Characterization of the auto-inhibitory sequence within the N-terminal domain of importin α

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 alpha and beta. An evolutionarily conserved auto- inhibitory sequence within the N- terminal importin beta binding ( IBB) domain of importin alpha regulates NLS- cargo binding to the NLS binding pocket on importin alpha. In this study, we have used site- directed mutagenesis coupled with in vitro binding assays and in vivo analyses to investigate the intramolecular interaction of the N- terminal IBB domain and the NLS binding pocket of Saccharomyces cerevisiae importin alpha, 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 alpha 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 alpha and other factors implicated in NLS- cargo release, Cse1p and Nup2p. We propose that the N- terminal IBB domain of importin alpha and Cse1p function together in NLS- cargo release, whereas Nup2p contributes to cargo release/ importin alpha recycling through a distinct mechanism.