The Mub1/Ubr2 Ubiquitin Ligase Complex Regulates the Conserved Dsn1 Kinetochore Protein

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The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-C^Mif2 protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.

Vyšlo v časopise: The Mub1/Ubr2 Ubiquitin Ligase Complex Regulates the Conserved Dsn1 Kinetochore Protein. PLoS Genet 9(2): e32767. doi:10.1371/journal.pgen.1003216
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003216

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1. HollandAJ, ClevelandDW (2012) Losing balance: the origin and impact of aneuploidy in cancer. EMBO Rep 13: 501–514.

2. WestermannS, DrubinDG, BarnesG (2007) Structures and functions of yeast kinetochore complexes. Annu Rev Biochem 76: 563–591.

3. CheesemanIM, DesaiA (2008) Molecular architecture of the kinetochore-microtubule interface. Nat Rev Mol Cell Biol 9: 33–46.

4. SantaguidaS, MusacchioA (2009) The life and miracles of kinetochores. EMBO J 28: 2511–2531.

5. BloomK, JoglekarA (2010) Towards building a chromosome segregation machine. Nature 463: 446–456.

6. LampertF, WestermannS (2011) A blueprint for kinetochores - new insights into the molecular mechanics of cell division. Nat Rev Mol Cell Biol 12: 407–412.

7. AllshireRC, KarpenGH (2008) Epigenetic regulation of centromeric chromatin: old dogs, new tricks? Nat Rev Genet 9: 923–937.

8. BlackBE, ClevelandDW (2011) Epigenetic Centromere Propagation and the Nature of CENP-A Nucleosomes. Cell 144: 471–479.

9. McAinshAD, TytellJD, SorgerPK (2003) Structure, function, and regulation of budding yeast kinetochores. Annu Rev Cell Dev Biol 19: 519–539.

10. TakeuchiK, FukagawaT (2012) Molecular architecture of vertebrate kinetochores. Exp Cell Res 318: 1367–1374.

11. TanakaK, ChangHL, KagamiA, WatanabeY (2009) CENP-C functions as a scaffold for effectors with essential kinetochore functions in mitosis and meiosis. Dev Cell 17: 334–343.

12. OkadaM, CheesemanIM, HoriT, OkawaK, McLeodIX, et al. (2006) The CENP-H-I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres. Nat Cell Biol 8: 446–457.

13. CarrollCW, StraightAF (2006) Centromere formation: from epigenetics to self-assembly. Trends Cell Biol 16: 70–78.

14. CheesemanIM, ChappieJS, Wilson-KubalekEM, DesaiA (2006) The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127: 983–997.

15. AkiyoshiB, SarangapaniKK, PowersAF, NelsonCR, ReichowSL, et al. (2010) Tension directly stabilizes reconstituted kinetochore-microtubule attachments. Nature 468: 576–579.

16. KlineSL, CheesemanIM, HoriT, FukagawaT, DesaiA (2006) The human Mis12 complex is required for kinetochore assembly and proper chromosome segregation. J Cell Biol 173: 9–17.

17. CheesemanIM, HoriT, FukagawaT, DesaiA (2008) KNL1 and the CENP-H/I/K complex coordinately direct kinetochore assembly in vertebrates. Mol Biol Cell 19: 587–594.

18. DaviesAE, KaplanKB (2010) Hsp90-Sgt1 and Skp1 target human Mis12 complexes to ensure efficient formation of kinetochore-microtubule binding sites. J Cell Biol 189: 261–274.

19. MaskellDP, HuXW, SingletonMR (2010) Molecular architecture and assembly of the yeast kinetochore MIND complex. J Cell Biol 190: 823–834.

20. PetrovicA, PasqualatoS, DubeP, KrennV, SantaguidaS, et al. (2010) The MIS12 complex is a protein interaction hub for outer kinetochore assembly. J Cell Biol 190: 835–852.

21. PrzewlokaMR, VenkeiZ, Bolanos-GarciaVM, DebskiJ, DadlezM, et al. (2011) CENP-C is a structural platform for kinetochore assembly. Curr Biol 21: 399–405.

22. ScrepantiE, De AntoniA, AlushinGM, PetrovicA, MelisT, et al. (2011) Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore. Curr Biol 21: 391–398.

23. DeshaiesRJ, JoazeiroCA (2009) RING domain E3 ubiquitin ligases. Annu Rev Biochem 78: 399–434.

24. HershkoA (2005) The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Differ 12: 1191–1197.

25. KavsakP, RasmussenRK, CausingCG, BonniS, ZhuH, et al. (2000) Smad7 binds to Smurf2 to form an E3 ubiquitin ligase that targets the TGF beta receptor for degradation. Mol Cell 6: 1365–1375.

26. JuD, WangX, XuH, XieY (2008) Genome-wide analysis identifies MYND-domain protein Mub1 as an essential factor for Rpn4 ubiquitylation. Mol Cell Biol 28: 1404–1412.

27. Moreno-MorenoO, Torras-LlortM, AzorinF (2006) Proteolysis restricts localization of CID, the centromere-specific histone H3 variant of Drosophila, to centromeres. Nucleic Acids Res 34: 6247–6255.

28. CollinsKA, FuruyamaS, BigginsS (2004) Proteolysis contributes to the exclusive centromere localization of the yeast Cse4/CENP-A histone H3 variant. Curr Biol 14: 1968–1972.

29. KaplanKB, HymanAA, SorgerPK (1997) Regulating the yeast kinetochore by ubiquitin-dependent degradation and Skp1p-mediated phosphorylation. Cell 91: 491–500.

30. KopskiKM, HuffakerTC (1997) Suppressors of the ndc10-2 mutation: a role for the ubiquitin system in Saccharomyces cerevisiae kinetochore function. Genetics 147: 409–420.

31. WangL, MaoX, JuD, XieY (2004) Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase. J Biol Chem 279: 55218–55223.

32. JuD, WangL, MaoX, XieY (2004) Homeostatic regulation of the proteasome via an Rpn4-dependent feedback circuit. Biochem Biophys Res Commun 321: 51–57.

33. AndresonBL, GuptaA, GeorgievaBP, RothsteinR (2010) The ribonucleotide reductase inhibitor, Sml1, is sequentially phosphorylated, ubiquitylated and degraded in response to DNA damage. Nucleic Acids Res 38: 6490–6501.

34. NillegodaNB, TheodorakiMA, MandalAK, MayoKJ, RenHY, et al. (2010) Ubr1 and Ubr2 function in a quality control pathway for degradation of unfolded cytosolic proteins. Mol Biol Cell 21: 2102–2116.

35. AkiyoshiB, NelsonCR, RanishJA, BigginsS (2009) Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit. Genes Dev 23: 2887–2899.

36. EverettRD, EarnshawWC, FindlayJ, LomonteP (1999) Specific destruction of kinetochore protein CENP-C and disruption of cell division by herpes simplex virus immediate-early protein Vmw110. EMBO J 18: 1526–1538.

37. WestermannS, CheesemanIM, AndersonS, YatesJR3rd, DrubinDG, et al. (2003) Architecture of the budding yeast kinetochore reveals a conserved molecular core. J Cell Biol 163: 215–222.

38. WelburnJP, VleugelM, LiuD, YatesJR3rd, LampsonMA, et al. (2010) Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface. Mol Cell 38: 383–392.

39. RanjitkarP, PressMO, YiX, BakerR, MacCossMJ, BigginsS (2010) An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain. Mol Cell 40: 455–464.

40. GoshimaG, YanagidaM (2000) Establishing biorientation occurs with precocious separation of the sister kinetochores, but not the arms, in the early spindle of budding yeast. Cell 100: 619–633.

41. StraightAF, BelmontAS, RobinettCC, MurrayAW (1996) GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion. Curr Biol 6: 1599–1608.

42. GohPY, KilmartinJV (1993) NDC10: a gene involved in chromosome segregation in Saccharomyces cerevisiae. J Cell Biol 121: 503–512.

43. WiggePA, JensenON, HolmesS, SouesS, MannM, et al. (1998) Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. J Cell Biol 141: 967–977.

44. HeX, AsthanaS, SorgerPK (2000) Transient sister chromatid separation and elastic deformation of chromosomes during mitosis in budding yeast. Cell 101: 763–775.

45. PearsonCG, YehE, GardnerM, OddeD, SalmonED, et al. (2004) Stable kinetochore-microtubule attachment constrains centromere positioning in metaphase. Curr Biol 14: 1962–1967.

46. PinskyBA, KungC, ShokatKM, BigginsS (2006) The Ipl1-Aurora protein kinase activates the spindle checkpoint by creating unattached kinetochores. Nat Cell Biol 8: 78–83.

47. PengY, WongCC, NakajimaY, TyersRG, SarkeshikAS, et al. (2011) Overlapping kinetochore targets of CK2 and Aurora B kinases in mitotic regulation. Mol Biol Cell 22: 2680–2689.

48. LiR, MurrayAW (1991) Feedback control of mitosis in budding yeast. Cell 66: 519–531.

49. HoytMA, TotisL, RobertsBT (1991) S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66: 507–517.

50. MarstonAL, ThamWH, ShahH, AmonA (2004) A genome-wide screen identifies genes required for centromeric cohesion. Science 303: 1367–1370.

51. CorbettKD, YipCK, EeLS, WalzT, AmonA, et al. (2010) The monopolin complex crosslinks kinetochore components to regulate chromosome-microtubule attachments. Cell 142: 556–567.

52. BettingJ, SeufertW (1996) A yeast Ubc9 mutant protein with temperature-sensitive in vivo function is subject to conditional proteolysis by a ubiquitin- and proteasome-dependent pathway. J Biol Chem 271: 25790–25796.

53. BordalloJ, PlemperRK, FingerA, WolfDH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol Biol Cell 9: 209–222.

54. BaysNW, GardnerRG, SeeligLP, JoazeiroCA, HamptonRY (2001) Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation. Nat Cell Biol 3: 24–29.

55. BrewCT, HuffakerTC (2002) The yeast ubiquitin protease, Ubp3p, promotes protein stability. Genetics 162: 1079–1089.

56. GardnerRG, NelsonZW, GottschlingDE (2005) Degradation-mediated protein quality control in the nucleus. Cell 120: 803–815.

57. EmanueleMJ, LanW, JwaM, MillerSA, ChanCS, et al. (2008) Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly. J Cell Biol 181: 241–254.

58. YangY, WuF, WardT, YanF, WuQ, et al. (2008) Phosphorylation of HsMis13 by Aurora B kinase is essential for assembly of functional kinetochore. J Biol Chem 283: 26726–26736.

59. KiyomitsuT, MurakamiH, YanagidaM (2011) Protein interaction domain mapping of human kinetochore protein Blinkin reveals a consensus motif for binding of spindle assembly checkpoint proteins Bub1 and BubR1. Mol Cell Biol 31: 998–1011.

60. AmannJM, NipJ, StromDK, LutterbachB, HaradaH, et al. (2001) ETO, a target of t(8;21) in acute leukemia, makes distinct contacts with multiple histone deacetylases and binds mSin3A through its oligomerization domain. Mol Cell Biol 21: 6470–6483.

61. IsobeT, UchidaC, HattoriT, KitagawaK, OdaT, et al. (2006) Ubiquitin-dependent degradation of adenovirus E1A protein is inhibited by BS69. Biochem Biophys Res Commun 339: 367–374.

62. ChoiKO, LeeT, LeeN, KimJH, YangEG, et al. (2005) Inhibition of the catalytic activity of hypoxia-inducible factor-1alpha-prolyl-hydroxylase 2 by a MYND-type zinc finger. Mol Pharmacol 68: 1803–1809.

63. HessonLB, CooperWN, LatifF (2007) Evaluation of the 3p21.3 tumour-suppressor gene cluster. Oncogene 26: 7283–7301.

64. Rose MD, Winston F, Heiter P (1990) Methods in yeast genetics. Cold Spring Harbor, N. Y.: Cold Spring Harbor Laboratory Press. p. 198 p.

65. BrownMT, GoetschL, HartwellLH (1993) MIF2 is required for mitotic spindle integrity during anaphase spindle elongation in Saccharomyces cerevisiae. J Cell Biol 123: 387–403.

66. GhislainM, UdvardyA, MannC (1993) S. cerevisiae 26S protease mutants arrest cell division in G2/metaphase. Nature 366: 358–362.

67. NekrasovVS, SmithMA, Peak-ChewS, KilmartinJV (2003) Interactions between centromere complexes in Saccharomyces cerevisiae. Mol Biol Cell 14: 4931–4946.

68. OrtizJ, StemmannO, RankS, LechnerJ (1999) A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore. Genes Dev 13: 1140–1155.

69. Enquist-NewmanM, CheesemanIM, Van GoorD, DrubinDG, MeluhPB, et al. (2001) Dad1p, third component of the Duo1p/Dam1p complex involved in kinetochore function and mitotic spindle integrity. Mol Biol Cell 12: 2601–2613.

70. LiY, BachantJ, AlcasabasAA, WangY, QinJ, et al. (2002) The mitotic spindle is required for loading of the DASH complex onto the kinetochore. Genes Dev 16: 183–197.

71. BigginsS, BhallaN, ChangA, SmithDL, MurrayAW (2001) Genes Involved in Sister Chromatid Separation and Segregation in the Budding Yeast Saccharomyces cerevisiae. Genetics 159: 453–470.

72. LongtineMS, McKenzieA3rd, DemariniDJ, ShahNG, WachA, et al. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953–961.

73. GelbartME, RechsteinerT, RichmondTJ, TsukiyamaT (2001) Interactions of Isw2 chromatin remodeling complex with nucleosomal arrays: analyses using recombinant yeast histones and immobilized templates. Mol Cell Biol 21: 2098–2106.

74. GoldsteinAL, McCuskerJH (1999) Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15: 1541–1553.

75. PinskyBA, TatsutaniSY, CollinsKA, BigginsS (2003) An Mtw1 complex promotes kinetochore biorientation that is monitored by the Ipl1/Aurora protein kinase. Dev Cell 5: 735–745.

76. SikorskiRS, HieterP (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27.

77. AkiyoshiB, BigginsS (2010) Cdc14-dependent dephosphorylation of a kinetochore protein prior to anaphase in Saccharomyces cerevisiae. Genetics 186: 1487–1491.

78. BigginsS, SeverinFF, BhallaN, SassoonI, HymanAA, et al. (1999) The conserved protein kinase Ipl1 regulates microtubule binding to kinetochores in budding yeast. Genes Dev 13: 532–544.

79. SwerdlowPS, FinleyD, VarshavskyA (1986) Enhancement of immunoblot sensitivity by heating of hydrated filters. Anal Biochem 156: 147–153.