Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Mar 2;17(5):1228–1235. doi: 10.1093/emboj/17.5.1228

Nuclear localization is required for function of the essential clock protein FRQ.

C Luo 1, J J Loros 1, J C Dunlap 1
PMCID: PMC1170471  PMID: 9482720

Abstract

The frequency (frq) gene in Neurospora encodes central components of a circadian oscillator, a negative feedback loop involving frq mRNA and two forms of FRQ protein. Here we report that FRQ is a nuclear protein and nuclear localization is essential for its function. Deletion of the nuclear localization signal (NLS) renders FRQ unable to enter into the nucleus and abolishes overt circadian rhythmicity, while reinsertion of the NLS at a novel site near the N-terminus of FRQ restores its function. Each form of FRQ enters the nucleus soon after its synthesis in the early subjective day; there is no evidence for regulated sequestration in the cytoplasm prior to nuclear entry. The kinetics of the nuclear entry are consistent with previous data showing rapid depression of frq transcript levels following the synthesis of FRQ, and suggest that early in each circadian cycle, when FRQ is synthesized, it enters the nucleus and depresses the level of its own transcript.

Full Text

The Full Text of this article is available as a PDF (276.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aronson B. D., Johnson K. A., Dunlap J. C. Circadian clock locus frequency: protein encoded by a single open reading frame defines period length and temperature compensation. Proc Natl Acad Sci U S A. 1994 Aug 2;91(16):7683–7687. doi: 10.1073/pnas.91.16.7683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baum J. A., Giles N. H. Genetic control of chromatin structure 5' to the qa-x and qa-2 genes of Neurospora. J Mol Biol. 1985 Mar 5;182(1):79–89. doi: 10.1016/0022-2836(85)90029-4. [DOI] [PubMed] [Google Scholar]
  3. Crosthwaite S. K., Dunlap J. C., Loros J. J. Neurospora wc-1 and wc-2: transcription, photoresponses, and the origins of circadian rhythmicity. Science. 1997 May 2;276(5313):763–769. doi: 10.1126/science.276.5313.763. [DOI] [PubMed] [Google Scholar]
  4. Crosthwaite S. K., Loros J. J., Dunlap J. C. Light-induced resetting of a circadian clock is mediated by a rapid increase in frequency transcript. Cell. 1995 Jun 30;81(7):1003–1012. doi: 10.1016/s0092-8674(05)80005-4. [DOI] [PubMed] [Google Scholar]
  5. Curtin K. D., Huang Z. J., Rosbash M. Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock. Neuron. 1995 Feb;14(2):365–372. doi: 10.1016/0896-6273(95)90292-9. [DOI] [PubMed] [Google Scholar]
  6. Dunlap J. C. Genetic analysis of circadian clocks. Annu Rev Physiol. 1993;55:683–728. doi: 10.1146/annurev.ph.55.030193.003343. [DOI] [PubMed] [Google Scholar]
  7. Dunlap J. C. Genetics and molecular analysis of circadian rhythms. Annu Rev Genet. 1996;30:579–601. doi: 10.1146/annurev.genet.30.1.579. [DOI] [PubMed] [Google Scholar]
  8. Fu Y. H., Paietta J. V., Mannix D. G., Marzluf G. A. cys-3, the positive-acting sulfur regulatory gene of Neurospora crassa, encodes a protein with a putative leucine zipper DNA-binding element. Mol Cell Biol. 1989 Mar;9(3):1120–1127. doi: 10.1128/mcb.9.3.1120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Garceau N. Y., Liu Y., Loros J. J., Dunlap J. C. Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell. 1997 May 2;89(3):469–476. doi: 10.1016/s0092-8674(00)80227-5. [DOI] [PubMed] [Google Scholar]
  10. Garcia-Bustos J., Heitman J., Hall M. N. Nuclear protein localization. Biochim Biophys Acta. 1991 Mar 7;1071(1):83–101. doi: 10.1016/0304-4157(91)90013-m. [DOI] [PubMed] [Google Scholar]
  11. Gekakis N., Saez L., Delahaye-Brown A. M., Myers M. P., Sehgal A., Young M. W., Weitz C. J. Isolation of timeless by PER protein interaction: defective interaction between timeless protein and long-period mutant PERL. Science. 1995 Nov 3;270(5237):811–815. doi: 10.1126/science.270.5237.811. [DOI] [PubMed] [Google Scholar]
  12. Hall J. C. Are cycling gene products as internal zeitgebers no longer the zeitgeist of chronobiology? Neuron. 1996 Nov;17(5):799–802. doi: 10.1016/s0896-6273(00)80211-1. [DOI] [PubMed] [Google Scholar]
  13. Hunter-Ensor M., Ousley A., Sehgal A. Regulation of the Drosophila protein timeless suggests a mechanism for resetting the circadian clock by light. Cell. 1996 Mar 8;84(5):677–685. doi: 10.1016/s0092-8674(00)81046-6. [DOI] [PubMed] [Google Scholar]
  14. Jans D. A., Jans P. Negative charge at the casein kinase II site flanking the nuclear localization signal of the SV40 large T-antigen is mechanistically important for enhanced nuclear import. Oncogene. 1994 Oct;9(10):2961–2968. [PubMed] [Google Scholar]
  15. Kanaan M. N., Marzluf G. A. The positive-acting sulfur regulatory protein CYS3 of Neurospora crassa: nuclear localization, autogenous control, and regions required for transcriptional activation. Mol Gen Genet. 1993 Jun;239(3):334–344. doi: 10.1007/BF00276931. [DOI] [PubMed] [Google Scholar]
  16. Lee C., Parikh V., Itsukaichi T., Bae K., Edery I. Resetting the Drosophila clock by photic regulation of PER and a PER-TIM complex. Science. 1996 Mar 22;271(5256):1740–1744. doi: 10.1126/science.271.5256.1740. [DOI] [PubMed] [Google Scholar]
  17. Lewis M. T., Morgan L. W., Feldman J. F. Analysis of frequency (frq) clock gene homologs: evidence for a helix-turn-helix transcription factor. Mol Gen Genet. 1997 Jan 27;253(4):401–414. doi: 10.1007/s004380050338. [DOI] [PubMed] [Google Scholar]
  18. Liu X., Zwiebel L. J., Hinton D., Benzer S., Hall J. C., Rosbash M. The period gene encodes a predominantly nuclear protein in adult Drosophila. J Neurosci. 1992 Jul;12(7):2735–2744. doi: 10.1523/JNEUROSCI.12-07-02735.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Liu Y., Garceau N. Y., Loros J. J., Dunlap J. C. Thermally regulated translational control of FRQ mediates aspects of temperature responses in the neurospora circadian clock. Cell. 1997 May 2;89(3):477–486. doi: 10.1016/s0092-8674(00)80228-7. [DOI] [PubMed] [Google Scholar]
  20. Loros J. J., Denome S. A., Dunlap J. C. Molecular cloning of genes under control of the circadian clock in Neurospora. Science. 1989 Jan 20;243(4889):385–388. doi: 10.1126/science.2563175. [DOI] [PubMed] [Google Scholar]
  21. McNally M. T., Free S. J. Isolation and characterization of a Neurospora glucose-repressible gene. Curr Genet. 1988 Dec;14(6):545–551. doi: 10.1007/BF00434079. [DOI] [PubMed] [Google Scholar]
  22. Merrow M. W., Dunlap J. C. Intergeneric complementation of a circadian rhythmicity defect: phylogenetic conservation of structure and function of the clock gene frequency. EMBO J. 1994 May 15;13(10):2257–2266. doi: 10.1002/j.1460-2075.1994.tb06507.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Merrow M. W., Garceau N. Y., Dunlap J. C. Dissection of a circadian oscillation into discrete domains. Proc Natl Acad Sci U S A. 1997 Apr 15;94(8):3877–3882. doi: 10.1073/pnas.94.8.3877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mittnacht S., Weinberg R. A. G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment. Cell. 1991 May 3;65(3):381–393. doi: 10.1016/0092-8674(91)90456-9. [DOI] [PubMed] [Google Scholar]
  25. Myers M. P., Wager-Smith K., Rothenfluh-Hilfiker A., Young M. W. Light-induced degradation of TIMELESS and entrainment of the Drosophila circadian clock. Science. 1996 Mar 22;271(5256):1736–1740. doi: 10.1126/science.271.5256.1736. [DOI] [PubMed] [Google Scholar]
  26. Paietta J. V. Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa. Mol Cell Biol. 1992 Apr;12(4):1568–1577. doi: 10.1128/mcb.12.4.1568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Price J. L., Dembinska M. E., Young M. W., Rosbash M. Suppression of PERIOD protein abundance and circadian cycling by the Drosophila clock mutation timeless. EMBO J. 1995 Aug 15;14(16):4044–4049. doi: 10.1002/j.1460-2075.1995.tb00075.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Saez L., Young M. W. Regulation of nuclear entry of the Drosophila clock proteins period and timeless. Neuron. 1996 Nov;17(5):911–920. doi: 10.1016/s0896-6273(00)80222-6. [DOI] [PubMed] [Google Scholar]
  29. Sauman I., Reppert S. M. Circadian clock neurons in the silkmoth Antheraea pernyi: novel mechanisms of Period protein regulation. Neuron. 1996 Nov;17(5):889–900. doi: 10.1016/s0896-6273(00)80220-2. [DOI] [PubMed] [Google Scholar]
  30. Templeton D. J., Park S. H., Lanier L., Weinberg R. A. Nonfunctional mutants of the retinoblastoma protein are characterized by defects in phosphorylation, viral oncoprotein association, and nuclear tethering. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3033–3037. doi: 10.1073/pnas.88.8.3033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vosshall L. B., Price J. L., Sehgal A., Saez L., Young M. W. Block in nuclear localization of period protein by a second clock mutation, timeless. Science. 1994 Mar 18;263(5153):1606–1609. doi: 10.1126/science.8128247. [DOI] [PubMed] [Google Scholar]
  32. Wood K. V. Marker proteins for gene expression. Curr Opin Biotechnol. 1995 Feb;6(1):50–58. doi: 10.1016/0958-1669(95)80009-3. [DOI] [PubMed] [Google Scholar]
  33. Wuarin J., Falvey E., Lavery D., Talbot D., Schmidt E., Ossipow V., Fonjallaz P., Schibler U. The role of the transcriptional activator protein DBP in circadian liver gene expression. J Cell Sci Suppl. 1992;16:123–127. doi: 10.1242/jcs.1992.supplement_16.15. [DOI] [PubMed] [Google Scholar]
  34. Zeng H., Qian Z., Myers M. P., Rosbash M. A light-entrainment mechanism for the Drosophila circadian clock. Nature. 1996 Mar 14;380(6570):129–135. doi: 10.1038/380129a0. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES