title: Six classes of nuclear localization signals specific to different binding grooves of importin alpha.
abstract: The importin alpha/beta pathway mediates nuclear import of proteins containing the classical nuclear localization signals (NLSs). Although the consensus sequences of the classical NLSs have been defined, there are still many NLSs that do not match the consensus rule and many nonfunctional sequences that match the consensus. We report here six different NLS classes that specifically bind to distinct binding pockets of importin alpha. By screening of random peptide libraries using an mRNA display, we selected peptides bound by importin alpha and identified six classes of NLSs, including three novel classes. Two noncanonical classes (class 3 and class 4) specifically bound the minor binding pocket of importin alpha, whereas the classical monopartite NLSs (class 1 and class 2) bound to the major binding pocket. Using a newly developed universal green fluorescent protein expression system, we found that these NLS classes, including plant-specific class 5 NLSs and bipartite NLSs, fundamentally require the regions outside the core basic residues for their activity and have specific residues or patterns that confer the activities differently between yeast, plants, and mammals. Furthermore, amino acid replacement analyses revealed that the consensus basic patterns of the classical NLSs are not essential for activity, thereby generating more unconventional patterns, including redox-sensitive NLSs. These results explain the causes of the NLS diversity. The defined consensus patterns and properties of importin alpha-dependent NLSs provide useful information for identifying NLSs.
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan; Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Kosugi
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Masako
authors.initials: M
authors.lastname: Hasebe
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Matsumura
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Takashima
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Etsuko
authors.initials: E
authors.lastname: Miyamoto-Sato
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Tomita
authors.affiliation: Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan; Institute for Advanced Biosciences, Keio University, Tsuruoka 997-0017, Japan and the Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan. Electronic address: hyana@bio.keio.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yanagawa
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D019913:Nuclear Localization Signals / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: D019913:Nuclear Localization Signals; D010940:Plant Proteins; D029701:Saccharomyces cerevisiae Proteins; D028901:alpha Karyopherins