The N-terminal nuclear export sequence of IκBα is required for RanGTP-dependent binding to CRM1

Nuclear export of I kappaB alpha is mediated by the CRM1 nuclear export receptor. However, the identity of the nuclear export sequences NES(s) in I kappaB alpha that are responsible for binding of I kappaB alpha 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 I kappaB alpha. 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 I kappaB alpha to CRM1. I kappaB alpha 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 I kappaB alpha functions as a physical adaptor between CRM1 and NF kappaB/Rel proteins. Both free I kappaB alpha and Rel-associated I kappaB alpha have comparable affinities for CRM1, suggesting that CRM1 does not discriminate between free I kappaB alpha and Rel-associated I kappaB alpha. Nuclear export of c-Rel by I kappaB alpha requires the N-terminal NES-like sequence of I kappaB alpha but is not affected by alanine substitutions within the C-terminal NES-like sequence of I kappaB alpha. In contrast, nuclear export of the v-Rel oncoprotein by I kappaB alpha 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 I kappaB alpha for v-Rel, suggesting that loss of export function for this mutant is secondary to reduced association between I kappaB alpha and v-Rel, Taken together, our results demonstrate that the N-terminal NES-like sequence in I kappaB alpha is required for RanGTP-dependent binding of both free I kappaB alpha and NF kappaB/Rel-associated I kappaB alpha proteins to CRM1.