Antibodies against the SV40 large T antigen nuclear localization sequence.
Transport of large proteins into the nucleus requires both a nuclear localization signal (NLS) and exposure of that signal to components of the transport machinery. In this report, polyclonal and monoclonal antibodies were generated against the NLS of SV40 large T antigen. Several of these antibodies immunoprecipitated large T antigen produced by in vitro transcription-translation and recognized T antigen expressed in cultured cells. Binding of the antibodies to T antigen was quantified using an indirect radioimmunoassay and found to be specifically inhibited by peptides corresponding to the T antigen NLS. The ability of NLS-specific antibodies to recognize large T antigen suggests that the NLS is exposed on the surface of T antigen. When one of the NLS-specific monoclonal antibodies was introduced into the cytoplasm of cells expressing T antigen, the antibody remained cytoplasmic. These results suggested either that cytoplasmic components compete for binding to the NLS or that the antibody dissociates from T antigen during transport into the nucleus. When an antibody directed against an epitope distinct from the NLS was microinjected into the cytoplasm of cells expressing large T antigen, both the antibody and antigen were transported into the nucleus. The observed stability of the antigen-antibody complex strongly suggest protein unfolding is not required for nuclear protein transport.
Wolff
B
B
Laboratory of Biochemistry and Metabolism, NIDDK National Institutes of Health, Bethesda, Maryland 20892.
Park
M K
MK
Klima
E
E
Hanover
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000378:metabolism; D000914:Antibodies, Viral / Q000008:administration & dosage / Q000378:metabolism; D000957:Antigens, Viral, Tumor / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D001693:Biological Transport, Active; D002460:Cell Line; D002467:Cell Nucleus / Q000276:immunology; D003593:Cytoplasm / Q000276:immunology; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000276:immunology; D014176:Protein Biosynthesis; D013539:Simian virus 40 / Q000276:immunology*; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D000957:Antigens, Viral, Tumor; D009842:Oligopeptides