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 depolar ization assay to measure the interaction of a nuclear localization signal ( NLS) substrate with its receptors, This assay relies on the change in fluor escence depolarization of an NLS fused to the green fluorescent protein upo n binding to receptor. No binding is observed in the absence of a functiona l NLS, and binding affinities measured correlate with previous in vivo stud ies of NLS function. We have used this assay to test an auto-inhibitory mod el 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-inhibit ory sequence within the N terminus of importin alpha, which is displaced by importin beta binding. Consistent with this model, NLS substrates bind tig htly to an N-terminally truncated importin alpha lacking the autoinhibitory domain (K-d similar to 10 nM), but measurable binding to full-length impor tin alpha is only observed upon addition of importin beta, Our quantitative results support the autoinhibitory model and suggest a mechanism for a swi tch between a cytoplasmic, high affinity and a nuclear, low affinity NLS re ceptor. This predicted mode of interaction would facilitate binding of subs trate in the cytoplasm and its subsequent release into the nucleus.