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 sequences in the environment of the living cell. Our approach uses retroviral gene transfer to create combinatorial expression libraries of trafficking...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 2001-02, Vol.98 (5), p.2431-2436 |
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| container_title | Proceedings of the National Academy of Sciences - PNAS |
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| creator | Zerangue, Noa Malan, Michael J. Fried, Sharon R. Dazin, Paul F. Jan, Yuh Nung Jan, Lily Yeh Schwappach, Blanche |
| description | 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 α-COP. |
| doi_str_mv | 10.1073/pnas.051630198 |
| format | Article |
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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. 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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. 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| subjects | 3T3 cells Amino Acid Sequence Amino acids Animals Antibodies Biological Sciences Cell lines Cellular biology Combinatorial Chemistry Techniques COS cells Endoplasmic Reticulum - metabolism Flow Cytometry Fluorescent Antibody Technique Genes, Reporter Golgi Apparatus - metabolism HEK293 cells Libraries Mammals Membrane proteins Membranes Molecular Sequence Data Proteins Signal Transduction Two-Hybrid System Techniques Yeasts |
| title | Analysis of Endoplasmic Reticulum Trafficking Signals by Combinatorial Screening in Mammalian Cells |
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