ALL 4 HOMOCHIRAL ENANTIOMERS OF A NUCLEAR-LOCALIZATION SEQUENCE DERIVED FROM C-MYC SERVE AS FUNCTIONAL IMPORT SIGNALS

The information that targets a protein to the nucleus often consists o f a short cluster of basic amino acids called a nuclear localization s equence (NLS). Since a wide range of sequences rich in basic amino aci d residues function as NLSs, we postulated that an NLS-like sequence c omposed exclusively of D-amino acids might have biological activity. W e synthesized peptides corresponding to the c-Myc NLS composed of eith er all L or D-amino acids, both in the forward and reverse order. We t ested these peptides for nuclear import activity in a digitonin-permea bilized cell assay. All four peptide-bovine serum albumin conjugates l ocalized to the nucleus with similar efficiency, and each conjugate co mpeted for import with an SV40 large T antigen-derived NLS conjugate. Cross-linking experiments with free NLS peptides in HeLa cytosol indic ated that each peptide bound to a protein that migrated at the molecul ar weight of importin alpha. Recombinant importin a, importin beta, Ra n, and NTF2 alone were sufficient to support the import of both L-form and D-form conjugates in permeabilized cells. This indicates that bot h D- and L-form NLS peptides use the same import machinery. Although t he free D-forms of the NLS were proteolytically resistant in cytosol, the L-forms were rapidly degraded. To our knowledge, this is the first example of an intracellular pathway in which the receptor is insensit ive to the chirality of the ligand.