KINETIC CHARACTERIZATION OF THE HUMAN RETINOBLASTOMA PROTEIN BIPARTITE NUCLEAR-LOCALIZATION SEQUENCE (NLS) IN-VIVO AND IN-VITRO - A COMPARISON WITH THE SV40 LARGE T-ANTIGEN NLS

The retinoblastoma (RE) tumor suppressor is a nuclear phosphoprotein i mportant for cell growth control and able to bind specifically to vira l oncoproteins such as the SV40 large tumor antigen (T-ag), Human RE p ossesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860-877, also pr esent in mouse and Xenopus homologs, which resembles that of nucleopla smin, The T-ag NLS represents a different type of NLS, consisting of o nly one stretch of basic amino acids, To compare the nuclear import ki netics conferred by the bipartite NLS of RE to those conferred by the T-ag NLS, we used beta-galactosidase fusion proteins containing the NL Ss of either RE or T-ag, The RE NLS was able to target beta-galactosid ase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells), M utational substitution of the proximal basic residues of the NLS aboli shed nuclear targeting activity, confirming its bipartite character, N uclear accumulation of the RE fusion protein was half-maximal within a bout 8 min in vivo maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by t he T-ag fusion protein, while the initial rate of nuclear import of th e RE protein was also less than half that of T-ag, Nuclear import conf erred by both NLSs in vitro was dependent on cytosol and ATP and inhib ited by the nonhydrolyzable GTP analog GTP gamma S. Using an ELISA-bas ed binding assay, we determined that the RE bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity h eterodimeric NLS-binding protein complex importin 58/97, this differen ce presumably representing the basis of the reduced maximal nuclear ac cumulation and import rate in vivo, The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is criti cal in determining the kinetics of nuclear protein import.