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Biologia

蛋白质降解的放线菌酮大通分析<em>酿酒酵母</em

Published: April 18, 2016
doi:
10.3791/53975
, , ,
1Department of Biology,Ball State University, 2Bioproduct Research & Development,Eli Lilly and Company

Summary

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).

Abstract

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。

Introduction

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。

Protocol

酵母细胞的生长1和收获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。 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。 <…

Representative Results

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?…

Discussion

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…

Declarações

The authors have nothing to disclose.

Acknowledgements

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.

Materials

Desired yeast strains, plasmids, standard medium and buffer components
Disposable borosilicate glass tubes Fisher Scientific 14-961-32 Available from a variety of manufacturers
Temperature-regulated incubator (e.g. Heratherm Incubator Model IMH180) Dot Scientific 51028068 Available from a variety of manufacturers
New Brunswick Interchangeable Drum for 18 mm tubes (tube roller) New Brunswick M1053-0450 A 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 H New Brunswick M1053-4004 For use with tube roller
SmartSpec Plus Spectrophotometer Bio-Rad 170-2525 Available from a variety of manufacturers
Centrifuge 5430 Eppendorf 5427 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-Place Eppendorf F-35-6-30
15-ml screen printed screw cap tube 17 x 20 mm conical, polypropylene Sarstedt 62.554.205 Available from a variety of manufacturers
1.5-ml flex-tube, PCR clean, natural microcentrifuge tubes Eppendorf 22364120 Available from a variety of manufacturers
Analog Dri-Bath Heaters Fisher Scientific 1172011AQ 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 °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 tubes Fisher Scientific 11-473-70 For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.
Heating block for 20 x 1.5-ml conical tubes Fisher Scientific 11-718-9Q For 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 membrane Will vary by lab and application
Western blot imaging system (e.g. Li-Cor Odyssey CLx scanner and Image Studio Software) Li-Cor 9140-01 Will vary by lab and application
EMD Millipore Immobilon PVDF Transfer Membranes Fisher Scientific IPFL00010 Will vary by lab and application
Primary antibodies (e.g. Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8)) Life Technologies 459250 Will vary by lab and application
Secondary antibodies (e.g. Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H+L)) Life Technologies A-21065 Will vary by lab and application
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Tags

Keywords: Protein DegradationSaccharomyces CerevisiaeCycloheximide ChaseEndogenous ProteinPlasmid-expressed ProteinOptical DensityMid-logarithmic GrowthHeat BlockStop MixCell LysisProtein Denaturation
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Buchanan, B. W., Lloyd, M. E., Engle, S. M., Rubenstein, E. M. Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae. J. Vis. Exp. (110), e53975, doi:10.3791/53975 (2016).
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