Determination of the Functional Domain Organization of the Importin α Nuclear Import Factor

Abstract

Although importin α (Imp α) has been shown to act as the receptor for basic nuclear localization signals (NLSs) and to mediate their recruitment to the importin β nuclear import factor, little is known about the functional domains present in Imp α, with the exception that importin β binding is known to map close to the Imp α NH₂ terminus. Here, we demonstrate that sequences essential for binding to the CAS nuclear export factor are located near the Imp α COOH terminus and include a critical acidic motif. Although point mutations introduced into this acidic motif inactivated both CAS binding and Imp α nuclear export, a putative leucine-rich nuclear export signal proved to be neither necessary nor sufficient for Imp α nuclear export. Analysis of sequences within Imp α that bind to the SV-40 T antigen NLS or to the similar LEF-1 NLS revealed that both NLSs interact with a subset of the eight degenerate armadillo (Arm) repeats that form the central part of Imp α. However, these two NLS-binding sites showed only minimal overlap, thus suggesting that the degeneracy of the Arm repeat region of Imp α may serve to facilitate binding to similar but nonidentical basic NLSs. Importantly, the SV-40 T NLS proved able to specifically inhibit the interaction of Imp α with CAS in vitro, thus explaining why the SV-40 T NLS is unable to also function as a nuclear export signal.