53. The Nuclear Localization Signal of the Human Ku70 Is a Variant Bipartite-type Recognized by the Two Components of Nuclear Pore-targeting Complex

Manabu Koike, Togo Ikuta^*, Yoshi-nobu Harada, and Tadahiro Shiomi
(^*Saitama Cancer Center Reserch Institute)

Keywords: GFP, Ku70, microinjection, NLS, PTAC58/97

Ku protein is a complex of two subunits, Ku70 and Ku80. Ku is suspected of participating in both DNA double-strand break repair and transcription. Since both of these processes take place in the cell nucleus, we have been investigating the subcellular localization and nuclear transport of Ku proteins. In the present study, we analyzed the subcellular localization and nuclear localization signal (NLS) of Ku70. Fusion proteins of Ku70 and green fluorescent protein (GFP) transiently expressed in cells were clearly localized in the nuclei of interphase cells. Ku70 staining was distributed throughout both the nucleus and the cytoplasm in late telophase to early G1 phase cells. The NLS of Ku70 was located at the region composed of 18 amino acid residues. This region overlapped with the Ku80-independent DNA-binding domain reported previously. The Ku70 NLS consisted of two basic subregions and a nonbasic intervening region. All the subregions were necessary for complete NLS activity. All of the basic amino acid residues in the basic subregions were conserved among mammalian and avian homologues, confirming their importance in the nuclear translocation of Ku70. The structure of the Ku70 NLS was a variant bipartite-type. The Ku70 NLS was mediated to target to the nuclear rim by two components of the nuclear pore-targeting complex, PTAC58 and PTAC97.

Publications:
1) Koike, M., Miyasaka, T., Mimori, T., and Shiomi, T. : Biochem. Biophys. Res. Commun., 252; 679-685, 1998.
2) Koike, M., Ikuta, T., Miyasaka, T., Shiomi, T. : Exp. Cell Res., 250, 401-413, 1999.