分钟平衡酵母细胞悬浮液。
  2. 准备一个计时器,从0:00计数。
  3. 要开始追酮,按"开始"计时器。很快,不过仔细,请执行下列步骤:
    1. 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。
    2. 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。
    3. 返回酵母细胞悬浮液至30℃。
  4. 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 )。
  5. 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。
    1. 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。
  6. 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。

3.后碱性蛋白提取(从16,40修改)

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. 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℃。

代表性的SDS-PAGE和Western印迹程序(从16改编)

  1. 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。
  2. 在200V运行凝胶直到染料前沿到达凝胶的底部边缘。
  3. 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。
  4. 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。
  5. 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。
  6. 在室温下用TBS / T洗膜3×5分钟,摇摆。
  7. 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。
  8. 在室温下用TBS / T洗膜3×5分钟,摇摆。
  9. 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。
  10. 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。
  11. 在室温下用TBS / T洗膜3×5分钟,摇摆。
  12. 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。
  13. 在室温下用TBS / T洗膜3×5分钟,摇摆。
  14. 图像膜如在步骤4.9。

结果

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?...

讨论

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

披露声明

The authors have nothing to disclose.

致谢

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.

材料

NameCompanyCatalog NumberComments
Desired yeast strains, plasmids, standard medium and buffer components
Disposable borosilicate glass tubesFisher Scientific14-961-32Available from a variety of manufacturers
Temperature-regulated incubator (e.g. Heratherm Incubator Model IMH180)Dot Scientific51028068Available from a variety of manufacturers
New Brunswick Interchangeable Drum for 18 mm tubes (tube roller)New BrunswickM1053-0450A tube roller is recommended to maintain overnight  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.
New Brunswick TC-7 Roller Drum 120V 50/60 HNew BrunswickM1053-4004For use with tube roller
SmartSpec Plus SpectrophotometerBio-Rad170-2525Available from a variety of manufacturers
Centrifuge 5430Eppendorf5427 000.216 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
Fixed-Angle Rotor F-35-6-30 with Lid and Adapters for Centrifuge Model 5430/R, 15/50 mL Conical Tubes, 6-PlaceEppendorfF-35-6-30
15-ml screen printed screw cap tube 17 x 20 mm conical, polypropyleneSarstedt62.554.205Available from a variety of manufacturers
1.5-ml flex-tube, PCR clean, natural microcentrifuge tubesEppendorf22364120Available from a variety of manufacturers
Analog Dri-Bath HeatersFisher Scientific1172011AQIt is recomended that two heaters are available (one for incubating cells during cycloheximide treatment and one for boiling lysates to denature proteins). Alternatively, 30 °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.
Heating block for 12 x 15-ml conical tubesFisher Scientific11-473-70For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.
Heating block for 20 x 1.5-ml conical tubesFisher Scientific11-718-9QFor use with Dri-Bath Heater to boil lysates for protein denaturation.
SDS-PAGE running and transfer apparatuses, power supplies, and imaging equipment or darkrooms for SDS-PAGE and transfer to membraneWill vary by lab and application
Western blot imaging system (e.g. Li-Cor Odyssey CLx scanner and Image Studio Software)Li-Cor9140-01Will vary by lab and application
EMD Millipore Immobilon PVDF Transfer MembranesFisher ScientificIPFL00010Will vary by lab and application
Primary antibodies (e.g. Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8))Life Technologies459250Will vary by lab and application
Secondary antibodies (e.g. Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H+L))Life TechnologiesA-21065Will vary by lab and application

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