Synthesis and conjugation of peptides and radioiodination of substrates

The correct localization of nuclear proteins is essential for proper growth and development of a eucaryotic cell. Nuclear protein import is a multistep process that eventually results in transport of the polypeptide from its cytoplasmic location of synthesis into the nucleus (for a recent review see Silver, 1991). The translocation into the nuclear interior occurs at the nuclear pore (Feldherr et al., 1984). An early step of nuclear protein import involves the specific recognition of nuclear localization sequences (NLS) in the protein destined for the nucleus. Polypeptides that associate with NLSs might recognize nuclear proteins in the cytosol and at the nuclear pore to facilitate the import reaction. NLS-binding proteins have been identified for various organisms by different techniques such as crosslinking or ligand binding assays (Adam et al., 1989; Li and Thomas, 1989; Silver et al., 1989; Lee and Melese, 1989). Such NLS-binding proteins were shown to distinguish between wild-type and mutant forms of the NLS derived from SV40 T-antigen (Adam et al., 1989; Silver et al., 1989). We have shown recently that a 70 kDa NLS-binding protein in yeast and higher eucaryotes, NBP70, is involved in nuclear protein import (Stochaj et al., 1991; Stochaj and Silver, 1992). Here we describe a different approach for the identification and analysis of binding proteins for nuclear localization sequences, i.e. the generation of anti-idiotype antibodies to NLSs. This technique has allowed us to verify the function of NBP70 as an NLS-binding protein. Two additional proteins of approx. 50 kDa and 95 kDa that specifically recognized functional NLSs were also identified by this procedure. Immunolocalization of antigens recognized by anti-idiotype antibodies revealed that potential NLS-binding proteins are at least in part located at the nuclear pore.