DFF45 contains a classical NLS in its C-terminal region

The identification of positively charged amino acid residues that are essential for the nuclear transport of the DFF complex in each subunit raised the question whether both basic clusters represent independent cNLSs. To answer this question, the nuclear transport of monomeric DFF subunits was analyzed focussing in particular on the capability of the C-terminal regions to mediate nuclear uptake. Hence, the monomeric DFF45 mutants described above (Fig. 11A), along with wild type DFF45 were expressed as EGFP-GST fusion proteins in HeLa P4 cells.

While wild type DFF45 showed a clear nuclear localization, deletion of the amino acids 306-331 (∆306-331) or substitution of the basic cluster KRxR (mutB) blocked nuclear uptake (Fig. 13A).

Mutation of R307 and K313 only had a minor effect on nuclear accumulation, leading to a homogenous distribution of monomeric DFF45 (Fig. 13A, mutA). This demonstrates that the C-terminal region, in particular the basic cluster KRxR, is essential for nuclear transport of DFF45.

To analyze whether this region is also sufficient for nuclear accumulation of a cytoplasmic protein we fused it to EGFP-EGFP-GST (EEG). The exclusively cytoplasmic localization of the EGFP-EGFP-GST fusion protein changed upon fusion to amino acids 306-331 of DFF45 [EEG-DFF45(aa306-331)], now being largely nuclear (Fig. 13B). These results clearly show that the C-terminal region of DFF45 exhibits a NLS that is necessary and sufficient for nuclear uptake of monomeric DFF45. In more detail, besides playing an essential role for importin α/β-mediated nuclear import of the DFF complex, the basic cluster KRxR was also identified as key element of the NLS in DFF45 itself. Based on these results, it was reasonable to assume that importin α/β is also the functional import receptor for DFF45. To verify this assumption, pull down assays with immobilized GST-DFF45 and recombinant importins were performed. Importin α/β specifically bound to GST-DFF45 and this binding was abolished in the presence of RanGTP (Fig. 13C).

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binding of iα/iβto GST-DFF45 (aa296-331)

binding of iα/iβto GST-DFF45 (aa296-331)

binding of iα/iβto GST-DFF45 (aa296-331)

FIG. 13: The C-terminal tail of DFF45 exhibits a classical NLS that is necessary and sufficient for nuclear uptake of monomeric DFF45. A and B, HeLa P4 cells were transiently transfected with plasmid DNA encoding wild type, truncated and mutated EGFP-GST-DFF45 (A) or EGFP-EGFP-GST (EEG) and amino acids 306-331 of DFF45 fused to EEG (B). The subcellular distribution was examined 24 h after transfection by direct fluorescence. The DNA was counterstained with Hoechst. Scale bars represent 10 µm. A, the dominant nuclear localization of wild type DFF45 was blocked when either amino acids 306-331 were deleted (∆306-331) or the basic cluster KRxR was mutated (DFF45mutB, see Fig. 11A). Substitution of the two positively charged amino acids at position 307 and 313 (DFF45mutA) affected the nuclear transport only moderately. B, the cytoplasmic localization of EEG changed upon fusion to amino acids 306-331 of DFF45 [EEG-DFF45(aa306-331)], the localization now becoming largely nuclear. C and D, amino acids 296-331 of DFF45 are responsible for the specific binding of importin α/β to DFF45. GST-pull down assays were performed as described in the legend to Fig. 7. Immobilized GST-DFF45 (C) and GST-fused amino acids 296-331 of DFF45 [GST-DFF45(aa296-331)] (D) were incubated with importin α/β in the absence or presence of 2 µM RanGTP. Bound fractions were analyzed by SDS-PAGE followed by Coomassie staining. MW, molecular weight in kilodalton; aa, amino acids; imp (i), importin.

Furthermore, in vitro binding studies with the C-terminus of DFF45 fused to GST [GST-DFF45(aa296-331)] confirmed the specific interaction of importin α/β with this region, since importin α/β was retained (Fig. 13D). In conclusion, DFF45 harbors a classical, presumably monopartite cNLS in its C-terminal region.

However, binding of importin α/β to DFF45 was less efficient than binding of importin α/β to the DFF complex. In other words, much more GST-DFF45 had to be immobilized to detect importin α/β-binding by Coomassie staining (compare Fig. 7).

Because of the weak binding of importin α/β we analyzed whether other import receptors are involved in nuclear transport of monomeric DFF45. For that purpose, GST-pull down assays were performed with importin α, importin α/β, importin β, transportin, importin 5, importin 7, importin 9 and importin 13, all from bacterial lysates (Fig. 14A). Besides importin α/β, none of the other import receptors interacted with GST-DFF45, strongly suggesting that DFF45 enters the nucleus via the importin α/β-pathway.

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binding of importins to GST-DFF45 binding of importins to

His-DFF40/ binding of importins to

GST-DFF40/

binding of importins to GST-DFF45

binding of importins to GST-DFF45 iα iα/iβ iβ trn i5 i7 i9 i13 45

binding of importins to GST-DFF45 binding of importins to

His-DFF40/ binding of importins to

His-DFF40/

binding of importins to His-DFF40/ binding of importins to

GST-DFF40/ binding of importins to

GST-DFF40/

His-DFF45∆306-331 ++ −

− +

− impβimpα binding of importins to

GST-DFF40/

FIG. 14: The C-terminus of DFF45 is necessary for the interaction between the DFF complex and importin α/β. A, Besides importin α/β none of the other import receptors was bound to GST-DFF45. Immobilized GST-DFF45 (45) was incubated with importin α, importin α/β, importin β, transportin (trn), importin 5, importin 7, importin 9 or importin 13, all from bacterial lysates. Bound fractions were analyzed by SDS-PAGE followed by Coomassie staining. B and C, epitope-tagged C-terminally truncated DFF45 (DFF45∆306-331; DFF45∆C) was coexpressed with epitope-tagged DFF40 in E. coli. The purified GST-DFF40/His-DFF45∆306-331 complex (B) or His-DFF40/GST-DFF45∆306-331 complex (C) was immobilized on glutathione-Sepharose and incubated with importin α, importin α/β and importin β as indicated. Bound fractions were analyzed by SDS-PAGE followed by Coomassie staining. None of the used import receptors was bound efficiently to the truncated DFF40/DFF45∆306-331 complex. MW, molecular weight in kilodalton; imp (i), importin.

Despite the weak binding of importin α/β to immobilized GST-DFF45, in vivo experiments had revealed an essential basic cluster for nuclear accumulation of the DFF complex (Fig. 11D and E). Therefore, in vitro binding studies with DFF complexes containing C-terminally truncated DFF45 (DFF45∆306-331) were performed (Fig. 14). In contrast to wild type DFF, binding of imortin α/β to GST-DFF40/His-DFF45∆306-331 was abolished (Fig. 14B). The same observation was made with a complex in which the affinity tags were exchanged among the subunits (Fig. 14C). The results of these in vitro binding studies again demonstrate the necessity of the C-terminal tail of DFF45 for the nuclear targeting of the complex.

2.8 The C-terminal tail of DFF40 interacts with importins and also functions as NLS