Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells
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Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells. / Zerangue, N; Malan, M J; Fried, S R; Dazin, P F; Jan, Y N; Jan, L Y; Schwappach, B.
In: P NATL ACAD SCI USA, Vol. 98, No. 5, 27.02.2001, p. 2431-6.Research output: SCORING: Contribution to journal › SCORING: Journal article › Research › peer-review
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TY - JOUR
T1 - Analysis of endoplasmic reticulum trafficking signals by combinatorial screening in mammalian cells
AU - Zerangue, N
AU - Malan, M J
AU - Fried, S R
AU - Dazin, P F
AU - Jan, Y N
AU - Jan, L Y
AU - Schwappach, B
PY - 2001/2/27
Y1 - 2001/2/27
N2 - 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.
AB - 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.
KW - Amino Acid Sequence
KW - Animals
KW - Combinatorial Chemistry Techniques
KW - Endoplasmic Reticulum/metabolism
KW - Flow Cytometry
KW - Fluorescent Antibody Technique
KW - Genes, Reporter
KW - Golgi Apparatus/metabolism
KW - Molecular Sequence Data
KW - Signal Transduction
KW - Two-Hybrid System Techniques
U2 - 10.1073/pnas.051630198
DO - 10.1073/pnas.051630198
M3 - SCORING: Journal article
C2 - 11226256
VL - 98
SP - 2431
EP - 2436
JO - P NATL ACAD SCI USA
JF - P NATL ACAD SCI USA
SN - 0027-8424
IS - 5
ER -