Subunits of the heterotrimeric transcription factor NF-Y are imported into the nucleus by distinct pathways involving importin beta and importin 13.
The transcriptional activator NF-Y is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC, which specifically binds the CCAAT consensus present in about 30% of eukaryotic promoters. All three subunits contain evolutionarily conserved core regions, which comprise a histone fold motif (HFM) in the case of NF-YB and NF-YC. Our results of in vitro binding studies and nuclear import assays reveal two different transport mechanisms for NF-Y subunits. While NF-YA is imported by an importin beta-mediated pathway, the NF-YB/NF-YC heterodimer is translocated into the nucleus in an importin 13-dependent manner. We define a nonclassical nuclear localization signal (ncNLS) in NF-YA, and mutational analysis indicates that positively charged amino acid residues in the ncNLS are required for nuclear targeting of NF-YA. Importin beta binding is restricted to the monomeric, uncomplexed NF-YA subunit. In contrast, the nuclear import of NF-YB and NF-YC requires dimer formation. Only the NF-YB/NF-YC dimer, but not the monomeric components, are recognized by importin 13 and are imported into the nucleus. Importin 13 competes with NF-YA for binding to the NF-YB/NF-YC dimer. Our data suggest that a distinct binding platform derived from the HFM of both subunits, NF-YB/NF-YC, mediates those interactions.
Kahle
Joerg
J
Institut für Biochemie und Molekulare Zellbiologie, Abteilung Molekularbiologie, Universität Göttingen, Humboldtallee 23, 37073 Göttingen, Germany.
Baake
Matthias
M
Doenecke
Detlef
D
Albig
Werner
W
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D023081:CCAAT-Binding Factor / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D017124:Conserved Sequence; D019281:Dimerization; D005260:Female; D005982:Glutathione Transferase / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000378:metabolism*; D008845:Microinjections; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis; D028961:beta Karyopherins / Q000378:metabolism*
D023081:CCAAT-Binding Factor; D028884:Karyopherins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C108121:TNPO1 protein, human; D028961:beta Karyopherins; D005982:Glutathione Transferase