A systems analysis of importin-α–β mediated nuclear protein import

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Abstract
Importin-β (Impβ) is a major transport receptor for Ran-dependent import of nuclear cargo. Impβ can bind cargo directly or through an adaptor such as Importin-α (Impα). Factors involved in nuclear transport have been well studied, but systems analysis can offer further insight into regulatory mechanisms. We used computer simulation and real-time assays in intact cells to examine Impα–β-mediated import. The model reflects experimentally determined rates for cargo import and correctly predicts that import is limited principally by Impα and Ran, but is also sensitive to NTF2. The model predicts that CAS is not limiting for the initial rate of cargo import and, surprisingly, that increased concentrations of Impβ and the exchange factor, RCC1, actually inhibit rather than stimulate import. These unexpected predictions were all validated experimentally. The model revealed that inhibition by RCC1 is caused by sequestration of nuclear Ran. Inhibition by Impβ results from depletion nuclear RanGTP, and, in support of this mechanism, expression of mRFP-Ran reversed the inhibition.