Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells

To improve the accuracy of predicting membrane protein sorting signals, we developed a general methodology for defining trafficking signal consensus s equences in the environment of the living cell. Our approach uses retrovira l 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 e fficacy of individual signals. Using this strategy to analyze arginine- and lysine-based endoplasmic reticulum localization signals, we demonstrate th at small changes in the local sequence context dramatically alter signal st rength, generating a broad spectrum of trafficking phenotypes. Finally, usi ng sequences from our screen, we found that the potency of di-lysine, but n ot di-arginine, mediated endoplasmic reticulum localization was correlated with the strength of interaction with alpha -COP.