Cohen Itamar, Geffen Yifat, Ravid Guy, Ravid Tommer
Department of Biological Chemistry, The Hebrew University of Jerusalem.
Department of Biological Chemistry, The Hebrew University of Jerusalem;
J Vis Exp. 2014 Nov 6(93):e52021. doi: 10.3791/52021.
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.
泛素-蛋白酶体系统（UPS）介导的蛋白质降解是所有真核生物中蛋白质稳态的主要调节机制。确定细胞内蛋白质降解的标准方法依赖于生化分析来追踪蛋白质减少的动力学过程。这类方法通常费力且耗时，因此不适用于旨在评估多种底物和降解条件的实验。作为一种替代方法，基于细胞生长的分析方法已经被开发出来，但其传统形式是终点分析，无法定量确定蛋白质水平的相对变化。在此，我们描述了一种通过将蛋白质降解速率与酵母细胞生长动力学相耦合来准确测定其变化的方法。该方法基于一个已建立的选择系统，在该系统中，缺失URA3的酵母细胞的尿嘧啶营养缺陷型可通过外源表达的报告蛋白得到挽救，该报告蛋白由必需的URA3基因与一个降解决定簇（降解决定子）融合而成。报告蛋白的设计使其合成速率恒定，而其降解速率由降解决定子决定。由于在缺乏尿嘧啶的培养基中细胞生长与Ura3的相对水平成正比，生长动力学完全依赖于报告蛋白的降解。这种方法能够准确测量细胞内蛋白质降解动力学的变化。它被应用于：（a）评估已知泛素结合因子对蛋白水解的相对贡献；（b）E2结合酶的结构-功能分析；（c）新型降解决定子的鉴定和表征。基于降解决定子-URA3的系统的应用超越了蛋白质降解领域，因为它也可用于监测与其他细胞途径功能相关的蛋白质水平变化。
