Cdc5-Dependent Asymmetric Localization of Bfa1 Fine-Tunes Timely Mitotic Exit
In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: 452S, 453S, 454S, and 559S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa14A) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa14D) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa14D was asymmetrically localized. BFA14A cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA14D cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.
Vyšlo v časopise:
Cdc5-Dependent Asymmetric Localization of Bfa1 Fine-Tunes Timely Mitotic Exit. PLoS Genet 8(1): e32767. doi:10.1371/journal.pgen.1002450
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002450
Souhrn
In budding yeast, the major regulator of the mitotic exit network (MEN) is Tem1, a GTPase, which is inhibited by the GTPase-activating protein (GAP), Bfa1/Bub2. Asymmetric Bfa1 localization to the bud-directed spindle pole body (SPB) during metaphase also controls mitotic exit, but the molecular mechanism and function of this localization are not well understood, particularly in unperturbed cells. We identified four novel Cdc5 target residues within the Bfa1 C-terminus: 452S, 453S, 454S, and 559S. A Bfa1 mutant in which all of these residues had been changed to alanine (Bfa14A) persisted on both SPBs at anaphase and was hypo-phosphorylated, despite retaining its GAP activity for Tem1. A Bfa1 phospho-mimetic mutant in which all of these residues were switched to aspartate (Bfa14D) always localized asymmetrically to the SPB. These observations demonstrate that asymmetric localization of Bfa1 is tightly linked to its Cdc5-dependent phosphorylation, but not to its GAP activity. Consistent with this, in kinase-defective cdc5-2 cells Bfa1 was not phosphorylated and localized to both SPBs, whereas Bfa14D was asymmetrically localized. BFA14A cells progressed through anaphase normally but displayed delayed mitotic exit in unperturbed cell cycles, while BFA14D cells underwent mitotic exit with the same kinetics as wild-type cells. We suggest that Cdc5 induces the asymmetric distribution of Bfa1 to the bud-directed SPB independently of Bfa1 GAP activity at anaphase and that Bfa1 asymmetry fine-tunes the timing of MEN activation in unperturbed cell cycles.
Zdroje
1. NiggEA 2001 Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2 21 32
2. MorganDO 1999 Regulation of the APC and the exit from mitosis. Nat Cell Biol 1 E47 53
3. QueraltEUhlmannF 2008 Cdk-counteracting phosphatases unlock mitotic exit. Curr Opin Cell Biol 20 661 668
4. BardinAJAmonA 2001 Men and sin: what's the difference? Nat Rev Mol Cell Biol 2 815 826
5. WangYHuFElledgeSJ 2000 The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit. Curr Biol 10 1379 1382
6. BloecherAVenturiGMTatchellK 2000 Anaphase spindle position is monitored by the BUB2 checkpoint. Nat Cell Biol 2 556 558
7. LeeSEFrenzLMWellsNJJohnsonALJohnstonLH 2001 Order of function of the budding-yeast mitotic exit-network proteins Tem1, Cdc15, Mob1, Dbf2, and Cdc5. Curr Biol 11 784 788
8. HuFWangYLiuDLiYQinJ 2001 Regulation of the Bub2/Bfa1 GAP complex by Cdc5 and cell cycle checkpoints. Cell 107 655 665
9. GeymonatMSpanosAWalkerPAJohnstonLHSedgwickSG 2003 In vitro regulation of budding yeast Bfa1/Bub2 GAP activity by Cdc5. J Biol Chem 278 14591 14594
10. KimJJangSSSongK 2008 Different levels of Bfa1/Bub2 GAP activity are required to prevent mitotic exit of budding yeast depending on the type of perturbations. Mol Biol Cell 19 4328 4340
11. ShirayamaMMatsuiYTohEA 1994 The yeast TEM1 gene, which encodes a GTP-binding protein, is involved in termination of M phase. Mol Cell Biol 14 7476 7482
12. GeymonatMSpanosAde BettigniesGSedgwickSG 2009 Lte1 contributes to Bfa1 localization rather than stimulating nucleotide exchange by Tem1. J Cell Biol 187 497 511
13. Valerio-SantiagoMMonje-CasasF 2011 Tem1 localization to the spindle pole bodies is essential for mitotic exit and impairs spindle checkpoint function. J Cell Biol 192 599 614
14. PereiraGHofkenTGrindlayJMansonCSchiebelE 2000 The Bub2p spindle checkpoint links nuclear migration with mitotic exit. Mol Cell 6 1 10
15. MolkJNSchuylerSCLiuJYEvansJGSalmonED 2004 The differential roles of budding yeast Tem1p, Cdc15p, and Bub2p protein dynamics in mitotic exit. Mol Biol Cell 15 1519 1532
16. PereiraGTanakaTUNasmythKSchiebelE 2001 Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex. EMBO J 20 6359 6370
17. FraschiniRD'AmbrosioCVenturettiMLucchiniGPiattiS 2006 Disappearance of the budding yeast Bub2-Bfa1 complex from the mother-bound spindle pole contributes to mitotic exit. J Cell Biol 172 335 346
18. Monje-CasasFAmonA 2009 Cell polarity determinants establish asymmetry in MEN signaling. Dev Cell 16 132 145
19. CaydasiAKPereiraG 2009 Spindle alignment regulates the dynamic association of checkpoint proteins with yeast spindle pole bodies. Dev Cell 16 146 156
20. CharlesJFJaspersenSLTinker-KulbergRLHwangLSzidonA 1998 The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae. Curr Biol 8 497 507
21. ShirayamaMZachariaeWCioskRNasmythK 1998 The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae. EMBO J 17 1336 1349
22. KimJJeongJSongK 2004 The C-terminus of Bfa1p in budding yeast is essential to induce mitotic arrest in response to diverse checkpoint-activating signals. Genes Cells 9 399 418
23. Cohen-FixOPetersJMKirschnerMWKoshlandD 1996 Anaphase initiation in Saccharomyces cerevisiae is controlled by the APC-dependent degradation of the anaphase inhibitor Pds1p. Genes Dev 10 3081 3093
24. ZachariaeWNasmythK 1999 Whose end is destruction: cell division and the anaphase-promoting complex. Genes Dev 13 2039 2058
25. ShouWSeolJHShevchenkoABaskervilleCMoazedD 1999 Exit from mitosis is triggered by Tem1-dependent release of the phosphatase Cdc14 from nucleolar RENT complex. Cell 97 233 244
26. WäschRCrossFR 2002 APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit. Nature 418 495 6
27. LiYYYehEHaysTBloomK 1993 Disruption of mitotic spindle orientation in a yeast dynein mutant. Proc Natl Acad Sci U S A 90 10096 10100
28. LeeLTirnauerJSLiJSchuylerSCLiuJY 2000 Positioning of the mitotic spindle by a cortical-microtubule capture mechanism. Science 287 2260 2262
29. AdamesNROberleJRCooperJA 2001 The surveillance mechanism of the spindle position checkpoint in yeast. J Cell Biol 153 159 168
30. LiakopoulosDKuschJGravaSVogelJBarralY 2003 Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment. Cell 112 561 574
31. GravaSSchaererFFatyMPhilippsenPBarralY 2006 Asymmetric recruitment of dynein to spindle poles and microtubules promotes proper spindle orientation in yeast. Dev Cell 10 425 439
32. KonigCMaekawaHSchiebelE 2010 Mutual regulation of cyclin-dependent kinase and the mitotic exit network. J Cell Biol 188 351 368
33. BertazziDTKurtulmusBPereiraG 2011 The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4. J Cell Biol 193 1033 1048
34. MorganD 2007 The Cell Cycle: Principle of control University of Oxford press 6
35. DrapkinBJLuYProckoALTimneyBLCrossFR 2009 Analysis of the mitotic exit control system using locked levels of stable mitotic cyclin. Mol Syst Biol 5 328
36. PereiraGSchiebelE 2005 Kin4 kinase delays mitotic exit in response to spindle alignment defects. Mol Cell 19 209 221
37. LoweryDMLimDYaffeMB 2005 Structure and function of Polo-like kinases. Oncogene 24 248 259
38. MaekawaHPriestCLechnerJPereiraGSchiebelE 2007 The yeast centrosome translates the positional information of the anaphase spindle into a cell cycle signal. J Cell Biol 179 423 436
39. CeruttiLSimanisV 1999 Asymmetry of the spindle pole bodies and spg1p GAP segregation during mitosis in fission yeast. J Cell Sci 112 2313 2321
40. LongtineMSMcKenzieA3rdDemariniDJShahNGWachA 1998 Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14 953 961
41. JankeCMagieraMMRathfelderNTaxisCReberS 2004 A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21 947 962
42. KinoshitaEKinoshita-KikutaETakiyamaKKoikeT 2006 Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5 749 757
43. LeeJMKimSLeeJYYooEYChoMC 2006 A differentially expressed proteomic analysis in placental tissues in relation to pungency during the pepper fruit development. Proteomics 6 5248 5259
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2012 Číslo 1
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