Molecular interactions between the importin alpha/beta heterodimer and proteins involved in vertebrate nuclear protein import.

We have used in vitro binding assays to examine specific interactions between a number of cytoplasmic and nuclear pore proteins involved in nuclear protein import in vertebrates. We demonstrate that nuclear transport factor 2 (NTF2), nucleoporin p62 and the Ras-like GTPase Ran bind to the importin heterodimer via its beta subunit. The binding behaviour of p62 truncation mutants indicated that importin-beta interacts primarily with the alpha-helical coiled-coil rod domain of nucleoporin p62 and not with the N-terminal domain that contains a number of degenerate repeats based on the xFxFG sequence motif. The binding of Ran to importin-beta was sensitive to its nucleotide state, with RanGTP binding strongly, whereas RanGDP binding could not be detected using our assay conditions. RanGTP, but not RanGDP, was able to displace p62 bound to the importin alpha/beta complex, suggesting that the binding sites for p62 and RanGTP on importin-beta overlap. Moreover, RanGTP, but not RanGDP, weakened the interaction between importin-alpha and importin-beta in a concentration-dependent manner. NTF2 bound to the importin heterodimer but did not displace p62, suggesting that the NTF2 and p62 binding sites on importin-beta do not overlap. The set of interactions we observed was not altered by the binding of NLS-containing substrates such as transcription factor IIIA to the importin heterodimer. Our results are consistent with models for nuclear protein import in which Ran nucleotide exchange modulates the binding of the importin-substrate complexes during translocation through nuclear pore complexes.