We previously reported that the nuclear import of substrates containing SV40 T antigen nuclear localization signal (NLS) was suppressed in a temperature-sensitive RCC1 mutant cell line, tsBN2, at non-permissive temperature. Moreover, it was shown that import into wild type BHK21 cell-derived nuclei gradually decreased in heterokaryons between the tsBN2 and BHK21 cells, although the BHK21 nuclei retained wild type RCC1 and should contain RanGTP (Tachibana et al., 1994). In this study, it was found that in the heterokaryons cultured at non-permissive temperature, endogenous importin α was not detected immunocytochemically in the cytoplasm or BHK21 nuclei but only in the tsBN2 nuclei, suggesting that importin α cannot be exported from the RCC1-depleted nuclei. In fact, importin α microinjected into the nucleus of tsBN2 cells at non-permissive temperature remained in the nucleus. These results strongly support the hypothesis that the recycling of importin α from the nucleus requires nuclear RanGTP. Moreover, it was found that cytoplasmic injection of importin α restored the import of SV40 T-NLS substrates in the BHK21 nuclei but not the tsBN2 nuclei in the heterokaryons. This indicates that the decrease of importin α from the cytoplasm in the heterokaryons leads to a suppression of the efficiency of nuclear import of the T-NLS substrate and provides support for the view that nuclear RanGTP is essential for the nuclear entry of the substrates.
2000 by Japan Society for Cell Biology