In a previous study, it was found that the microinjection of purified SV40 large T antigen into the cytoplasm of BALB/c 3T3 cells significantly increased both the relative rate of signal-mediated nuclear transport and the functional size of the transport channels that are located within the pores. In this investigation, a series of mutants were employed to identify the region of large T responsible for the permeability increase. Plasmids encoding wild-type or mutant forms of large T were injected into the nucleoplasm of proliferating BALB/c 3T3 cells, and the nuclear import of nucleoplasmin-coated gold particles was analyzed approximately 18 h later. The large T mutants that were not effective in inducing the increase in nuclear transport capacity were also unable to bind p53. Further evidence that transport activity and p53 binding localize to the same region of large T was obtained by simultaneously injecting plasmids that overexpress wild- type or mutant p53 and plasmids that encode active forms of large T. It was found that wild-type p53 prevented the large T-induced transport increase; however, mutant p53, which is unable to bind to large T, had no effect. Decreasing the concentration of endogenous p53 in cells that do not contain large T, by injecting anti-p53 antibodies or plasmids that express mutant p53, resulted in a significant increase in the nuclear import of nucleoplasmin-coated gold. The latter results suggest that p53 might normally act as a transport suppressor.
ASJC Scopus subject areas
- Cell Biology