title: Functional characterization of importin α8 as a classical nuclear localization signal receptor.
abstract: Importin α8 has recently been identified as an importin α family member based on its primary structure and binding ability to importin β1 and to several karyophilic proteins. However, there has been no experimental evidence that importin α8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin α8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin β1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin α8-binding proteins, only 62 of which overlap with those of importin α1, the closest importin α family member. Among the importin α8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin α8/β1 pathway. Furthermore, we found that the other subtypes of importin α, which were also identified as importin α8-binding candidates, indeed form heterodimers with importin α8. Notably, we found that these importin α8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin α1. From these findings, we propose that importin α8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin α8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin β binding domain.
authors.affiliation: Biomolecular Dynamics Laboratory, Department of Frontier Bioscience, Graduate School of Frontier Bioscience, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan; Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Kimoto
authors.affiliation: Biomolecular Dynamics Laboratory, Department of Frontier Bioscience, Graduate School of Frontier Bioscience, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan; Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Moriyama
authors.affiliation: Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Tsujii
authors.affiliation: Laboratory of Toxicogenomics Informatics, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan.
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Igarashi
authors.affiliation: Graduate School of Life Science Hokkaido University, Kita-21, Nishi-11, Sapporo, Hokkaido 001-0021, Japan.
authors.forename: Chikashi
authors.initials: C
authors.lastname: Obuse
authors.affiliation: Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Electronic address: ymiyamoto@nibiohn.go.jp.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Miyamoto
authors.affiliation: Laboratory of Nuclear Transport Dynamics, National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan; Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address: moka@nibiohn.go.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Oka
authors.affiliation: Biomolecular Dynamics Laboratory, Department of Frontier Bioscience, Graduate School of Frontier Bioscience, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan; Laboratory of Biomedical Innovation, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan; National Institutes of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan. Electronic address: y-yoneda@nibiohn.go.jp.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Yoneda
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015398:Signal Transduction / Q000502:physiology*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000952:Antigens, Polyomavirus Transforming; C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; C107282:IPO8 protein, human; C424166:KPNB1 protein, human; D028961:beta Karyopherins
keywords: DDB2; Dimer; Importin α; KPNA; Mass spectrometry; Nuclear localization signal