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

the binding of importin α/β to DFF45 is much weaker than to the DFF40/DFF45 complex, we further analyzed the function of DFF40 in nuclear transport of DFF. Firstly, GST-pull down assays with a DFF complex containing C-terminally truncated DFF40 were performed. For that purpose, GST-DFF40∆324-338 was coexpressed with His-DFF45 in E. coli. The proper folding of the subunits and complex formation was analyzed in a plasmid cleavage assay (Fig. 15A) as described previously (for details see Materials and Methods). Upon activation of the purified GST-DFF40∆324-338/His-DFF45 complex by caspase-3, GST-DFF40∆324-338 was able to cleave plasmid DNA (Fig. 15A, lane 3-5). This demonstrates that the GST-DFF40∆324-338/His-DFF45 complex is functional. For in vitro binding studies GST-DFF40∆324-338/His-GST-DFF40∆324-338/His-DFF45 was immobilized on glutathione-Sepharose and incubated with importin α and importin β from bacterial lysates. As shown in Fig. 15B, deletion of amino acids 324-338 of DFF40 abolished the binding of importin α/β to the DFF complex (compare Fig. 7A). Similar results were obtained for His-DFF40∆324-338/GST-DFF45 complex (Fig. 15C and D) excluding the influence of affinity-tags. Thus, these observations support the results of the in vivo transfection experiments (see Fig.

10C and D) underlining the importance of the C-terminus of DFF40 for nuclear import. To examine whether the C-terminal region of DFF40 is directly involved in importin α/β-binding we next performed pull down assays with DFF40 alone. To this end, immobilized DFF40/His-DFF45 was treated with purified caspase-3 to cleave off DFF45. The activated GST-DFF40 was then incubated with import receptors. Among them, importin α/β was bound specifically to DFF40 (Fig. 16A) but importin α/β-binding was less efficient than to the DFF complex (for comparison see Fig. 7A). In contrast to the DFF complex and monomeric DFF45, immobilized GST-DFF40 was additionally bound by importin β with nearly the same efficiency

D

GST-DFF40∆324-338/ binding of importins to

His-DFF40∆324-338/ binding of importins to

GST-DFF40∆324-338/ binding of importins to

GST-DFF40∆324-338/ binding of importins to

GST-DFF40∆324-338/ binding of importins to

His-DFF40∆324-338/ binding of importins to

His-DFF40∆324-338/

binding of importins to His-DFF40∆324-338/

FIG. 15: The C-terminal tail of DFF40 contributes to the interaction between the DFF complex and importin α/β. A and C, the GST-DFF40∆324-338/His-DFF45 complex (A) and the His-DFF40∆324-338/GST-DFF45 complex (C) can be activated by caspase-3 leading to the release of nucleolytically active DFF40∆324-338 or His-DFF40∆324-DFF40∆324-338 respectively. Plasmid DNA was incubated with increasing amounts of GST-DFF40/His-DFF45 (lane 3-5 in panel A) or His-DFF40/GST-DFF45 (lane 3-5 in panel C) in the presence or absence of caspase-3 for 2 h at 37°C. Neither caspase-3 (lane 2 and 7) nor epitope-tagged DFF complex (lane 6) alone were able to cleave plasmid DNA. After phenol-chloroform extraction, the DNA was analyzed on a 1% agarose gel. The bands in lane 1 (plasmid control) represent the three topological forms of circular plasmid DNA. B and C, epitope-tagged C-terminally deleted DFF40 (DFF40∆324-338) was coexpressed with epitope-epitope-tagged DFF45 in E. coli. The purified GST-DFF40∆324-338/His-DFF45 complex (B) or His-DFF40∆324-338/GST-DFF45 complex (D) 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∆324-338/DFF45 complex (for comparison with wild type DFF see also Fig 7B and D). MW, molecular weight in kilodalton; aa, amino acids; imp (i), importin.

as by importin α/β (Fig. 16A). Furthermore, the C-terminal region of DFF40 fused to GST [GST-DFF40(aa314-338)] interacts with importin α/β as well as with importin β in a RanGTP-sensitive fashion (Fig. 16B). The binding of importin β alone to the immobilized C-terminus of DFF40 was even stronger compared to importin α/β-binding. These data suggest that the C-terminal tail of DFF40 exhibits a NLS. However, this NLS alone can not account for the strong importin α/β binding to the DFF complex because this signal is hardly recognized within a DFF40/DFF45 complex containing C-terminally truncated DFF45 (see again Fig. 14B and C).

D

(aa314-338) binding of importins to GST-DFF40

(aa314-338) binding of importins to GST-DFF40

(aa314-338)

− + RanGTP

iα/iβ

40C − +

iβ binding of importins to GST-DFF40

(aa314-338)

FIG. 16: The C-terminal tail of DFF40 contains a functional NLS. A and B, amino acids 314-338 of DFF40 are responsible for the specific binding of importin α/β and importin β to DFF40. Immobilized GST-DFF40/His-DFF45 complex (40/45) was incubated with caspase-3 for 30 min at 30°C to cleave off DFF45 and thus activate DFF40. The immobilized activated GST-DFF40 (40) (A) and GST-fused amino acids 314-338 of DFF40 [GST-DFF40(aa314-338), 40C] (B) were incubated with importin α/β and importin β in the absence or presence of 2 µM RanGTP. Bound fractions were analyzed by SDS-PAGE followed by Coomassie staining. C and D, HeLa P4 cells were transiently transfected with (C) wild type EGFP-GST-DFF40 and (D) amino acids 301-338 of DFF40 fused to EGFP-EGFP-GST (EEG). The subcellular distribution was examined 24 h after transfection by direct fluorescence and the DNA was counterstained with Hoechst. C, exclusively cytoplasmic localization of EGFP-GST-DFF40. D, the dominant cytoplasmic distribution of EEG (left panel) changed upon fusion to the C-terminus (aa301-338) of DFF40 (right panel), the localization now becoming largely nuclear. Scale bars represent 10 µm.

MW, molecular weight in kilodalton; aa, amino acids; imp (i), importin.

To characterize the function of the NLS of DFF40, in vivo transfection studies with EGFP-GST-tagged DFF40 were performed in HeLa P4 cells. Surprisingly, EGFP-GST-DFF40 was not transported into the nucleus as shown in Fig. 16C. However, the cytoplasmic EGFP-EGFP-GST fusion protein accumulated in the nucleus of transfected cells upon fusion to the C-terminal amino acids 301-338 of DFF40 [EGFP-EGFP-GST-DFF40(aa301-338); Fig. 16D]. This confirms that the C-terminus of DFF40 harbors a functional NLS. The cytoplasmic localization of monomeric EGFP-GST-DFF40 may be due to misfolding of DFF40 overexpressed in the absence of exogenous DFF45-chaperone.

In conclusion, in vivo transfection experiments with C-terminal regions of DFF40 and DFF45 indicate that both subunits exhibit functional NLSs. However, in pull down assays importin α/β was shown to bind the DFF40/DFF45 complex more strongly than the individual components.

To explain the high affinity of importin α/β to the DFF complex compared to the single subunits, the interaction between the DFF complex and importin α/β was analyzed in more detail.

2.9 Basic clusters in the C-terminal tails of DFF40 and DFF45 together presumably form