Protein cargoes that contain a classic nuclear localization signal (NLS) are transported into the nucleus through binding to a heterodimeric receptor comprised of importin/karyopherin ␣ and ␤. An evolutionarily conserved auto-inhibitory sequence within the N-terminal importin ␤ binding (IBB) domain of importin ␣ regulates NLS-cargo binding to the NLS binding pocket on importin ␣. In this study, we have used site-directed mutagenesis coupled with in vitro binding assays and in vivo analyses to

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... vestigate the intramolecular interaction of the N-terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin ␣, Srp1p. We find that mutations within the IBB domain that decrease the binding affinity of the auto-inhibitory sequence for the NLS binding pocket impact importin ␣ function in vivo. In addition, the severity of the in vivo phenotype is directly correlated to the reduction of auto-inhibition measured in vitro, suggesting that the in vivo phenotypes are directly related to the loss of auto-inhibitory function. We exploit a conditional auto-inhibitory mutant, srp1-55, to study the in vivo functional overlap between the N-terminal IBB domain of importin ␣ and other factors implicated in NLS-cargo release, Cse1p and Nup2p. We propose that the N-terminal IBB domain of importin ␣ and Cse1p function together in NLS-cargo release, whereas Nup2p contributes to cargo release/ importin ␣ recycling through a distinct mechanism.