Functional characterization of a plant importin alpha homologue. Nuclear localization signal (NLS)-selective binding and mediation of nuclear import of nls proteins in vitro.
Nuclear import of most nuclear proteins is initiated by recognition of the nuclear localization signal (NLS) by importin alpha. We recently isolated an importin alpha homologue from rice (rice importin alpha1) and demonstrated that transcription of the gene is down-regulated by light in rice leaves. To address the function of rice importin alpha1 in the process of nuclear import of proteins, we performed in vitro binding and nuclear import assays. The rice importin alpha1 showed specific binding to fusion proteins containing either monopartite or bipartite NLSs, but not to a fusion protein containing a Matalpha-2-type NLS, suggesting that there exists selective binding of rice importin alpha1 to different plant NLSs. The rice importin alpha1 is also capable of forming a complex with mouse importin beta and NLS protein in vitro. An in vitro nuclear import assay using permeabilized HeLa cells revealed that rice importin alpha1, in conjunction with other vertebrate transport factors, mediates the nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data provide strong, direct evidence suggesting that rice importin alpha1 functions as a component of the NLS receptor in plant cells.
Jiang
C J
CJ
National Institute of Agrobiological Resources, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
Imamoto
N
N
Matsuki
R
R
Yoneda
Y
Y
Yamamoto
N
N
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D012275:Oryza / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism
D017931:DNA Primers; D028884:Karyopherins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins