A long synthetic peptide containing a nuclear localization signal and its flanking sequences of SV40 T-antigen directs the transport of IgM into the nucleus efficiently.
Synthetic short peptides containing only the nuclear localization signal (NLS) direct the transport of nonnuclear proteins into the nucleus. As a conjugate of the synthetic peptide with immunoglobulin M (IgM) did not enter the nucleus, there was believed to be a size limit for nuclear transport of NLS-conjugated proteins. However, we found that IgM conjugated with purified nucleoplasmin, a nuclear protein of Xenopus oocytes, rapidly accumulated in the nucleus. For direct comparison with the short peptide, we prepared a long peptide containing the NLS and its flanking sequences of SV40 large T-antigen and its mutated long peptide, in which possible phosphorylation sites located at the amino terminal of the NLS were changed to alanine. Kinetic experiments showed that wild-type long peptide-IgM conjugates were almost entirely taken up into the nucleus within 30 min after their injection, whereas almost 60 min was required for the mutated long peptide-IgM conjugates to enter the nucleus of all the cells examined, and there was no apparent accumulation of short peptide-IgM conjugates in the nucleus within 60 min. These results indicate that even when the kinetics of transport are affected by amino acid substitutions, the long peptide directs the transport of large molecules such as IgM into the nucleus.
Yoneda
Y
Y
Institute for Molecular and Cellular Biology, Osaka University, Japan.
Semba
T
T
Kaneda
Y
Y
Noble
R L
RL
Matsuoka
Y
Y
Kurihara
T
T
Okada
Y
Y
Imamoto
N
N
D000595:Amino Acid Sequence; D000957:Antigens, Viral, Tumor / Q000032:analysis*; D001692:Biological Transport; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D006801:Humans; D007075:Immunoglobulin M / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D056489:Nucleoplasmins; D010750:Phosphoproteins; D021382:Protein Sorting Signals / Q000138:chemical synthesis; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013539:Simian virus 40 / Q000276:immunology
D000957:Antigens, Viral, Tumor; D007075:Immunoglobulin M; D009687:Nuclear Proteins; D056489:Nucleoplasmins; D010750:Phosphoproteins; D021382:Protein Sorting Signals; D012710:Serum Albumin, Bovine