Plant importin alpha binds nuclear localization sequences with high affinity and can mediate nuclear import independent of importin beta.
Nuclear import of conventional nuclear localization sequence (NLS)-containing proteins initially involves recognition by the importin (IMP) alpha/beta heterodimer, where IMPalpha binds the NLS and IMPbeta targets the IMPalpha/NLS-containing protein complex to the nuclear pore. Here we examine IMPalpha from the plant Arabidopsis thaliana (At-IMPalpha), which exhibits nuclear envelope localization typical of IMPbeta rather than IMPalpha in other eukaryotic cell systems. We show that At-IMPalpha recognizes conventional NLSs of two different types with high affinity (K(d) of 5-10 nM), in contrast to mouse IMPalpha (m-IMPalpha), which exhibits much lower affinity (K(d) of 50-70 nM) and only achieves high affinity in the presence of m-IMPbeta. Unlike m-IMPalpha, At-IMPalpha is thus a high affinity NLS receptor in the absence of IMPbeta. Interestingly, At-IMPalpha was also able to bind with high affinity to NLSs recognized specifically by m-IMPbeta and not m-IMPalpha, including that of the maize transcription factor Opaque-2. Reconstitution of nuclear import in vitro indicated that in the absence of exogenous IMPbeta subunit but dependent on RanGDP and NTF2, At-IMPalpha was able to mediate nuclear accumulation to levels comparable with those mediated by m-IMPalpha/beta. Neither m-IMPalpha nor -beta was able to mediate nuclear import in the absence of the other subunit. At-IMPalpha's novel NLS recognition and nuclear transport properties imply that plants may possess an IMPalpha-mediated nuclear import pathway independent of IMPbeta in addition to that mediated by IMPalpha/beta.
Hübner
S
S
Nuclear Signalling Laboratory, Division for Biochemistry and Molecular Biology, John Curtin School of Medical Research, Canberra ACT 2601, Australia.
Smith
H M
HM
Hu
W
W
Chan
C K
CK
Rihs
H P
HP
Paschal
B M
BM
Raikhel
N V
NV
Jans
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D028884:Karyopherins; D051379:Mice; D008969:Molecular Sequence Data; D019913:Nuclear Localization Signals / Q000502:physiology*; D009687:Nuclear Proteins / Q000378:metabolism*; D029543:Nucleocytoplasmic Transport Proteins; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D020931:ran GTP-Binding Protein
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D028884:Karyopherins; D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; C503613:Nutf2 protein, mouse; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C067402:opaque-2 protein, Zea mays; D019204:GTP-Binding Proteins; D020931:ran GTP-Binding Protein