All four 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 of a short cluster of basic amino acids called a nuclear localization sequence (NLS). Since a wide range of sequences rich in basic amino acid residues function as NLSs, we postulated that an NLS-like sequence composed exclusively of D-amino acids might have biological activity. We synthesized peptides corresponding to the c-Myc NLS composed of either all L or D-amino acids, both in the forward and reverse order. We tested these peptides for nuclear import activity in a digitonin-permeabilized cell assay. All four peptide-bovine serum albumin conjugates localized to the nucleus with similar efficiency, and each conjugate competed for import with an SV40 large T antigen-derived NLS conjugate. Cross-linking experiments with free NLS peptides in HeLa cytosol indicated that each peptide bound to a protein that migrated at the molecular weight of importin alpha. Recombinant importin alpha, importin beta, Ran, and NTF2 alone were sufficient to support the import of both L-form and D-form conjugates in permeabilized cells. This indicates that both D- and L-form NLS peptides use the same import machinery. Although the 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 insensitive to the chirality of the ligand.
Saphire
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The Scripps Research Institute, La Jolla, California 92037, USA.
Bark
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SJ
Gerace
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D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry; D001692:Biological Transport; D006367:HeLa Cells; D006801:Humans; D019913:Nuclear Localization Signals; D016271:Proto-Oncogene Proteins c-myc / Q000737:chemistry* / Q000378:metabolism; D013539:Simian virus 40 / Q000276:immunology; D013237:Stereoisomerism
D000952:Antigens, Polyomavirus Transforming; D019913:Nuclear Localization Signals; D016271:Proto-Oncogene Proteins c-myc