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 ␤-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 ␤ 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.The existence of a cell nucleus in eukaryotes implies the spatial separation of transcription and translation and therefore requires bidirectional intracellular trafficking of macromolecules. The site of exchange is the nuclear pore complex (NPC), one of the largest macromolecular assemblies in a eukaryotic cell, which can be traversed in a passive or a facilitated manner. The passive mode applies for small molecules but becomes ineffective for proteins with a molecular mass greater than 40 kDa. In addition, molecules which may potentially passively diffuse are often actively translocated, since this allows a more efficient and regulated transport (for reviews, see references 17, 25, 47, 64, and 77).Most nuclear transport processes are mediated by soluble transport receptors that recognize specific sequences or structural characteristics of their cargoes and facilitate the passage of receptor-cargo complexes through the NPC. Transport receptors constantly shuttle between the nucleus and cytoplasm, thereby rapidly crossing the permeability barrier of nuclear pores (59). The largest class of nuclear transport receptors is the superfamily of importin ␤-like factors (also named karyopherins) that can be classified as importins (import karyopherin) and exportins (export karyopherin) depending on the direction in which they transport the cargo (reviewed in references 25, 32, 43, 72, and 80). Cargo binding and release of importins and exportins is controlled by a steep RanGTP gradient, which is maintained across the nuclear envelope through the asymmetric distribution of factors that regulate the guanine nucleotide-bound state of Ran (25,41,43,47,76). The exchange factor, RanGEF (also called RCC1), is exclusively nuclear, while the GTPase-ac...

Regulation of Nuclear Import and Export of Negative Cofactor 2

“…Additionally, the affinity of importin 13 to the NC2 complex might be low in vivo due to phosphorylation of the NC2 subunits (3). As recently described for its closest relative, the NF-YB/NF-YC histone-fold heterodimer, only the dimerized NC2 subunits were recognized by importin 13 (52). Hence, despite their functional divergence these two histone-fold heterodimers rely on the same import mechanism to translocate through the NPC.…”

“…In that way, the NC2 complex behaves like its closest relative, the NF-YB/NF-YC histone-fold heterodimer. Only the heterodimerized NF-YB/NF-YC subunits were specifically recognized by importin 13, as shown previously (52). Because heterodimerization of both NC2␣ with NC2␤ and NF-YB with NF-YC is a prerequisite for importin 13 binding, the minimal sequence elements recognized by importin 13 comprise the histone-fold domains of both NC2 subunits.…”

Nuclear Factor-Y and Epstein Barr Virus in Nasopharyngeal Cancer

“…which normally resides in the cytoplasm, rapidly translocates to the nucleus through a dual mechanism where NF-YA is transported in an importin h-dependent pathway, whereas the NF-YB and NF-YC subunits are translocated through a distinct pathway involving importin 13 (44). Our investigation of all three NF-Y subunits in response to cisplatin and radiation showed an increase in NF-YA and NF-YB transcript levels beginning at 8 h reaching maximal levels 48 h posttreatment (Fig.…”

“…7) where, upon stress treatment with cisplatin and radiation, NF-Y localizes to the nucleus (44) and transcriptionally activates the expression of the EBV lytic genes BRLF1 and BZLF1 within 30 min. NF-Y activation of BRLF1 may be mediated by the physical interaction of NF-Y with one or a combination of the three consensus sequences within the distal promoter region.…”

NF-YC Functions as a Corepressor of Agonist-bound Mineralocorticoid Receptor

“…The presence of histone fold motifs in the NF-YB and NF-YC implicates these proteins as being likely to have multiple protein-protein interactions with other transcription factors as well as being given protein modifications. The NF-YA and NF-YB/NF-YC heterodimer were shown to be separately imported to the nucleus through Importins ␤ and 13, respectively (36). However, our coimmunoprecipitation and ChIP assays showed that NF-YC alone, not a complex of NF-YA, NF-YB, and NF-YC, is recruited to a MR-responsive ENaC gene promoter, thus resulting in MR-mediated transcriptional repression.…”