Whole Genome Sequencing Identifies a Deletion in Protein Phosphatase 2A That Affects Its Stability and Localization in

English version České info

Whole genome sequencing is a powerful tool in the discovery of single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) among mutant strains, which simplifies forward genetics approaches. However, identification of the causative mutation among a large number of non-causative SNPs in a mutant strain remains a big challenge. In the unicellular biflagellate green alga Chlamydomonas reinhardtii, we generated a SNP/indel library that contains over 2 million polymorphisms from four wild-type strains, one highly polymorphic strain that is frequently used in meiotic mapping, ten mutant strains that have flagellar assembly or motility defects, and one mutant strain, imp3, which has a mating defect. A comparison of polymorphisms in the imp3 strain and the other 15 strains allowed us to identify a deletion of the last three amino acids, Y₃₁₃F₃₁₄L₃₁₅, in a protein phosphatase 2A catalytic subunit (PP2A3) in the imp3 strain. Introduction of a wild-type HA-tagged PP2A3 rescues the mutant phenotype, but mutant HA-PP2A3 at Y₃₁₃ or L₃₁₅ fail to rescue. Our immunoprecipitation results indicate that the Y₃₁₃, L₃₁₅, or YFLΔ mutations do not affect the binding of PP2A3 to the scaffold subunit, PP2A-2r. In contrast, the Y₃₁₃, L₃₁₅, or YFLΔ mutations affect both the stability and the localization of PP2A3. The PP2A3 protein is less abundant in these mutants and fails to accumulate in the basal body area as observed in transformants with either wild-type HA-PP2A3 or a HA-PP2A3 with a V₃₁₀T change. The accumulation of HA-PP2A3 in the basal body region disappears in mated dikaryons, which suggests that the localization of PP2A3 may be essential to the mating process. Overall, our results demonstrate that the terminal YFL tail of PP2A3 is important in the regulation on Chlamydomonas mating.

Vyšlo v časopise: Whole Genome Sequencing Identifies a Deletion in Protein Phosphatase 2A That Affects Its Stability and Localization in. PLoS Genet 9(9): e32767. doi:10.1371/journal.pgen.1003841
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003841

Souhrn

Zdroje

1. Lawson NathanD, Wolfe ScotA (2011) Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish. Developmental cell 21: 48–64.

2. KileBT, HiltonDJ (2005) The art and design of genetic screens: mouse. Nature Reviews Genetics 6: 557–567.

3. PazourGJ, DickertBL, VucicaY, SeeleyES, RosenbaumJL, et al. (2000) Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene Tg737, are required for assembly of cilia and flagella. The Journal of Cell Biology 151: 709–718.

4. DutcherSK, TrabucoEC (1998) The UNI3 gene is required for assembly of basal bodies of Chlamydomonas and encodes δ-Tubulin, a new member of the tubulin superfamily. Molecular Biology of the Cell 9: 1293–1308.

5. TaxFE, VernonDM (2001) T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics. Plant Physiology 126: 1527–1538.

6. AmsterdamA, BurgessS, GollingG, ChenW, SunZ, et al. (1999) A large-scale insertional mutagenesis screen in zebrafish. Genes & Development 13: 2713–2724.

7. AlonsoJM, StepanovaAN, LeisseTJ, KimCJ, ChenH, et al. (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301: 653–657.

8. GollingG, AmsterdamA, SunZ, AntonelliM, MaldonadoE, et al. (2002) Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nature Genetics 31: 135–140.

9. EsparzaJM, O'TooleE, LiL, GiddingsTHJr, KozakB, et al. (2013) Katanin localization requires triplet microtubules in Chlamydomonas reinhardtii. PLoS ONE 8: e53940.

10. JanderG, NorrisSR, RounsleySD, BushDF, LevinIM, et al. (2002) Arabidopsis map-based cloning in the post-genome era. Plant Physiology 129: 440–450.

11. MutoA, OrgerMB, WehmanAM, SmearMC, KayJN, et al. (2005) Forward genetic analysis of visual behavior in zebrafish. PLoS Genetics 1: e66.

12. WansleebenC, van GurpL, FeitsmaH, KroonC, RieterE, et al. (2011) An ENU-mutagenesis screen in the mouse: identification of novel developmental gene functions. PLoS ONE 6: e19357.

13. DutcherSK, MorrissetteNS, PrebleAM, RackleyC, StangaJ (2002) ε-Tubulin is an essential component of the centriole. Molecular Biology of the Cell 13: 3859–3869.

14. IominiC, LiL, EsparzaJM, DutcherSK (2009) Retrograde intraflagellar transport mutants identify Complex A proteins With multiple genetic interactions in Chlamydomonas reinhardtii. Genetics 183: 885–896.

15. GerholdAR, RichterDJ, YuAS, HariharanIK (2011) Identification and characterization of genes required for compensatory growth in Drosophila. Genetics 189: 1309–1326.

16. DoitsidouM, PooleRJ, SarinS, BigelowH, HobertO (2010) C. elegans mutant identification with a one-step whole-genome-sequencing and SNP mapping strategy. PLoS ONE 5: e15435.

17. MinevichG, ParkDS, BlankenbergD, PooleRJ, HobertO (2012) CloudMap: a cloud-based pipeline for analysis of mutant genome sequences. Genetics 192: 1249–1269.

18. DutcherSK, LiL, LinH, MeyerL, GiddingsTH, et al. (2012) Whole-genome sequencing to identify mutants and polymorphisms in Chlamydomonas reinhardtii. G3: Genes|Genomes|Genetics 2: 15–22.

19. OssowskiS, SchneebergerK, ClarkRM, LanzC, WarthmannN, et al. (2008) Sequencing of natural strains of Arabidopsis thaliana with short reads. Genome Research 18: 2024–2033.

20. SilflowCD, LefebvrePA (2001) Assembly and motility of eukaryotic cilia and flagella. Lessons from Chlamydomonas reinhardtii. Plant Physiology 127: 1500–1507.

21. GoodenoughUW, HwangC, MartinH (1976) Isolation and genetic analysis of mutant strains of Chlamydomonas reinhardi defective in gametic differentiation. Genetics 82: 169–186.

22. HwangCJ, MonkBC, GoodenoughUW (1981) Linkage of mutations affecting minus flagellar membrane agglutinability to the mt− mating-type locus of Chlamydomonas. Genetics 99: 41–47.

23. GoodenoughU, LinH, LeeJ-H (2007) Sex determination in Chlamydomonas. Semin Cell Dev Biol 18: 350–361.

24. FerrisPJ, WaffenschmidtS, UmenJG, LinH, LeeJ-H, et al. (2005) Plus and minus sexual agglutinins from Chlamydomonas reinhardtii. Plant Cell 17: 597–615.

25. VallonO, WollmanFA (1995) Mutations affecting O-glycosylation in Chlamydomonas reinhardtii cause delayed cell wall degradation and sex-limited sterility. Plant Physiology 108: 703–712.

26. FerrisPJ, WoessnerJP, GoodenoughUW (1996) A sex recognition glycoprotein is encoded by the plus mating-type gene fus1 of Chlamydomonas reinhardtii. Molecular Biology of the Cell 7: 1235–1248.

27. MisamoreMJ, GuptaS, SnellWJ (2003) The Chlamydomonas Fus1 protein is present on the mating type plus fusion organelle and required for a critical membrane adhesion event during fusion with minus gametes. Molecular Biology of the Cell 14: 2530–2542.

28. FerrisPJ, GoodenoughUW (1997) Mating type in Chlamydomonas is specified by mid, the minus-dominance gene. Genetics 146: 859–869.

29. LinH, GoodenoughUW (2007) Gametogenesis in the Chlamydomonas reinhardtii minus mating type is controlled by two genes, MID and MTD1. Genetics 176: 913–925.

30. SaitoT, SmallL, GoodenoughU (1993) Activation of adenylyl cyclase in Chlamydomonas reinhardtii by adhesion and by heat. The Journal of Cell Biology 122: 137–147.

31. JanssensV, GorisJ (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem J 353: 417–439.

32. ShiY (2009) Serine/threonine phosphatases: mechanism through structure. Cell 139: 468–484.

33. JanssensV, LonginS, GorisJ (2008) PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends in Biochemical Sciences 33: 113–121.

34. ElamCA, WirschellM, YamamotoR, FoxLA, YorkK, et al. (2011) An axonemal PP2A B-subunit is required for PP2A localization and flagellar motility. Cytoskeleton 68: 363–372.

35. Harris E, Stern D, Witman G (2009) The Chlamydomonas sourcebook: introduction to Chlamydomonas and its laboratory use: Academic Press.

36. MerchantSS, ProchnikSE, VallonO, HarrisEH, KarpowiczSJ, et al. (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318: 245–250.

37. FerrisPJ, ArmbrustEV, GoodenoughUW (2002) Genetic Structure of the Mating-Type Locus of Chlamydomonas reinhardtii. Genetics 160: 181–200.

38. HuangB, RifkinMR, LuckDJ (1977) Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii. The Journal of Cell Biology 72: 67–85.

39. PröscholdT, HarrisEH, ColemanAW (2005) Portrait of a Species: Chlamydomonas reinhardtii. Genetics 170: 1601–1610.

40. CohenP, KlumppS, SchellingDL (1989) An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Letters 250: 596–600.

41. ChenJ, MartinB, BrautiganD (1992) Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257: 1261–1264.

42. LiM, DamuniZ (1994) Okadaic acid and microcystin-LR directly inhibit the methylation of protein phosphatase 2A by its specific methyltransferase. Biochemical and Biophysical Research Communications 202: 1023–1030.

43. FloerM, StockJ (1994) Carboxyl methylation of protein phosphatase 2A from Xenopus eggs is stimulated by cAMP and inhibited by okadaic acid. Biochemical and Biophysical Research Communications 198: 372–379.

44. TakaiA, SasakiK, NagaiH, MieskesG, IsobeM, et al. (1995) Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein. Biochemical Journal 306: 657.

45. LiY, WangX, YueP, TaoH, RamalingamSS, et al. (2013) Protein phosphatase 2A and DNA-dependent protein kinase are involved in mediating rapamycin-induced Akt phosphorylation. Journal of Biological Chemistry 288: 13215–13224.

46. FavreB, TurowskiP, HemmingsBA (1997) Differential inhibition and posttranslational modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic acid, and tautomycin. Journal of Biological Chemistry 272: 13856–13863.

47. NamboodiripadAN, JenningsML (1996) Permeability characteristics of erythrocyte membrane to okadaic acid and calyculin A. American Journal of Physiology - Cell Physiology 270: C449–C456.

48. WagnerV, GeßnerG, HeilandI, KaminskiM, HawatS, et al. (2006) Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways. Eukaryotic Cell 5: 457–468.

49. PazourGJ, AgrinN, LeszykJ, WitmanGB (2005) Proteomic analysis of a eukaryotic cilium. The Journal of Cell Biology 170: 103–113.

50. WangQ, PanJ, SnellWJ (2006) Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas. Cell 125: 549–562.

51. BlumenstielJP, NollAC, GriffithsJA, PereraAG, WaltonKN, et al. (2009) Identification of EMS-induced mutations in Drosophila melanogaster by whole-genome sequencing. Genetics 182: 25–32.

52. BamshadMJ, NgSB, BighamAW, TaborHK, EmondMJ, et al. (2011) Exome sequencing as a tool for Mendelian disease gene discovery. Nature Reviews Genetics 12: 745–755.

53. Genomes Project Consortium (2012) An integrated map of genetic variation from 1,092 human genomes. Nature 491: 56–65.

54. CaoJ, SchneebergerK, OssowskiS, GuntherT, BenderS, et al. (2011) Whole-genome sequencing of multiple Arabidopsis thaliana populations. Nature Genetics 43: 956–963.

55. HuangB, PipernoG, RamanisZ, LuckDJ (1981) Radial spokes of Chlamydomonas flagella: genetic analysis of assembly and function. The Journal of Cell Biology 88: 80–88.

56. AlbeeAJ, KwanAL, LinH, GranasD, StormoGD, et al. (2013) Identification of cilia genes that affect cell cycle progression using whole genome transcriptome analysis in Chlamydomonas reinhardtti. G3: Genes|Genomes|Genetics 979–91 doi: 10.1534/g3.113.006338

57. OrgadS, BrewisND, AlpheyL, AxtonJM, DudaiY, et al. (1990) The structure of protein phosphatase 2A is as highly conserved as that of protein phosphatase I. FEBS Letters 275: 44–48.

58. KitagawaD, FlückigerI, PolanowskaJ, KellerD, ReboulJ, et al. (2011) PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans embryos. Developmental cell 20: 550–562.

59. BallesterosI, DomínguezT, SauerM, ParedesP, DupratA, et al. (2013) Specialized functions of the PP2A subfamily II catalytic subunits PP2A-C3 and PP2A-C4 in the distribution of auxin fluxes and development in Arabidopsis. The Plant Journal 73: 862–872.

60. PernasM, García-CasadoG, RojoE, SolanoR, Sánchez-SerranoJJ (2007) A protein phosphatase 2A catalytic subunit is a negative regulator of abscisic acid signalling1. The Plant Journal 51: 763–778.

61. TangW, YuanM, WangR, YangY, WangC, et al. (2011) PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1. Nature Cell Biology 13: 124–131.

62. XuY, XingY, ChenY, ChaoY, LinZ, et al. (2006) Structure of the protein phosphatase 2A holoenzyme. Cell 127: 1239–1251.

63. LonginS, ZwaenepoelK, LouisJV, DilworthS, GorisJ, et al. (2007) Selection of protein phosphatase 2A regulatory subunits is mediated by the C terminus of the catalytic subunit. Journal of Biological Chemistry 282: 26971–26980.

64. Nunbhakdi-CraigV, SchuechnerS, SontagJ-M, MontgomeryL, PallasDC, et al. (2007) Expression of protein phosphatase 2A mutants and silencing of the regulatory Bα subunit induce a selective loss of acetylated and detyrosinated microtubules. Journal of Neurochemistry 101: 959–971.

65. WeiH, AshbyDG, MorenoCS, OgrisE, YeongFM, et al. (2001) Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits Cdc55p and Rts1p. Journal of Biological Chemistry 276: 1570–1577.

66. GentryMS, LiY, WeiH, SyedFF, PatelSH, et al. (2005) A novel assay for protein phosphatase 2A (PP2A) complexes in vivo reveals differential effects of covalent modifications on different Saccharomyces cerevisiae PP2A heterotrimers. Eukaryotic Cell 4: 1029–1040.

67. SchlaitzA-L, SraykoM, DammermannA, QuintinS, WielschN, et al. (2007) The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly. Cell 128: 115–127.

68. SontagE, Nunbhakdi-CraigV, BloomGS, MumbyMC (1995) A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. The Journal of Cell Biology 128: 1131–1144.

69. FleggCP, SharmaM, Medina-PalazonC, JamiesonC, GaleaM, et al. (2010) Nuclear export and centrosome targeting of the protein phosphatase 2A subunit B56α: role of B56α in nuclear export of the catalytic subunit. Journal of Biological Chemistry 285: 18144–18154.

70. DutcherSK (2003) Elucidation of basal body and centriole functions in Chlamydomonas reinhardtii. Traffic 4: 443–451.

71. HuangK, DienerDR, MitchellA, PazourGJ, WitmanGB, et al. (2007) Function and dynamics of PKD2 in Chlamydomonas reinhardtii flagella. The Journal of Cell Biology 179: 501–514.

72. BelzileO, Hernandez-Lara CarmenI, WangQ, Snell WilliamJ (2013) Regulated membrane protein entry into flagella is facilitated by cytoplasmic microtubules and does not require IFT. Current Biology 23: 1460–1465.

73. LiH, HandsakerB, WysokerA, FennellT, RuanJ, et al. (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25: 2078–2079.

74. CingolaniP, PlattsA, WangLL, CoonM, NguyenT, et al. (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome ofDrosophila melanogaster strain w1118; iso-2; iso-3. Fly 6: 80–92.

75. EdgarR (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32: 1792–1797.

76. LinH, KwanAL, DutcherSK (2010) Synthesizing and salvaging NAD+: lessons learned from Chlamydomonas reinhardtii. PLoS Genetics 6: e1001105.

77. SizovaI, FuhrmannM, HegemannP (2001) A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii. Gene 277: 221–229.

78. PiaseckiBP, LaVoieM, TamL-W, LefebvrePA, SilflowCD (2008) The Uni2 phosphoprotein is a cell cycle–regulated component of the basal body maturation pathway in Chlamydomonas reinhardtii. Molecular Biology of the Cell 19: 262–273.

79. DutcherSK, HuangB, LuckDJ (1984) Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii. The Journal of Cell Biology 98: 229–236.

80. LeeJ-H, LinH, JooS, GoodenoughU (2008) Early sexual origins of homeoprotein heterodimerization and evolution of the plant KNOX/BELL family. Cell 133: 829–840.

81. OlsonBJSC, OberholzerM, LiY, ZonesJM, KohliHS, et al. (2010) Regulation of the Chlamydomonas cell cycle by a stable, chromatin-associated retinoblastoma tumor suppressor complex. The Plant Cell Online 22: 3331–3347.

82. MortzE, KroghTN, VorumH, GörgA (2001) Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 1: 1359–1363.