[The characteristics of the transport into the nucleus of conjugates with normal and mutant nucleophilic sequences]. / Kharakteristika transporta v iadro kon''iugatov s normal'noi i mutantnoi nukleofil'nymi posledovatel'nostiami.

A study was made of the transport of conjugates of a fluorescently labeled protein and a synthetic peptide, corresponding to the nuclear localization sequence of the SV 40 large T-antigen, into the nuclei of cultured human (HeLa and A431) and murine (HER14) cells. A possibility for such conjugates to be transported into the nuclei of digitonin-permeabilized cells, without addition of exogenous cytosol, was demonstrated. A quantitative comparison of the transport levels of constructions with normal or altered (K128T) peptide sequence was performed, and a low selectivity of nuclear transport with this alteration in the digitonin-permeabilized cell system was revealed, whereas constructions with the mutant sequence, when injected into live cells, remained in the cytoplasm. ATP-dependent transport of constructions with the mutant sequence into permeabilized cell nuclei was demonstrated, with a considerable part of this transport being NEM-insensitive. A suggestion is put forward that there are several variants of the nucleophilic sequence containing protein transport in the digitonin-permeabilized cell system.

Comparison of diverse transport signals in synthetic peptide-induced nuclear transport.

Several investigations have demonstrated the ability of synthetic peptides homologous to the nuclear transport signal of simian virus 40 large T antigen to induce the nuclear transport of nonnuclear carrier proteins. To determine the generality of peptide-induced transport, six peptides with sequences derived from four previously identified nuclear transport signals were synthesized and examined for their ability to induce the transport of mouse immunoglobulin G following microinjection into the cytoplasm of mammalian cells. Peptides containing transport signals from simian virus 40 T antigen, Xenopus nucleoplasmin, and adenovirus E1A proteins were highly efficient at peptide-induced transport, while a peptide homologous to yeast MAT alpha 2 protein was incapable of inducing transport. A short nucleoplasmin peptide that contained only the basic amino acid domain was capable of inducing transport but yielded a much slower rate of transport than a long nucleoplasmin peptide encompassing the previously identified minimal transport signal. The short nucleoplasmin signal exhibited a greater capacity for transport than a peptide homologous to the cytoplasmic mutant T antigen signal when conjugates with a low number of signals coupled per carrier protein were examined. However, the short nucleoplasmin peptide was only marginally more effective than the T antigen mutant peptide when conjugates with a high number of signals coupled per carrier protein were examined.