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. 1989 Dec 1;109(6):2623–2632. doi: 10.1083/jcb.109.6.2623

Identification of a human protein that interacts with nuclear localization signals

PMCID: PMC2115907  PMID: 2592400

Abstract

Through a series of label transfer experiments, we have identified a HeLa cell nuclear protein that interacts with nuclear localization signals (NLSs). The protein has a molecular weight of 66,000 and an isoelectric point of approximately 6. It associates with a synthetic peptide that contains the SV-40 T antigen NLS peptide but not with an analogous peptide in which an asparagine is substituted for an essential lysine (un-NLS peptide). In addition to these peptides, several proteins have been tested as label donors. With the proteins, there is a correlation between nuclear localization (assayed with lysolecithin-permeabilized cells) and label transfer to the 66-kD protein. The NLS peptide (but not the un-NLS peptide) competes with the proteins in label transfer experiments, but neither wheat germ agglutinin nor ATP has an effect. These results suggest that the 66-kD protein functions as an NLS receptor in the first step of nuclear localization. In the course of this work, we have observed that the Staphylococcus aureus protein A is a strongly karyophilic protein. Its dramatic nuclear localization properties suggest that it may have multiple copies of an NLS.

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Selected References

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  1. Adam S. A., Lobl T. J., Mitchell M. A., Gerace L. Identification of specific binding proteins for a nuclear location sequence. Nature. 1989 Jan 19;337(6204):276–279. doi: 10.1038/337276a0. [DOI] [PubMed] [Google Scholar]
  2. Allen R. A., Tolley J. O., Jesaitis A. J. Preparation and properties of an improved photoaffinity ligand for the N-formyl peptide receptor. Biochim Biophys Acta. 1986 Jul 16;882(3):271–280. doi: 10.1016/0304-4165(86)90248-5. [DOI] [PubMed] [Google Scholar]
  3. Bürglin T. R., De Robertis E. M. The nuclear migration signal of Xenopus laevis nucleoplasmin. EMBO J. 1987 Sep;6(9):2617–2625. doi: 10.1002/j.1460-2075.1987.tb02552.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casadaban M. J., Martinez-Arias A., Shapira S. K., Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. doi: 10.1016/0076-6879(83)00063-4. [DOI] [PubMed] [Google Scholar]
  5. Colledge W. H., Richardson W. D., Edge M. D., Smith A. E. Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen. Mol Cell Biol. 1986 Nov;6(11):4136–4139. doi: 10.1128/mcb.6.11.4136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dabauvalle M. C., Schulz B., Scheer U., Peters R. Inhibition of nuclear accumulation of karyophilic proteins in living cells by microinjection of the lectin wheat germ agglutinin. Exp Cell Res. 1988 Jan;174(1):291–296. doi: 10.1016/0014-4827(88)90163-2. [DOI] [PubMed] [Google Scholar]
  7. Dang C. V., Lee W. M. Identification of the human c-myc protein nuclear translocation signal. Mol Cell Biol. 1988 Oct;8(10):4048–4054. doi: 10.1128/mcb.8.10.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dingwall C., Laskey R. A. Protein import into the cell nucleus. Annu Rev Cell Biol. 1986;2:367–390. doi: 10.1146/annurev.cb.02.110186.002055. [DOI] [PubMed] [Google Scholar]
  9. Dworetzky S. I., Lanford R. E., Feldherr C. M. The effects of variations in the number and sequence of targeting signals on nuclear uptake. J Cell Biol. 1988 Oct;107(4):1279–1287. doi: 10.1083/jcb.107.4.1279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Finlay D. R., Newmeyer D. D., Price T. M., Forbes D. J. Inhibition of in vitro nuclear transport by a lectin that binds to nuclear pores. J Cell Biol. 1987 Feb;104(2):189–200. doi: 10.1083/jcb.104.2.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Goldfarb D. S., Gariépy J., Schoolnik G., Kornberg R. D. Synthetic peptides as nuclear localization signals. Nature. 1986 Aug 14;322(6080):641–644. doi: 10.1038/322641a0. [DOI] [PubMed] [Google Scholar]
  12. Greenspan D., Palese P., Krystal M. Two nuclear location signals in the influenza virus NS1 nonstructural protein. J Virol. 1988 Aug;62(8):3020–3026. doi: 10.1128/jvi.62.8.3020-3026.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hall M. N., Hereford L., Herskowitz I. Targeting of E. coli beta-galactosidase to the nucleus in yeast. Cell. 1984 Apr;36(4):1057–1065. doi: 10.1016/0092-8674(84)90055-2. [DOI] [PubMed] [Google Scholar]
  14. Jones I. M., Reay P. A., Philpott K. L. Nuclear location of all three influenza polymerase proteins and a nuclear signal in polymerase PB2. EMBO J. 1986 Sep;5(9):2371–2376. doi: 10.1002/j.1460-2075.1986.tb04506.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kalderon D., Roberts B. L., Richardson W. D., Smith A. E. A short amino acid sequence able to specify nuclear location. Cell. 1984 Dec;39(3 Pt 2):499–509. doi: 10.1016/0092-8674(84)90457-4. [DOI] [PubMed] [Google Scholar]
  16. Knutson V. P. The covalent tagging of the cell surface insulin receptor in intact cells with the generation of an insulin-free, functional receptor. A new approach to the study of receptor dynamics. J Biol Chem. 1987 Feb 15;262(5):2374–2383. [PubMed] [Google Scholar]
  17. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  18. Lanford R. E., Kanda P., Kennedy R. C. Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal. Cell. 1986 Aug 15;46(4):575–582. doi: 10.1016/0092-8674(86)90883-4. [DOI] [PubMed] [Google Scholar]
  19. Lanford R. E., White R. G., Dunham R. G., Kanda P. Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals. Mol Cell Biol. 1988 Jul;8(7):2722–2729. doi: 10.1128/mcb.8.7.2722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lee B. A., Maher D. W., Hannink M., Donoghue D. J. Identification of a signal for nuclear targeting in platelet-derived-growth-factor-related molecules. Mol Cell Biol. 1987 Oct;7(10):3527–3537. doi: 10.1128/mcb.7.10.3527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lyons R. H., Ferguson B. Q., Rosenberg M. Pentapeptide nuclear localization signal in adenovirus E1a. Mol Cell Biol. 1987 Jul;7(7):2451–2456. doi: 10.1128/mcb.7.7.2451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Miller M. R., Ulrich R. G., Wang T. S., Korn D. Monoclonal antibodies against human DNA polymerase-alpha inhibit DNA replication in permeabilized human cells. J Biol Chem. 1985 Jan 10;260(1):134–138. [PubMed] [Google Scholar]
  23. Moreland R. B., Langevin G. L., Singer R. H., Garcea R. L., Hereford L. M. Amino acid sequences that determine the nuclear localization of yeast histone 2B. Mol Cell Biol. 1987 Nov;7(11):4048–4057. doi: 10.1128/mcb.7.11.4048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moreland R. B., Nam H. G., Hereford L. M., Fried H. M. Identification of a nuclear localization signal of a yeast ribosomal protein. Proc Natl Acad Sci U S A. 1985 Oct;82(19):6561–6565. doi: 10.1073/pnas.82.19.6561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Newmeyer D. D., Forbes D. J. Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation. Cell. 1988 Mar 11;52(5):641–653. doi: 10.1016/0092-8674(88)90402-3. [DOI] [PubMed] [Google Scholar]
  26. Newport J. W., Forbes D. J. The nucleus: structure, function, and dynamics. Annu Rev Biochem. 1987;56:535–565. doi: 10.1146/annurev.bi.56.070187.002535. [DOI] [PubMed] [Google Scholar]
  27. Nilsson B., Abrahmsén L., Uhlén M. Immobilization and purification of enzymes with staphylococcal protein A gene fusion vectors. EMBO J. 1985 Apr;4(4):1075–1080. doi: 10.1002/j.1460-2075.1985.tb03741.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  29. Picard D., Yamamoto K. R. Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 1987 Nov;6(11):3333–3340. doi: 10.1002/j.1460-2075.1987.tb02654.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pinkham J. L., Olesen J. T., Guarente L. P. Sequence and nuclear localization of the Saccharomyces cerevisiae HAP2 protein, a transcriptional activator. Mol Cell Biol. 1987 Feb;7(2):578–585. doi: 10.1128/mcb.7.2.578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Richardson W. D., Mills A. D., Dilworth S. M., Laskey R. A., Dingwall C. Nuclear protein migration involves two steps: rapid binding at the nuclear envelope followed by slower translocation through nuclear pores. Cell. 1988 Mar 11;52(5):655–664. doi: 10.1016/0092-8674(88)90403-5. [DOI] [PubMed] [Google Scholar]
  32. Richardson W. D., Roberts B. L., Smith A. E. Nuclear location signals in polyoma virus large-T. Cell. 1986 Jan 17;44(1):77–85. doi: 10.1016/0092-8674(86)90486-1. [DOI] [PubMed] [Google Scholar]
  33. Roberts B. L., Richardson W. D., Smith A. E. The effect of protein context on nuclear location signal function. Cell. 1987 Jul 31;50(3):465–475. doi: 10.1016/0092-8674(87)90500-9. [DOI] [PubMed] [Google Scholar]
  34. Shephard E. G., de Beer F. C., von Holt C., Hapgood J. P. The use of sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate as a crosslinking reagent to identify cell surface receptors. Anal Biochem. 1988 Feb 1;168(2):306–313. doi: 10.1016/0003-2697(88)90323-5. [DOI] [PubMed] [Google Scholar]
  35. Silver P. A., Keegan L. P., Ptashne M. Amino terminus of the yeast GAL4 gene product is sufficient for nuclear localization. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5951–5955. doi: 10.1073/pnas.81.19.5951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Slot J. W., Geuze H. J. A new method of preparing gold probes for multiple-labeling cytochemistry. Eur J Cell Biol. 1985 Jul;38(1):87–93. [PubMed] [Google Scholar]
  37. Sorensen P., Farber N. M., Krystal G. Identification of the interleukin-3 receptor using an iodinatable, cleavable, photoreactive cross-linking agent. J Biol Chem. 1986 Jul 15;261(20):9094–9097. [PubMed] [Google Scholar]
  38. Stacey D. W., Allfrey V. G. Microinjection studies of protein transit across the nuclear envelope of human cells. Exp Cell Res. 1984 Sep;154(1):283–292. doi: 10.1016/0014-4827(84)90687-6. [DOI] [PubMed] [Google Scholar]
  39. Stone J., de Lange T., Ramsay G., Jakobovits E., Bishop J. M., Varmus H., Lee W. Definition of regions in human c-myc that are involved in transformation and nuclear localization. Mol Cell Biol. 1987 May;7(5):1697–1709. doi: 10.1128/mcb.7.5.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ullmann A. One-step purification of hybrid proteins which have beta-galactosidase activity. Gene. 1984 Jul-Aug;29(1-2):27–31. doi: 10.1016/0378-1119(84)90162-8. [DOI] [PubMed] [Google Scholar]
  41. Vanin E. F., Ji T. H. Synthesis and application of cleavable photoactivable heterobifunctional reagents. Biochemistry. 1981 Nov 24;20(24):6754–6760. doi: 10.1021/bi00527a003. [DOI] [PubMed] [Google Scholar]
  42. Wychowski C., Benichou D., Girard M. A domain of SV40 capsid polypeptide VP1 that specifies migration into the cell nucleus. EMBO J. 1986 Oct;5(10):2569–2576. doi: 10.1002/j.1460-2075.1986.tb04536.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yoneda Y., Arioka T., Imamoto-Sonobe N., Sugawa H., Shimonishi Y., Uchida T. Synthetic peptides containing a region of SV 40 large T-antigen involved in nuclear localization direct the transport of proteins into the nucleus. Exp Cell Res. 1987 Jun;170(2):439–452. doi: 10.1016/0014-4827(87)90319-3. [DOI] [PubMed] [Google Scholar]
  44. Yoneda Y., Imamoto-Sonobe N., Yamaizumi M., Uchida T. Reversible inhibition of protein import into the nucleus by wheat germ agglutinin injected into cultured cells. Exp Cell Res. 1987 Dec;173(2):586–595. doi: 10.1016/0014-4827(87)90297-7. [DOI] [PubMed] [Google Scholar]

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