TIM Binds Importin α1, and Acts as an Adapter to Transport PER to the Nucleus

English version České info

In Drosophila, circadian rhythms are driven by a negative feedback loop that includes the key regulators, period (per) and timeless (tim). To generate this feedback loop, PER and TIM proteins first accumulate in the cytoplasm and then translocate to the nucleus where PER represses transcription. Thus, the nuclear import of PER-TIM proteins is a critical step to separate the phases of activation and repression of mRNA synthesis. In this study, we discovered that a member of the nuclear import machinery, importin α1 is an essential component of this feedback loop. Flies lacking importin α1 (IMPα1) display arrhythmic behavior and cytoplasmic expression of both PER and TIM at all times. In cultured S2 cells, IMPα1 expression directly facilitates nuclear import of TIM, but the effect on PER appears to be indirect. TIM expression is detected at the nuclear envelope and it interacts with other components of the nuclear transport machinery, which we show are also required for nuclear expression of TIM-PER and for behavioral rhythms. Our results thus suggest that TIM functions to link PER to the nuclear import machinery through IMPα1. Altogether, this study provides the mechanistic basis of a crucial step in the circadian clock mechanism.

Vyšlo v časopise: TIM Binds Importin α1, and Acts as an Adapter to Transport PER to the Nucleus. PLoS Genet 11(2): e32767. doi:10.1371/journal.pgen.1004974
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004974

Souhrn

Zdroje

1. Zheng X, Sehgal A (2012) Speed control: cogs and gears that drive the circadian clock. Trends Neurosci 35: 574–585. doi: 10.1016/j.tins.2012.05.007 22748426

2. Hardin PE (2011) Molecular genetic analysis of circadian timekeeping in Drosophila. Adv Genet 74: 141–173. doi: 10.1016/B978-0-12-387690-4.00005-2 21924977

3. Rothenfluh A, Young MW, Saez L (2000) A TIMELESS-independent function for PERIOD proteins in the Drosophila clock. Neuron 26: 505–514. 10839368

4. Curtin KD, Huang ZJ, Rosbash M (1995) Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock. Neuron 14: 365–372. 7857645

5. Ko HW, Kim EY, Chiu J, Vanselow JT, Kramer A, et al. (2010) A hierarchical phosphorylation cascade that regulates the timing of PERIOD nuclear entry reveals novel roles for proline-directed kinases and GSK-3beta/SGG in circadian clocks. J Neurosci 30: 12664–12675. doi: 10.1523/JNEUROSCI.1586-10.2010 20861372

6. Chang DC, Reppert SM (2003) A novel C-terminal domain of drosophila PERIOD inhibits dCLOCK:CYCLE-mediated transcription. Curr Biol 13: 758–762. 12725734

7. Nawathean P, Rosbash M (2004) The doubletime and CKII kinases collaborate to potentiate Drosophila PER transcriptional repressor activity. Mol Cell 13: 213–223. 14759367

8. Cyran SA, Yiannoulos G, Buchsbaum AM, Saez L, Young MW, et al. (2005) The double-time protein kinase regulates the subcellular localization of the Drosophila clock protein period. J Neurosci 25: 5430–5437. 15930393

9. Meyer P, Saez L, Young MW (2006) PER-TIM interactions in living Drosophila cells: an interval timer for the circadian clock. Science 311: 226–229. 16410523

10. Price JL, Dembinska ME, Young MW, Rosbash M (1995) Suppression of PERIOD protein abundance and circadian cycling by the Drosophila clock mutation timeless. EMBO J 14: 4044–4049. 7664743

11. Suri V, Lanjuin A, Rosbash M (1999) TIMELESS-dependent positive and negative autoregulation in the Drosophila circadian clock. EMBO J 18: 675–686. 9927427

12. Ashmore LJ, Sathyanarayanan S, Silvestre DW, Emerson MM, Schotland P, et al. (2003) Novel insights into the regulation of the timeless protein. J Neurosci 23: 7810–7819. 12944510

13. Mason DA, Goldfarb DS (2009) The nuclear transport machinery as a regulator of Drosophila development. Semin Cell Dev Biol 20: 582–589. doi: 10.1016/j.semcdb.2009.02.006 19508860

14. Goldfarb DS, Corbett AH, Mason DA, Harreman MT, Adam SA (2004) Importin alpha: a multipurpose nuclear-transport receptor. Trends Cell Biol 14: 505–514. 15350979

15. Lange A, Mills RE, Lange CJ, Stewart M, Devine SE, et al. (2007) Classical nuclear localization signals: definition, function, and interaction with importin alpha. J Biol Chem 282: 5101–5105. 17170104

16. Cook A, Bono F, Jinek M, Conti E (2007) Structural biology of nucleocytoplasmic transport. Annu Rev Biochem 76: 647–671. 17506639

17. Stewart M (2007) Molecular mechanism of the nuclear protein import cycle. Nat Rev Mol Cell Biol 8: 195–208. 17287812

18. Xu D, Farmer A, Chook YM (2010) Recognition of nuclear targeting signals by Karyopherin-beta proteins. Curr Opin Struct Biol 20: 782–790. doi: 10.1016/j.sbi.2010.09.008 20951026

19. Chook YM, Suel KE (2011) Nuclear import by karyopherin-betas: recognition and inhibition. Biochim Biophys Acta 1813: 1593–1606. doi: 10.1016/j.bbamcr.2010.10.014 21029754

20. Kotera I, Sekimoto T, Miyamoto Y, Saiwaki T, Nagoshi E, et al. (2005) Importin alpha transports CaMKIV to the nucleus without utilizing importin beta. EMBO J 24: 942–951. 15719015

21. Fagotto F, Gluck U, Gumbiner BM (1998) Nuclear localization signal-independent and importin/karyopherin-independent nuclear import of beta-catenin. Curr Biol 8: 181–190. 9501980

22. Saez L, Young MW (1996) Regulation of nuclear entry of the Drosophila clock proteins period and timeless. Neuron 17: 911–920. 8938123

23. Saez L, Derasmo M, Meyer P, Stieglitz J, Young MW (2011) A key temporal delay in the circadian cycle of Drosophila is mediated by a nuclear localization signal in the timeless protein. Genetics 188: 591–600. doi: 10.1534/genetics.111.127225 21515571

24. Shafer OT, Rosbash M, Truman JW (2002) Sequential nuclear accumulation of the clock proteins period and timeless in the pacemaker neurons of Drosophila melanogaster. J Neurosci 22: 5946–5954. 12122057

25. Hara T, Koh K, Combs DJ, Sehgal A (2011) Post-translational regulation and nuclear entry of TIMELESS and PERIOD are affected in new timeless mutant. J Neurosci 31: 9982–9990. doi: 10.1523/JNEUROSCI.0993-11.2011 21734289

26. Garbe DS, Fang Y, Zheng X, Sowcik M, Anjum R, et al. (2013) Cooperative interaction between phosphorylation sites on PERIOD maintains circadian period in Drosophila. PLoS Genet 9: e1003749. doi: 10.1371/journal.pgen.1003749 24086144

27. Sledz CA, Williams BR (2005) RNA interference in biology and disease. Blood 106: 787–794. 15827131

28. Ratan R, Mason DA, Sinnot B, Goldfarb DS, Fleming RJ (2008) Drosophila importin alpha1 performs paralog-specific functions essential for gametogenesis. Genetics 178: 839–850. doi: 10.1534/genetics.107.081778 18245351

29. Vosshall LB, Price JL, Sehgal A, Saez L, Young MW (1994) Block in nuclear localization of period protein by a second clock mutation, timeless. Science 263: 1606–1609. 8128247

30. Meissner RA, Kilman VL, Lin JM, Allada R (2008) TIMELESS is an important mediator of CK2 effects on circadian clock function in vivo. J Neurosci 28: 9732–9740. doi: 10.1523/JNEUROSCI.0840-08.2008 18815259

31. Mosammaparast N, Pemberton LF (2004) Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol 14: 547–556. 15450977

32. Cingolani G, Petosa C, Weis K, Muller CW (1999) Structure of importin-beta bound to the IBB domain of importin-alpha. Nature 399: 221–229. 10353244

33. Catimel B, Teh T, Fontes MR, Jennings IG, Jans DA, et al. (2001) Biophysical characterization of interactions involving importin-alpha during nuclear import. J Biol Chem 276: 34189–34198. 11448961

34. Fanara P, Hodel MR, Corbett AH, Hodel AE (2000) Quantitative analysis of nuclear localization signal (NLS)-importin alpha interaction through fluorescence depolarization. Evidence for auto-inhibitory regulation of NLS binding. J Biol Chem 275: 21218–21223. 10806202

35. Harreman MT, Hodel MR, Fanara P, Hodel AE, Corbett AH (2003) The auto-inhibitory function of importin alpha is essential in vivo. J Biol Chem 278: 5854–5863. 12486120

36. Sakakida Y, Miyamoto Y, Nagoshi E, Akashi M, Nakamura TJ, et al. (2005) Importin alpha/beta mediates nuclear transport of a mammalian circadian clock component, mCRY2, together with mPER2, through a bipartite nuclear localization signal. J Biol Chem 280: 13272–13278. 15689618

37. Vodovar N, Clayton JD, Costa R, Odell M, Kyriacou CP (2002) The Drosophila clock protein Timeless is a member of the Arm/HEAT family. Curr Biol 12: R610–611. 12372263

38. Andrade MA, Petosa C, O'Donoghue SI, Muller CW, Bork P (2001) Comparison of ARM and HEAT protein repeats. J Mol Biol 309: 1–18. 11491282

39. Sehgal A, Price JL, Man B, Young MW (1994) Loss of circadian behavioral rhythms and per RNA oscillations in the Drosophila mutant timeless. Science 263: 1603–1606. 8128246

40. Chiu JC, Vanselow JT, Kramer A, Edery I (2008) The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock. Genes Dev 22: 1758–1772. doi: 10.1101/gad.1682708 18593878

41. Park JH, Helfrich-Forster C, Lee G, Liu L, Rosbash M, et al. (2000) Differential regulation of circadian pacemaker output by separate clock genes in Drosophila. Proc Natl Acad Sci U S A 97: 3608–3613. 10725392

42. Zheng X, Sowcik M, Chen D, Sehgal A (2014) Casein kinase 1 promotes synchrony of the circadian clock network. Mol Cell Biol.

43. Martinek S, Inonog S, Manoukian AS, Young MW (2001) A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock. Cell 105: 769–779. 11440719

44. Lin JM, Kilman VL, Keegan K, Paddock B, Emery-Le M, et al. (2002) A role for casein kinase 2alpha in the Drosophila circadian clock. Nature 420: 816–820. 12447397

45. Sathyanarayanan S, Zheng X, Xiao R, Sehgal A (2004) Posttranslational regulation of Drosophila PERIOD protein by protein phosphatase 2A. Cell 116: 603–615. 14980226

46. Sun WC, Jeong EH, Jeong HJ, Ko HW, Edery I, et al. (2010) Two distinct modes of PERIOD recruitment onto dCLOCK reveal a novel role for TIMELESS in circadian transcription. J Neurosci 30: 14458–14469. doi: 10.1523/JNEUROSCI.2366-10.2010 20980603

47. Ko HW, Jiang J, Edery I (2002) Role for Slimb in the degradation of Drosophila Period protein phosphorylated by Doubletime. Nature 420: 673–678. 12442174

48. Koh K, Zheng X, Sehgal A (2006) JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science 312: 1809–1812. 16794082

49. Peschel N, Chen KF, Szabo G, Stanewsky R (2009) Light-dependent interactions between the Drosophila circadian clock factors cryptochrome, jetlag, and timeless. Curr Biol 19: 241–247. doi: 10.1016/j.cub.2008.12.042 19185492

50. Koizumi K, Stivers C, Brody T, Zangeneh S, Mozer B, et al. (2001) A search for Drosophila neural precursor genes identifies ran. Dev Genes Evol 211: 67–75. 11455416

51. Bischof J, Maeda RK, Hediger M, Karch F, Basler K (2007) An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases. Proc Natl Acad Sci U S A 104: 3312–3317. 17360644

52. Williams JA, Su HS, Bernards A, Field J, Sehgal A (2001) A circadian output in Drosophila mediated by neurofibromatosis-1 and Ras/MAPK. Science 293: 2251–2256. 11567138

53. Chiu JC, Ko HW, Edery I (2011) NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed. Cell 145: 357–370. doi: 10.1016/j.cell.2011.04.002 21514639

54. Capelson M, Liang Y, Schulte R, Mair W, Wagner U, et al. (2010) Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes. Cell 140: 372–383. doi: 10.1016/j.cell.2009.12.054 20144761

55. Kelley LA, Sternberg MJ (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 4: 363–371. doi: 10.1038/nprot.2009.2 19247286

56. Kim DE, Chivian D, Baker D (2004) Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res 32: W526–531. 15215442

57. Roy A, Kucukural A, Zhang Y I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5: 725–738. doi: 10.1038/nprot.2010.5 20360767

58. Lobley A, Sadowski MI, Jones DT (2009) pGenTHREADER and pDomTHREADER: new methods for improved protein fold recognition and superfamily discrimination. Bioinformatics 25: 1761–1767. doi: 10.1093/bioinformatics/btp302 19429599

59. Soding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33: W244–248. 15980461

60. Kallberg M, Wang H, Wang S, Peng J, Wang Z, et al. Template-based protein structure modeling using the RaptorX web server. Nat Protoc 7: 1511–1522. doi: 10.1038/nprot.2012.085 22814390

61. Giarre M, Torok I, Schmitt R, Gorjanacz M, Kiss I, et al. (2002) Patterns of importin-alpha expression during Drosophila spermatogenesis. J Struct Biol 140: 279–290. 12490175

62. Buchan DW, Minneci F, Nugent TC, Bryson K, Jones DT Scalable web services for the PSIPRED Protein Analysis Workbench. Nucleic Acids Res 41: W349–357. doi: 10.1093/nar/gkt381 23748958