Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells
Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells
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DOI：
10.1073/pnas.051630198
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发表时间：
2001-02-27
影响因子：
11.1
通讯作者：
Schwappach, B
中科院分区：
文献类型：
--
作者：
Zerangue, N;Malan, MJ;Schwappach, B
为了提高预测膜蛋白分选信号的准确性，我们开发了一种用于定义活细胞环境中的运输信号共有序列的通用方法。我们的方法使用逆转录病毒基因转移在哺乳动物细胞中创建运输信号变体的组合表达文库，使用流式细胞术根据运输表型对细胞进行分类，并使用定量运输分析来测量单个信号的功效。使用这种策略来分析基于精氨酸和赖氨酸的内质网定位信号，我们证明局部序列背景的微小变化会显着改变信号强度，产生广泛的运输表型。最后，使用我们筛选的序列，我们发现二赖氨酸（而非二精氨酸）介导的内质网定位的效力与与α-COP相互作用的强度相关。
To improve the accuracy of predicting membrane protein sorting signals, we developed a general methodology for defining trafficking signal consensus sequences in the environment of the living cell. Our approach uses retroviral gene transfer to create combinatorial expression libraries of trafficking signal variants in mammalian cells, flow cytometry to sort cells based on trafficking phenotype, and quantitative trafficking assays to measure the efficacy of individual signals. Using this strategy to analyze arginine- and lysine-based endoplasmic reticulum localization signals, we demonstrate that small changes in the local sequence context dramatically alter signal strength, generating a broad spectrum of trafficking phenotypes. Finally, using sequences from our screen, we found that the potency of di-lysine, but not di-arginine, mediated endoplasmic reticulum localization was correlated with the strength of interaction with alpha -COP.