Importin 5 mediates the nuclear import of PGC7/Stella but is dispensable

We next wanted to elucidate a possible role of importin 5 in the nuclear import of CHRAC-15/17 and to illuminate the discrepancy between the data from the in vitro import assay (Fig. 6) and the in vivo transfection experiments (Fig. 10A). Therefore, RNAi experiments with siRNA against importin 5 were performed in order to control a potential significance of importin 5 in the nuclear transport of CHRAC-15/17. For that purpose, RNAi duplex oligoribonucleotides against importin 5 were transfected in HeLa P4 cells. After three and seven days the depletion of

importin 5 was controlled by Western blotting (Fig. 12A) using a specific antibody against importin 5. Cells transfected with control siRNA and mock treated cells served as expression

FIG. 11: In vivo coexpression of importin 13 is necessary for the nuclear accumulation of differently tagged CHRAC-15/17 complexes. (A) HeLa P4 cells were transiently cotransfected with DNA coding for RFP-CHRAC-15 and EGFP-CHRAC-17. In addition, plasmid DNA coding for different flag-tagged transport receptors was added as indicated. The subcellular localization of the gene products was examined 24 hours post transfection by direct fluorescence. Using Hoechst the DNA was counterstained. The merged picture results from the overlap between the green EGFP and red RFP fluorescence.Coexpression of importin 13 leads to an exclusive nuclear accumulation of the CHRAC-15/17 complex. Neither importin β, importin 5, importin 7 nor importin 9 mediates the nuclear accumulation of the CHRAC-15/17 heterodimer when cotransfected. (B) CHRAC-15 carboxy-terminally fused to EGFP (upper panel) or RFP (lower panel) and CHRAC-17 likewise fused to EGFP or RFP were cotransfected with flag-importin 13 in HeLa P4 cells. The subcellular distribution of the heterodimeric complex was determined 24 hours post transfection. The DNA was counterstained with Hoechst. The exchange of EGFP and RFP in CHRAC-15 and CHRAC-17 from the amino-terminus to the carboxy-terminus did not influence the nuclear localization upon importin 13 coexpression. (C) Flag-tagged importins used in the study were overexpressed in HeLa P4 cells. The localization of each nuclear transport receptor was determined using an anti-flag antibody. The DNA was stained with Hoechst. Whereas flag-tagged importin 7 and importin 13 localized predominantly in the nucleus, flag-tagged importin β, importin 5, and importin 9 showed a strong rim staining. imp, importin.

controls. Comparing the band intensities of importin 5 in mock treated cells, set 100 %, with the cells transfected with siRNA a decrease in the importin level of 74 % after three days and 90 % after 7 days became evident. Importin 5 levels, however, was even slightly increased (9 %) in cells treated with control siRNA. To analyze the effect of importin 5 depletion on the nuclear import of CHRAC-15/17, antibodies against CHRAC-17 were applied to detect its subcellular localization (Fig. 12B). Despite the depletion of importin 5 in the cells, the localization of endogenous CHRAC-17 was not affected. This indicates that importin 5 has no significant role in the import of the heterodimeric CHRAC-15/17 complex in vivo. To further investigate the primary role of importin 13, we performed RNAi experiments with Stealth siRNA to deplete endogenous importin 13. Transfection of Stealth siRNA against importin 13 in HeLa P4 cells were performed using Oligofectamine and in addition, the electroporation system Nucleofector^® II Device. Unfortunately, the protein levels of importin 13, verified by Western blotting remained unchanged (data not shown).

However, since the flag-tagged importin 13 expression construct was able to translocate the CHRAC heterodimer from the cytoplasm into the nucleus, but flag-importin 5 was not (Fig. 10A), the functionality of the overexpressed importin 5 construct was controlled. Therefore, PGC7/Stella, a known importin 5 cargo protein (Nakamura et al., 2007) was transfected in HeLa P4 cells. PGC7/Stella, a maternal factor essential for early development, protects the DNA methylation state and is preferentially expressed in primordial germ cells (PGCs), early preimplantation embryos and oocytes (Bortvin et al., 2004; Sato et al., 2002). Because the primary structure of PGC7/Stella, consisting of 150 amino acids, contains a putative bipartite NLS (amino acid residues 42-59) and in addition a NES (amino acid residues 32-46), it is localized in the nucleus and the cytoplasm when expressed alone (Nakamura et al., 2007). In analogy to the results from Nakamura and colleagues, flag-tagged PGC7/Stella was localized homogenously within transfected cells, showing an even distribution between the nucleus and the cytoplasm (Fig. 12C). In contrast, EGFP-PGC7/Stella was mainly found in the cytoplasm (Fig. 12D), forming cytoplasmic aggregates, pointing towards an unfolded state of the EGFP-fusion proteins. Additional cotransfection of importin 5 consequently led to a predominantly nuclear localization of PGC7/Stella (Fig. 12C and D, middle panel), analogous to the increased nuclear uptake of CHRAC-15/17 upon importin 13 coexpression. Interestingly, importin 5 not only led to an increased nuclear import of PGC7/Stella, but also acted as a molecular chaperone for the EGFP-tagged protein by preventing its aggregation upon overexpression. This

‘anti-aggregation activity’ is in line with previous reports concerning the dual role of importins by mediating nuclear transport processes and possessing a general chaperone-like role in protein

FIG. 12: Influence of importin 5 on the endogenous distribution of CHRAC-17 and the known cargo protein PGC7/Stella. (A) RNAi experiments with siRNA against importin 5 were accomplished using Stealth™ RNAi duplex oligoribonucleotides. HeLa P4 cells were transfected and cells were assayed for gene knockdown by Western blotting analysis 3 and 7 days post transfection using a specific antibody against importin 5. Mock treated HeLa P4 cells served as expression control. The software 1Dscan Ex (Scanalytics, Inc) was used to analyze band intensities.

Mock treated cells were set 100 % and an importin 5 depletion of 74 % after 3 days and 90 % after 7 days was measured. An antibody against actin was used to control equal loading. (B) Importin 5 depleted HeLa P4 cells (3 and 7 days after siRNA treatment), cells treated with control siRNA (after 7 days) and mock treated cells were used to detect the subcellular distribution of endogenous CHRAC-17 using an anti-CHRAC-17 antibody. Hoechst was used to counterstain the DNA. Depletion of importin 5 did not affect the localization of endogenous CHRAC-17

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folding (Jäkel et al., 2002). The cotransfection of importin 13 had neither an influence on the subcellular localization of flag-tagged PGC7/Stella nor of the EGFP-PGC7/Stella (Fig. 12C and D, lower panel).

From these data, it can be concluded that, importin 5 has no functional significance in facilitating nuclear import of the CHRAC-15/17 heterodimer in vivo. Nuclear import of the CHRAC-15/17 complex overexpressed in HeLa P4 cells depends on the additional coexpression of importin 13.

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