A high-throughput screening of genes that encode proteins transported into the endoplasmic reticulum in mammalian cells.
The compartments of eukaryotic cells maintain a distinct protein composition to perform a variety of specialized functions. We developed a new method for identifying the proteins that are transported to the endoplasmic reticulum (ER) in living mammalian cells. The principle is based on the reconstitution of two split fragments of enhanced green fluorescent protein (EGFP) by protein splicing with DnaE from Synechocystis PCC6803. Complementary DNA (cDNA) libraries fused to the N-terminal halves of DnaE and EGFP are introduced in mammalian cells with retroviruses. If an expressed protein is transported into the ER, the N-terminal half of EGFP meets its C-terminal half in the ER, and full-length EGFP is reconstituted by protein splicing. The fluorescent cells are isolated using fluorescence-activated cell sorting and the cDNAs are sequenced. The developed method was able to accurately identify cDNAs that encode proteins transported to the ER. We identified 27 novel proteins as the ER-targeting proteins. The present method overcomes the limitation of the previous GFP- or epitope-tagged methods, using which it was difficult to identify the ER-targeting proteins in a high-throughput manner.
Ozawa
Takeaki
T
Department of Chemistry, School of Science, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Nishitani
Kengo
K
Sako
Yusuke
Y
Umezawa
Yoshio
Y
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D021382:Protein Sorting Signals; D019154:Protein Splicing; D021381:Protein Transport; D011506:Proteins / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D005456:Fluorescent Dyes; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
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