The N-terminal Nuclear Export Sequence of IκBα Is Required for RanGTP-dependent Binding to CRM1

Abstract

Nuclear export of IκBα is mediated by the CRM1 nuclear export receptor. However, the identity of the nuclear export sequences NES(s) in IκBα that are responsible for binding of IκBα to CRM1 is controversial. Both a N-terminal NES-like region (amino acids 45-54) and a C-terminal NES-like region (amino acids 265-280) have, in a number of reports from different laboratories, been implicated in CRM1-dependent nuclear export of IκBα. We now demonstrate that the N-terminal NES-like region, but not the C-terminal NES-like region, is required for RanGTP-dependent binding of IκBα to CRM1. IκBα is a relatively weak substrate for CRM1, with an affinity for CRM1 that is 100-fold less than the minute virus of mice NS2 protein, a high affinity cargo protein for CRM1. We also demonstrate that IκBα functions as a physical adaptor between CRM1 and NFκB/Rel proteins. Both free IκBα and Rel-associated IκBα have comparable affinities for CRM1, suggesting that CRM1 does not discriminate between free IκBα and Rel-associated IκBα. Nuclear export of c-Rel by IκBα requires the N-terminal NES-like sequence of IκBα but is not affected by alanine substitutions within the C-terminal NES-like sequence of IκBα. In contrast, nuclear export of the v-Rel oncoprotein by IκBα is disrupted by alanine substitutions within either the N-terminal or the C-terminal NES-like sequences. However, alanine substitutions within the C-terminal NES-like sequence significantly reduce the affinity of IκBα for v-Rel, suggesting that loss of export function for this mutant is secondary to reduced association between IκBα and v-Rel. Taken together, our results demonstrate that the N-terminal NES-like sequence in IκBα is required for RanGTP-dependent binding of both free IκBα and NFκB/Rel-associated IκBα proteins to CRM1.