Full length importin 13 is required to mediate efficient nuclear import of the

After verifying that importin 13 represents a functional import receptor for the CHRAC-15/17 complex in vivo, we next wanted to characterize the binding sites of the heterodimer in importin 13. For that purpose, different deletion constructs of importin 13 were generated and tested for their import capacity for CHRAC-15/17. Independent of the HEAT repeat prediction for importin 13 in Swiss-Prot (http://www.uniprot.org/uniprot/O94829), putative α-helices within the sequence of importin 13 were identified using the secondary structure prediction program PSIPRED (Jones, 1999; McGuffin et al., 2000). According to PSIPRED, importin 13 contains 38 α-helices. This is in line with the assumption that all transport receptors of the importin β-like family are comprised of 19 HEAT repeats (Petosa et al., 2004). Based on this prediction, we selectively deleted α-helices in importin 13 by using specific oligonucleotides (Fig. 13A). The resulting truncated flag-tagged importin 13 constructs (Fig. 13C) were coexpressed with EGFP-CHRAC-15 and RFP-CHRAC-17 in HeLa P4 cells. Among them, amino acids 1-784 and

FIG. 12: continued

flag-tagged PGC7/Stella. In addition, importin 5 and importin 13 were cotransfected as indicated 24 hours post transfection. The subcellular localization of flag-PGC7/Stella was determined using specific anti-PGC7/Stella antibody. The DNA was counterstained with Hoechst. Flag-PGC7/Stella localizes homogenously within transfected cells and accumulates predominantly in the nucleus upon importin 5 coexpression. Coexpression of flag-tagged importin 13 had no effect. The localization of the PGC7/Stella fusion protein was additionally quantified by measuring the fluorescence intensity in 20 cells, followed by a calculation of the ratio between nuclear (nucl.) and cytoplasmic (cyto.) distribution. (D) HeLa P4 cells were additionally transfected with EGFP-PGC7/Stella. The localization of the green fluorescent fusion protein was determined 24 hours post transfection. Strong overexpression of EGFP-tagged PGC7/Stella led to a cytoplasmic localization, which changed to a mainly nuclear localization when importin 5 was coexpressed. Coexpression of importin 13 had no influence on the subcellular distribution of PGC7/Stella. The percentage of cytoplasmic (cyto.) and nuclear (nucl.) localization of EGFP-PGC7/Stella was quantitatively analyzed using the ImageJ software. WB, Western blot; imp, importin.

FIG. 13: Full length importin 13 is required for efficient nuclear accumulation of the CHRAC-15/17 heterodimer. (A) Importin 13 presumably consists of 19 HEAT repeats. According to the prediction of α-helices by PSIPRED, HEAT repeats were selectively deleted. (B) Plasmid DNA encoding EGFP-CHRAC-15 and RFP-CHRAC-17 was cotransfected in HeLa P4 cells. Different flag-tagged importin 13 fragments were additionally coexpressed as indicated. The subcellular distribution of the EGFP-CHRAC-15/RFP-CHRAC-17 complex was determined 24 hours post transfection and is shown in yellow (merge). The addition of full length importin 13 (1-963 amino acids) results in a strict nuclear accumulation (see also Fig. 10A). In the absence of exogenous importin 13 (w/o) the CHRAC-15/17 heterodimer localizes mainly in the cytoplasm. (continued on page 82)

0 10 20 30 40 50 60 70 80 90 100

w/o 1-963

1-784 1-669

1-410 45-963

153-963 153-784

153-669 251-963

251-784 251-669

flag-importin 13 constructs

cotransfected cells

N>C N=C N<C

45-963 were still capable in facilitating import of the CHRAC-15/17 complex in the nucleus of transfected cells although to a lower degree than wild type importin 13 (Fig. 13B). All other importin 13 fragments were not able to facilitate nuclear import of the CHRAC-15/17 complex.

A quantitative analysis of the import is shown in Figure 13D. The same results were obtained when RFP-CHRAC-15 and EGFP-CHRAC-17 were coexpressed in HeLa P4 cells along with the different importin 13 fragments (Fig. 14A).

Indirect fluorescence microscopy of the importin 13 fragments revealed a dominant nuclear localization pattern. Hence, the subcellular localization of the importin 13 fragments is comparable to the full length importin 13 (see again Fig. 11C) and all importin 13 fragments retained their capacity to enter the nucleus (Fig. 14B). Based on these data, it can be concluded that the loss of nuclear uptake of the CHRAC-15/17 dimer is caused by the absence of importin 13 amino acid residues essential for the interaction with the heterodimeric CHRAC complex and not through the cytoplasmic retention of the importin 13 fragments. The deletion of one HEAT repeat at the amino-terminus and four HEAT repeats at the carboxy-terminus already negatively influenced the nuclear transport of the CHRAC-15/17 heterodimer. This indicates that, full length importin 13 is required for binding and subsequent nuclear uptake of the histone fold pair.

In addition, these results point towards a broad interaction surface between the nuclear transport factor importin 13 and the CHRAC-15/17 complex.

Recently, Tao and colleagues (2004) demonstrated the same localization pattern for full length (amino acid 1-963) and a carboxy-terminally deleted fragment of importin 13 (amino acid 1-488), referred to as lgl2 (late gestation lung 2 protein). However, upon deletion of the amino acid residues 1-488 of importin 13, Tao and coworkers observed a cytoplasmic retention. In contrast, in our study, the loss of the first 251 amino acids did not result in a cytoplasmic localization. This might indicate that the remaining 238 amino acid residues of importin 13 harbor binding pockets, which are essential for the interaction between importin 13 with

FIG. 13: continued

Relative to wild type importin 13 (1-963), the C-terminally truncated importin 13 fragment 1-784 and the N-terminally truncated importin 13 fragment 45-963 show a reduced potential to translocate the heterodimer into the

nucleus of transfected cells. All other importin 13 fragments were not able to import the CHRAC-15/17 complex in the nucleus. (C) Importin 13 constructs used in this assay are listed. The amino acids or the importin 13 fragments are indicated and are represented by grey bars. Deleted regions are depicted by thin lines. Construct 1-963 amino

acid represents wild type importin 13. (D) Quantification of the nuclear import of EGFP-CHRAC-15/

RFP-CHRAC-17 mediated by full length and truncated importin 13. 100 transfected cells were quantitatively analyzed and the nucleocytoplasmic colocalization of the CHRAC-15/17 heterodimer scored into the following three categories: N > C, N = C, and N < C. imp13, importin 13; aa, amino acid.

FIG. 14: Transport characteristics of different importin 13 constructs for the CHRAC-15/17 complex do not differ upon exchange of EGFP and RFP among the subunits. (A) Plasmid DNA coding for RFP-CHRAC-15 and EGFP-CHRAC-17 were cotransfected in HeLa P4 cells. As in Figure 13, importin 13 fragments were cotransfected as indicated. The subcellular distribution of the CHRAC-15/17 complex in transfected cells is shown in yellow (merge). Full length importin 13 (1-963aa) mediates nuclear uptake of the heterodimers, whereas the carboxy-terminally truncated 1-784 fragment and the (continued on page 84)

FG-containing nucleoporins. In fact, for importin β several binding pockets in the corresponding HEAT repeats 6, 7, and 8 have been identified (Bayliss et al., 2000; Liu and Stewart, 2005). For the importin 13 fragment 45-963 amino acid we still observed import capacity for the CHRAC-15/17 heterodimer. However, since this importin 13 fragment lacks at least parts of the putative RanGTP binding site, which is essential for the dissociation of receptor-cargo-complex, the nuclear release of the CHRAC-15/17 complex from this importin 13 fragment remains unclear. Conceivable for this particular last step of nuclear transport is however a direct competition for either the cargo or the nuclear transport receptor.

3.7 Importin 13 does not influence the nuclear localization of monomeric