Quantitative analysis of nuclear localization signal (NLS)-importin alpha interaction through fluorescence depolarization. Evidence for auto-inhibitory regulation of NLS binding.
We have developed a quantitative in vitro steady-state fluorescence depolarization assay to measure the interaction of a nuclear localization signal (NLS) substrate with its receptors. This assay relies on the change in fluorescence depolarization of an NLS fused to the green fluorescent protein upon binding to receptor. No binding is observed in the absence of a functional NLS, and binding affinities measured correlate with previous in vivo studies of NLS function. We have used this assay to test an auto-inhibitory model for the interaction of an NLS with the NLS receptor complex. This model suggests that NLS binding to importin alpha is modulated by an auto-inhibitory sequence within the N terminus of importin alpha, which is displaced by importin beta binding. Consistent with this model, NLS substrates bind tightly to an N-terminally truncated importin alpha lacking the auto-inhibitory domain (K(d) approximately 10 nm), but measurable binding to full-length importin alpha is only observed upon addition of importin beta. Our quantitative results support the auto-inhibitory model and suggest a mechanism for a switch between a cytoplasmic, high affinity and a nuclear, low affinity NLS receptor. This predicted mode of interaction would facilitate binding of substrate in the cytoplasm and its subsequent release into the nucleus.
Fanara
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Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Hodel
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Corbett
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Hodel
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D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D005454:Fluorescence Polarization / Q000379:methods; D049452:Green Fluorescent Proteins; D028884:Karyopherins; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000502:physiology*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013816:Thermodynamics
D028884:Karyopherins; D008164:Luminescent Proteins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins