Watts Sheldon G, Crowder Justin J, Coffey Samuel Z, Rubenstein Eric M
Department of Biology, Ball State University.
Department of Biology, Ball State University; Division of Nephrology, Cincinnati Children's Hospital.
J Vis Exp. 2015 Feb 16(96):e52428. doi: 10.3791/52428.
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.
受调控的蛋白质降解对于几乎所有细胞功能都至关重要。目前所知的许多关于真核生物蛋白质降解的分子机制和遗传需求，最初都是在酿酒酵母中建立起来的。蛋白质降解的经典分析依赖于生化脉冲追踪和环己酰亚胺追踪方法。虽然这些技术为观察蛋白质降解提供了灵敏的手段，但它们费力、耗时且通量低。这些方法不适合对阻止蛋白质降解的突变进行快速或大规模筛选。在此，描述了一种基于酵母生长的检测方法，用于轻松鉴定蛋白质降解的遗传需求。在该检测方法中，在特定选择性条件下生长所需的报告酶与一种不稳定蛋白融合。缺乏内源性报告酶但表达融合蛋白的细胞，只有在融合蛋白稳定时（即蛋白质降解受损时）才能在选择性条件下生长。在此处描述的生长检测中，将携带编码融合蛋白质粒的野生型和突变型酵母细胞的系列稀释液点种在选择性和非选择性培养基上。在选择性条件下的生长与特定突变导致的降解受损一致。蛋白质丰度增加应通过生化方法进行确认。还展示了一种以适合电泳和蛋白质印迹分析的形式快速提取酵母蛋白质的方法。基于生长的蛋白质稳定性读数，结合用于生化分析的简单蛋白质提取方案，有助于快速鉴定蛋白质降解的遗传需求。这些技术可用于监测各种短命蛋白质的降解。在给出的示例中，将组氨酸生物合成所需的His3酶与Deg1-Sec62融合。Deg1-Sec62在内质网易位子异常结合后会被靶向降解。当蛋白质稳定时，携带Deg1-Sec62-His3的细胞能够在选择性条件下生长。
