Form of human p53 protein during nuclear transport in Xenopus laevis embryos

The p53 protein binds DNA as a tetramer inside the nucleus, but a form of the p53 protein during nuclear transport has not been fully elucidated. To verify whether the human p53 protein passes through the nuclear pore as a monomer or oligomer, two different p53 mutants N1 and C1NLS- with or without a nuclear localization signal (NLS), respectively, were expressed in Xenopus laevis embryos. By the whole-mount immunostaining method, their intracellular distributions were observed to exist in an NLS-dependent manner. In a immunoprecipitation assay system, NLS-defective mutants formed oligomer in the cytoplasm. When coexpressed with NLS-containing N1, C1NLS- still stayed in the cytoplasm and did not inhibit N1 transport into the nucleus. Furthermore, when oligomerization-defective p53 mutant was expressed in Xenopus embryos, efficiency of its nuclear transport was demonstrated to be unchanged compared to that of the wild type. Assuming that NLS-defective p53 mutants have no dominant-negative effect on wild-type p53 in the nucleus of p53 heterozygous cells, we investigated the dominant-negative effect by CAT activity assay using human cell line Saos-2 and NLS-defective mutants. It was found that the NLS-defective p53 mutant did not have a dominant-negative effect on the function of wild-type p53 protein in the nucleus. Data indicate that each monomeric p53 protein independently passes through the nuclear pore; however, the possibility of homooligomeric p53 protein transport into the nucleus is not completely excluded.