Engineered mutants in the switch II loop of ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import

Nuclear protein import requires a precisely choreographed series of interac tions between nuclear pore components and soluble factors such as importin- beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal struc ture of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction . X-ray crystallography showed that the F72Y, F72W and R76E mutations did n ot introduce major structural changes into the mutant Ran. The GDP-bound fo rm of the switch ii mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Ph e72 are all crucial for the interaction between Ran and NTF2. Nuclear prote in accumulation in digitonin-permeabilized cells was impaired with Ran muta nts deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologu e Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. A lthough neither mutant was viable when integrated into the genome as a sing le copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72 Y on a centromeric plasmid were viable, confirming that this mutant retaine d the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, al though strains overexpressing the mutant to higher levels using an episomal 2 mu m plasmid were viable, indicating that the R76E mutation may also hav e interfered with other interactions made by Gsp1p. (C) 1999 Academic Press .