Subunits of the Heterotrimeric Transcription Factor NF-Y Are Imported into the Nucleus by Distinct Pathways Involving Importin β and Importin 13

Abstract

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 β-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 β 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.

ACKNOWLEDGMENTS

We gratefully thank Roberto Mantovani (Dipartimento di Biologia Animale, Università di Modena e Reggio Emilia, Modena, Italy) for providing the plasmid DNA for NF-YA, NF-YB, and NF-YC; Dirk Görlich, José-Manuel Mingot, and Stefan Jäkel (ZMBH, Heidelberg, Germany) for providing the expression plasmids for the import factors and rpL23a; Tomas Pieler and Susanne Loop (Developmental Biochemistry, University of Göttingen, Germany) for providing the plasmid DNA for myc-TFIIIA, myc-GST, Pax6, and GST-nucleoplasmin and for the opportunity to perform the oocyte injections in the Department of Developmental Biochemistry; Gideon Dreyfuss (HHMI, Philadelphia, Pa.) for providing the expression plasmid for GST-M9; Susanne Loop and Katja Horvay for preparation of oocytes and detailed introduction to oocytes injections; and Sonja Neimanis and Ralf Jauch for critical reading of the manuscript and inspiring discussions.

This work was supported by the DFG (Graduiertenkolleg 521: Protein-Protein-Interaktionen beim intrazellulären Transport von Makromolekülen) and the Fonds der Chemischen Industrie.