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 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.
Howard Hughes Medical Institute, Department of Physiology, University of California, San Francisco, CA 94143-0725, USA.
N
N
Zerangue
M J
MJ
Malan
S R
SR
Fried
P F
PF
Dazin
Y N
YN
Jan
L Y
LY
Jan
B
B
Schwappach
D000595:Amino Acid Sequence; D000818:Animals; D020650:Combinatorial Chemistry Techniques; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000378:metabolism; D008969:Molecular Sequence Data; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques