DataSheet5_Intra-FCY1: a novel system to identify mutations that cause protein misfolding.CSV

Protein misfolding is a common intracellular occurrence. Most mutations to coding sequences increase the propensity of the encoded protein to misfold. These misfolded molecules can have devastating effects on cells. Despite the importance of protein misfolding in human disease and protein evolution, there are fundamental questions that remain unanswered, such as, which mutations cause the most misfolding? These questions are difficult to answer partially because we lack high-throughput methods to compare the destabilizing effects of different mutations. Commonly used systems to assess the stability of mutant proteins in vivo often rely upon essential proteins as sensors, but misfolded proteins can disrupt the function of the essential protein enough to kill the cell. This makes it difficult to identify and compare mutations that cause protein misfolding using these systems. Here, we present a novel in vivo system named Intra-FCY1 that we use to identify mutations that cause misfolding of a model protein [yellow fluorescent protein (YFP)] in Saccharomyces cerevisiae. The Intra-FCY1 system utilizes two complementary fragments of the yeast cytosine deaminase Fcy1, a toxic protein, into which YFP is inserted. When YFP folds, the Fcy1 fragments associate together to reconstitute their function, conferring toxicity in media containing 5-fluorocytosine and hindering growth. But mutations that make YFP misfold abrogate Fcy1 toxicity, thus strains possessing misfolded YFP variants rise to high frequency in growth competition experiments. This makes such strains easier to study. The Intra-FCY1 system cancels localization of the protein of interest, thus can be applied to study the relative stability of mutant versions of diverse cellular proteins. Here, we confirm this method can identify novel mutations that cause misfolding, highlighting the potential for Intra-FCY1 to illuminate the relationship between protein sequence and stability.

蛋白质错误折叠（protein misfolding）是细胞内常见的现象。绝大多数编码序列突变都会提升编码蛋白发生错误折叠的倾向，这类错误折叠的蛋白质分子会对细胞造成毁灭性影响。尽管蛋白质错误折叠在人类疾病与蛋白质演化中具有重要意义，但仍有诸多基础问题尚未得到解答，例如哪些突变最易引发蛋白质错误折叠？
这类问题难以解答的部分原因在于，我们缺乏可用于比较不同突变的去稳定化效应的高通量方法。当前常用于活体内（in vivo）评估突变蛋白稳定性的系统，通常以必需蛋白作为传感元件，但错误折叠的蛋白质足以干扰必需蛋白的功能并导致细胞死亡，这使得利用这类系统难以识别并比较引发蛋白质错误折叠的突变。
本研究构建了一种全新的活体内系统，命名为Intra-FCY1，用于在酿酒酵母（Saccharomyces cerevisiae）中识别引发模式蛋白——黄色荧光蛋白（yellow fluorescent protein, YFP）——错误折叠的突变。Intra-FCY1系统将酵母胞嘧啶脱氨酶Fcy1（一种毒性蛋白）拆分为两个互补片段，并将YFP插入该拆分位点。当YFP正确折叠时，Fcy1的两个互补片段会相互结合并恢复其酶活性，在含有5-氟胞嘧啶（5-fluorocytosine）的培养基中产生毒性，抑制细胞生长；而引发YFP错误折叠的突变会消除Fcy1的毒性，因此在生长竞争实验中，携带错误折叠YFP变体的菌株会快速富集为优势种群，这使得这类菌株的研究更为便捷。
Intra-FCY1系统不受目标蛋白亚细胞定位的限制，因此可用于研究多种细胞蛋白的突变体相对稳定性。本研究验证了该方法可用于识别引发蛋白质错误折叠的新型突变，凸显了Intra-FCY1系统在解析蛋白质序列与稳定性之间关系方面的应用潜力。