Importin 13 Mediates Nuclear Import of Histone Fold-containing Chromatin Accessibility Complex Heterodimers

Abstract

The histone fold is a structural element that facilitates heterodimerization, and histone fold heterodimers play crucial roles in gene regulation. Here, we investigated the nuclear import of two human histone fold pairs, which belong to the H2A/H2B family: CHRAC-15/CHRAC-17 and p12/CHRAC-17. Our results from in vitro nuclear import assays with permeabilized cells and in vivo cotransfection experiments reveal that importin 13 facilitates nuclear import of both histone fold heterodimers. Using glutathione S-transferase pulldown experiments, we provide evidence that heterodimers are required for efficient binding of importin 13 because the monomers alone do not significantly interact. Mutational analysis shows that stepwise substitution of basic amino acid residues conserved among the histone fold subunits leads to a progressive loss of importin 13 binding and nuclear accumulation of CHRAC-15/CHRAC-17 and p12/CHRAC-17. The distribution of basic amino acid residues among the histone fold subunits essential for nuclear uptake suggests that heterodimerization of the histone fold motif-containing proteins forms an importin 13-specific binding platform.In eukaryotic cells, bidirectional exchange of macromolecules between the cytosolic and nuclear compartment occurs exclusively through the nuclear pore complexes (1, 2). Molecules larger than 40 kDa have to be actively transported via soluble transport receptors that bind specific amino acid residues or sequence elements within the cargo protein. Nuclear export signals are recognized by exportins (export karyopherins), whereas importins (import karyopherins) bind to nuclear localization signals (NLS) 3 that can be further differentiated in classical and nonclassical types. Proteins bearing a classical NLS are imported by a heterodimer of importin ␣ and ␤ (3, 4). Proteins with a nonclassical NLS are directly recognized by one import receptor without the help of the importin ␣ adapter molecule. In contrast to classical NLSs, the definition of a putative nonclassical NLS sequence is difficult because its length and the pattern of NLS-specific amino acids can vary strongly among proteins (5). Because of their structural flexibility, transport receptors can bind to very different signals, and some of them even function as import and export receptors. For instance, importin 13 recognizes the export substrate eIF1A and import cargoes such as hUbc9, RBM8 (6), paired type homeodomain transcriptions factors (7), the glucocorticoid receptor (8), and the actin-binding protein myopodin (9). In addition to the monomeric substrates, our recent studies have shown that importin 13 mediates nuclear import of the heterodimeric NF-YB/NF-YC complex of the transcriptional activator NF-Y (10). The subunits exhibit a histone fold motif (11, 12), the structural feature responsible for heterodimerization (13,14).In this study, we have analyzed the nuclear import of two related histone fold pairs, CHRAC-15/CHRAC-17 (CHRAC-15/17) and p12/CHRAC-17. The CHRAC-15/17 heterodimer is part of the chromat...

Nuclear import by karyopherin-βs: Recognition and inhibition

“…Pax6 (PDB 2CUE) and Arx both bind through their helical homeodomains [106,147]. Heterodimerization of histone-fold pairs NF-YB•NF-YC (transcription factors), CHRAC-15•CHRAC-17 (component of chromatin accessibility complex), CHRAC-12•CHRAC-17 (component of DNA polymerase ε) and NC2α•NC2β (transcriptional regulator) are necessary for Importin-13 recognition [148–150]. Almost the entire compact heterodimer of two α/β proteins Mago•Y14 is engulfed inside the Importin-13 ring (Figure 3) [151].…”

Structural basis for the nuclear export activity of Importin13

“…In Drosophila larvae, Imp13 affects neurotransmitter release at the neuromuscular junctions and homozygous imp13 mutations are lethal (Giagtzoglou et al , 2009). In humans, Imp13 has been involved in the import of the core exon junction complex components Mago‐Y14, the E2 SUMO‐conjugating enzyme Ubc9 (Mingot et al , 2001), histone fold‐containing and paired‐type homeodomain transcription factors (Ploski et al , 2004; Kahle et al , 2005; Walker et al , 2009), the glucocorticoid receptor (Tao et al , 2006) and the actin‐binding protein myopodin (Liang et al , 2008). The only export cargo of Imp13 known so far is the eukaryotic initiation factor 1A (eIF1A) (Mingot et al , 2001).…”

NEX‐TRAP, a Novel Method for In Vivo Analysis of Nuclear Export of Proteins

“…Exp6 (Xpo6) is responsible for nuclear export of actin, a protein that slowly leaks into the nucleus, independent of an import signal (8). Three transport factors, Exp4, Exp5 and Importin 13 (Imp13) are particularly versatile in that they transport a distinct set of cargoes either into or out of the nucleus (9)(10)(11)(12)(13)(14)(15)(16)(17). Finally, Exp5 and Exp-t recognize RNA-based export signals present in precursor microRNAs or mature tRNAs, respectively (18,19).…”