A role for the basic patch and the C terminus of RanGTP in regulating the dynamic interactions with importin beta, CRM1 and RanBP1

Transport of macromolecules between the nucleus and cytoplasm involves the recognition of intrinsic localization signals by either import or export re ceptors. The interaction of the receptors with their cargo is regulated by the small GTPase Ran in its GTP bound state. We have investigated the inter action of RanGTP with the import factor, importin beta, the export factor, CRM1, and the Ran binding protein, RanBP1, in solution. Importin beta speci fically protected residues in the switch regions and basic patch region of Ran against proteolytic cleavage, whereas RanBP1 protected the C terminus. Moreover, the binding of importin beta induced a conformational change in t he structure of Ran leading to an exposure of the C terminus and stimulated the binding of RanBP1. Mutating the basic patch (HRKK142) of Ran resulted in an increased binding of RanBP1 and weakened importin beta binding. In co ntrast to wild-type Ran, the mutant Ran could be released from importin bet a independently of importin a. These data provide experimental support for a model in which the accessibility of the C terminus of Ran is influenced b y an intramolecular interaction between the basic patch and the C-terminal acidic DEDDDL216 motif. Binding of importin beta probably disrupts this int eraction causing an exposure of the C-terminal extension, which is favorabl e for RanBP1 binding. Interestingly, basic patch mutations abolish CRM1 int eraction, indicating that the determinants for RanGTP binding to the export factor, CRM1, is different from the import factor, importin beta. (C) 2001 Academic Press.