Characterization of the auto-inhibitory sequence within the N-terminal domain of importin alpha.
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.
Harreman
Michelle T
MT
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road NE, Atlanta, GA 30322, USA.
Cohen
Pamela E
PE
Hodel
Mary R
MR
Truscott
Glyn J
GJ
Corbett
Anita H
AH
Hodel
Alec E
AE
D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D015151:Immunoblotting; D028884:Karyopherins / Q000737:chemistry; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature
D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C491115:TIR1 protein, S cerevisiae; D049452:Green Fluorescent Proteins