Heterodimerization as a prerequisite for nuclear access

Importin 13 was identified by Mingot and colleagues (2001) as a member of the importin β-like transport receptor family mediating the import and surprisingly also the export of several different proteins. The human SUMO-conjugating enzyme UBC9 and the RNA-binding motif protein 8 (RBM8; also referred to as Y14) were identified among the import cargoes, but importin 13 also accounts for nuclear import of the paired-type homeodomain transcriptions factors Pax6, Pax3, and Crx (Ploski et al., 2004). As export cargo, the translation initiation factor eIF1A was identified (Mingot et al., 2001). These cargoes are all monomeric proteins. In addition to these data, work in our lab revealed that the importin 13 mediated nuclear import of

the H2A/H2B family member NF-YB/NF-YC depends on complex formation (Kahle et al., 2005). A similar behaviour has been recently shown for another histone fold pair of the H2A/H2B-type, namely NC2α/NC2β (Kahle et al, 2009). In this case, binding of the complex to importin 13 was also strictly dependent on subunit dimerization, thus showing the same binding properties as the NF-YB/NF-YC complex. With the new data from this work, we have expanded the spectrum of importin 13 recognized heterodimers of the H2A/H2B family by two novel heterodimeric cargoes, namely CHRAC-15/CHRAC-17 and p12/CHRAC-17.

Binding of both histone fold heterodimers to importin 13 was identified by performing in vitro GST-Pulldown assays with immobilized CHRAC-15/17 and p12/CHRAC-17, respectively. The specific loss of binding capacity between the nuclear transport factor importin 13 and the histone fold heterodimers in the presence of RanGTP pointed towards importin 13 as a specific nuclear import factor for CHRAC-15/17 and p12/CHRAC-17. Hence, within the cell importin 13 binds to CHRAC-15/17 or p12/CHRAC-17 at low RanGTP concentration in the cytoplasm. The import complex is then transferred into the nucleus and the dimeric cargo finally dissociates from importin 13 in the nucleus via RanGTP, which is present at high concentrations. The results from the GST-Pulldown assays also ruled out that importin 13 may function as an export factor as shown for eIF1A (Mingot et al., 2001). However, the fact that at least CHRAC-17 is recognized by importin 13 is not new. Mingot and colleagues (2001) already showed that CHRAC-17, referred to as NF-YB-like protein, binds to importin 13 in a RanGTP dependent manner. In the study by Mingot and colleagues, importin 13 was immobilized and a cytosolic HeLa cell extract was used to identify importin 13 interacting proteins. However, it remains unclear if CHRAC-17 was bound to importin 13 as a monomer or complexed with another protein, since only selected binding proteins were analyzed by mass spectrometry. However, our data concerning the monomeric subunits demonstrate that regardless of the absence or presence of RanGTP neither CHRAC-15 nor CHRAC-17 specifically binds to importin 13. Hence, only the CHRAC-15/17 heterodimer but not the monomeric components are recognized by importin 13.

This type of cooperativity between the individual histone fold subunits for nuclear import was also observed in situ for the nuclear accumulation of the p12 and CHRAC-17 homologues Dpb3 and Dpb4 in the fission yeast Schizosaccharomyces pombe. In that case, the repression of Dpb3 expression abrogated nuclear localization of Dpb4 (Spiga and D'Urso, 2004). These results support our data that the monomeric subunits are only imported in the nucleus when complexed with their histone fold partner.

In line with our interaction data, recombinant importin 13 was also able to mediate nuclear uptake of CHRAC-15/17 in vitro. In the in vitro import assays, described by Adam et al. (1990),

digitonin permeabilized cells were used to reconstitute nuclear import processes by substituting endogenous cytosolic components. The addition of importin 13 to the recombinant CHRAC-15/17 heterodimer led to a nearly 100 % nuclear localization of the histone fold pair.

This nuclear localization was analogous to that of the endogenous CHRAC-15 and CHRAC-17 subunits. In addition, by using antibodies against the GST-tag fused to CHRAC-15 for detection, but the His-tag of CHRAC-17 for purification of the complex, we excluded the possibility of detecting monomeric subunits. We therefore conclude that the nuclear uptake of CHRAC-15/17 is energy-dependent and facilitated by importin 13.

The prerequisite of heterodimerization for nuclear uptake of the CHRAC-15/17 complex was further confirmed by the results of in vivo transfection experiments. The subcellular localization of monomeric CHRAC-15 and CHRAC-17 when fused to either RFP or EGFP-EGFP-GST (EEG) was strictly cytoplasmic. In contrast, EGFP-fused CHRAC-15 was localized homogeneously within the cell whereas EGFP-CHRAC-17 fusion protein was localized exclusively in the cytoplasm. The homogeneous localization of EGFP-CHRAC-15 may be explained by the tendency of EGFP to diffuse passively into the nucleus. Seibel et al. (2007) showed that even EGFP homohexamers diffuse into the nucleus of different mammalian cell lines. In addition to Seibel and coworkers, we show that RFP protein also tends to diffuse passively into the nucleus of HeLa P4 cells. However, these in vivo results implied that neither CHRAC-15 nor CHRAC-17 contain a NLS. Alternatively, the cytoplasmic retention of histone fold subunits could have also resulted from the masking of a NLS as shown by Wagstaff and Jans (2006). Therefore, we analyzed whether nuclear import of EEG alone or EEG fused to CHRAC-17 occurs in the presence of a non-classical or classical NLS. Thus, EEG was fused to the ribosomal protein L23a or was inserted between the bipartite cNLS of Nucleoplasmin and CHRAC-17.

The non-classical NLS in L23a, namely beta-like importin receptor binding (BIB) domain, consists of a highly basic stretch of 43 amino acids. It has been shown that L23a as well as the ribosomal proteins S7 and L5, are recognized by at least importin β, importin 5, importin 7, and transportin (Jäkel and Görlich, 1998). Fusions of the BIB alone (data not shown) or full length L23a to EEG therefore consequently result in a strict localization of the fusion proteins in the nucleoli, resembling the localization of ribosomal proteins. The NLS in Nucleoplasmin represents the prototype of a bipartite classical NLS with two basic clusters, separated by a stretch of 10-12 amino acids. Fusion of the bipartite cNLS between EEG and CHRAC-17 resulted also in a nuclear accumulation of the EEG-cNLS-CHRAC-17 fusion protein. Therefore, it was reasonable to assume that (i) neither the fluorescent EEG nor probably EGFP or RFP have

a significant influence on the interaction between a putative NLS in the monomeric CHRAC subunits and importin α/importin β or other transport receptors, (ii) both subunits, CHRAC-15 and CHRAC-17, do not possess a NLS for individual nuclear transport, and (iii) nuclear uptake therefore depends on the heterodimerization of the histone fold subunits.