Functional analysis of the hydrophobic patch on nuclear transport factor 2involved in interactions with the nuclear pore in vivo

Nuclear transport factor 2 (NTF2) is a small homodimeric protein that inter acts simultaneously with both RanGDP and FxFG nucleoporins. The interaction between NTF2 and Ran is essential for the import of Ran into the nucleus. Here we use mutational analysis to dissect the in vivo role of the interact ion between NTF2 and nucleoporins. We identify a series of surface residues that form a hydrophobic patch on NTF2, which when mutated disrupt the NTF2 -nucleoporin interaction. Analysis of these mutants in vivo demonstrates th at the strength of this interaction can be significantly reduced without af fecting cell viability. However, cells cease to be viable if the interactio n between NTF2 and nucleoporins is abolished completely, indicating that th is interaction is essential for the function of NTF2 in vivo. In addition, we have isolated a dominant negative mutant of NTF2, N77Y, which has increa sed affinity for nucleoporins. Overexpression of the N77Y protein blocks nu clear protein import and concentrates Ran at the nuclear rim. These data su pport a mechanism in which NTF2 interacts transiently with FxFG nucleoporin s to translocate through the pore and import RanGDP into the nucleus.