Zerangue N, Malan M J, Fried S R, Dazin P F, Jan Y N, Jan L Y, Schwappach B
Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco, CA 94143-0725, USA.
Proc Natl Acad Sci U S A. 2001 Feb 27;98(5):2431-6. doi: 10.1073/pnas.051630198.
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.
为提高预测膜蛋白分选信号的准确性，我们开发了一种在活细胞环境中定义转运信号共有序列的通用方法。我们的方法利用逆转录病毒基因转移在哺乳动物细胞中创建转运信号变体的组合表达文库，通过流式细胞术根据转运表型对细胞进行分选，并采用定量转运分析来测量单个信号的效力。利用该策略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著改变信号强度，产生广泛的转运表型。最后，利用我们筛选得到的序列，我们发现双赖氨酸（而非双精氨酸）介导的内质网定位效力与与α-COP的相互作用强度相关。
