Analysis of nuclear localization signals using a green fluorescent protein-fusion protein library.
We describe here an efficient method for identifying intracellular localization signals in proteins with stereospecific intracellular localizations in culture cells. The method involves rapid fluorescence screening of cells transfected with a cDNA library in which cDNAs are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP). We analyzed nuclear localization and nuclear localization signals (NLSs) in a model application of this method. As a result, we identified classical NLSs in 75% of nuclear localized proteins. We identified some novel NLS candidates among the classical NLS-negative sequences whose nuclear localization was also identified in another cell line and with other molecular tag sequences. This method will be useful for identifying intracellular localization signals and for more detailed analysis of intracellular architecture.
Fujii
G
G
ERATO, JST, Ibaraki, Tsukuba, 300-2635, Japan.
Tsuchiya
R
R
Ezoe
E
E
Hirohashi
S
S
D000595:Amino Acid Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D015723:Gene Library; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008099:Liver / Q000166:cytology; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000235:genetics / Q000302:isolation & purification*; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012641:Selection, Genetic; D001616:beta-Galactosidase / Q000235:genetics
D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C085706:FLAG peptide; D001616:beta-Galactosidase