Latent Effects of Hsp90 Mutants Revealed at Reduced Expression Levels

English version České info

In natural systems, selection acts on both protein sequence and expression level, but it is unclear how selection integrates over these two dimensions. We recently developed the EMPIRIC approach to systematically determine the fitness effects of all possible point mutants for important regions of essential genes in yeast. Here, we systematically investigated the fitness effects of point mutations in a putative substrate binding loop of yeast Hsp90 (Hsp82) over a broad range of expression strengths. Negative epistasis between reduced expression strength and amino acid substitutions was common, and the endogenous expression strength frequently obscured mutant defects. By analyzing fitness effects at varied expression strengths, we were able to uncover all mutant effects on function. The majority of mutants caused partial functional defects, consistent with this region of Hsp90 contributing to a mutation sensitive and critical process. These results demonstrate that important functional regions of proteins can tolerate mutational defects without experimentally observable impacts on fitness.

Vyšlo v časopise: Latent Effects of Hsp90 Mutants Revealed at Reduced Expression Levels. PLoS Genet 9(6): e32767. doi:10.1371/journal.pgen.1003600
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003600

Souhrn

Zdroje

1. StemmerWP (1994) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370: 389–391.

2. HughesAL, NeiM (1988) Pattern of nucleotide substitution at major histocompatibility complex class I loci reveals overdominant selection. Nature 335: 167–170.

3. YangZ, BielawskiJP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15: 496–503.

4. KingMC, WilsonAC (1975) Evolution at two levels in humans and chimpanzees. Science 188: 107–116.

5. ManceauM, DominguesVS, MallarinoR, HoekstraHE (2011) The developmental role of Agouti in color pattern evolution. Science 331: 1062–1065.

6. DekelE, AlonU (2005) Optimality and evolutionary tuning of the expression level of a protein. Nature 436: 588–592.

7. DrummondDA, BloomJD, AdamiC, WilkeCO, ArnoldFH (2005) Why highly expressed proteins evolve slowly. Proc Natl Acad Sci U S A 102: 14338–14343.

8. GoutJF, KahnD, DuretL (2010) The relationship among gene expression, the evolution of gene dosage, and the rate of protein evolution. PLoS Genet 6: e1000944.

9. HietpasRT, JensenJD, BolonDN (2011) Experimental illumination of a fitness landscape. Proc Natl Acad Sci U S A 108: 7896–7901.

10. PursellNW, MishraP, BolonDN (2012) Solubility-promoting function of hsp90 contributes to client maturation and robust cell growth. Eukaryot Cell 11: 1033–1041.

11. WayneN, MishraP, BolonDN (2011) Hsp90 and client protein maturation. Methods Mol Biol 787: 33–44.

12. WhitesellL, MimnaughEG, De CostaB, MyersCE, NeckersLM (1994) Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation. Proc Natl Acad Sci U S A 91: 8324–8328.

13. RennellD, BouvierSE, HardyLW, PoteeteAR (1991) Systematic mutation of bacteriophage T4 lysozyme. J Mol Biol 222: 67–88.

14. SanjuanR, MoyaA, ElenaSF (2004) The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus. Proc Natl Acad Sci U S A 101: 8396–8401.

15. CarrascoP, de la IglesiaF, ElenaSF (2007) Distribution of fitness and virulence effects caused by single-nucleotide substitutions in Tobacco Etch virus. J Virol 81: 12979–12984.

16. Domingo-CalapP, CuevasJM, SanjuanR (2009) The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages. PLoS Genet 5: e1000742.

17. PerisJB, DavisP, CuevasJM, NebotMR, SanjuanR (2010) Distribution of fitness effects caused by single-nucleotide substitutions in bacteriophage f1. Genetics 185: 603–609.

18. WylieCS, ShakhnovichEI (2011) A biophysical protein folding model accounts for most mutational fitness effects in viruses. Proc Natl Acad Sci U S A 108: 9916–9921.

19. ZeldovichKB, ChenP, ShakhnovichEI (2007) Protein stability imposes limits on organism complexity and speed of molecular evolution. Proc Natl Acad Sci U S A 104: 16152–16157.

20. TokurikiN, TawfikDS (2009) Stability effects of mutations and protein evolvability. Curr Opin Struct Biol 19: 596–604.

21. KacserH, FellDA (1995) The control of flux: 21 years on. Biochem Soc Trans 23: 341–366.

22. BorkovichKA, FarrellyFW, FinkelsteinDB, TaulienJ, LindquistS (1989) hsp82 is an essential protein that is required in higher concentrations for growth of cells at higher temperatures. Mol Cell Biol 9: 3919–3930.

23. LunzerM, GoldingGB, DeanAM (2010) Pervasive cryptic epistasis in molecular evolution. PLoS Genet 6: e1001162.

24. LunzerM, MillerSP, FelsheimR, DeanAM (2005) The biochemical architecture of an ancient adaptive landscape. Science 310: 499–501.

25. BershteinS, MuW, SerohijosAW, ZhouJ, ShakhnovichEI (2013) Protein quality control acts on folding intermediates to shape the effects of mutations on organismal fitness. Mol Cell 49: 133–144.

26. KacserH, BurnsJA (1981) The molecular basis of dominance. Genetics 97: 639–666.

27. KacserH, BurnsJA (1973) The control of flux. Symp Soc Exp Biol 27: 65–104.

28. HeinrichR, RapoportTA (1974) A linear steady-state treatment of enzymatic chains. General properties, control and effector strength. Eur J Biochem 42: 89–95.

29. HegrenessM, ShoreshN, HartlD, KishonyR (2006) An equivalence principle for the incorporation of favorable mutations in asexual populations. Science 311: 1615–1617.

30. LynchM, ConeryJS (2003) The origins of genome complexity. Science 302: 1401–1404.

31. TsaiIJ, BensassonD, BurtA, KoufopanouV (2008) Population genomics of the wild yeast Saccharomyces paradoxus: Quantifying the life cycle. Proc Natl Acad Sci U S A 105: 4957–4962.

32. HarrisSF, ShiauAK, AgardDA (2004) The crystal structure of the carboxy-terminal dimerization domain of htpG, the Escherichia coli Hsp90, reveals a potential substrate binding site. Structure 12: 1087–1097.

33. AdkarBV, TripathiA, SahooA, BajajK, GoswamiD, et al. (2012) Protein model discrimination using mutational sensitivity derived from deep sequencing. Structure 20: 371–381.

34. WhiteheadTA, ChevalierA, SongY, DreyfusC, FleishmanSJ, et al. (2012) Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing. Nat Biotechnol 30: 543–548.

35. FowlerDM, ArayaCL, FleishmanSJ, KelloggEH, StephanyJJ, et al. (2010) High-resolution mapping of protein sequence-function relationships. Nat Methods 7: 741–746.

36. HietpasR, RoscoeB, JiangL, BolonDN (2012) Fitness analyses of all possible point mutations for regions of genes in yeast. Nat Protoc 7: 1382–1396.

37. DebartoloJ, DuttaS, ReichL, KeatingAE (2012) Predictive bcl-2 family binding models rooted in experiment or structure. J Mol Biol 422: 124–144.

38. PittJN, Ferre-D'AmareAR (2010) Rapid construction of empirical RNA fitness landscapes. Science 330: 376–379.

39. McLaughlinRNJr, PoelwijkFJ, RamanA, GosalWS, RanganathanR (2012) The spatial architecture of protein function and adaptation. Nature 491: 138–142.

40. ArayaCL, FowlerDM, ChenW, MuniezI, KellyJW, et al. (2012) A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function. Proc Natl Acad Sci U S A 109: 16858–16863.

41. MumbergD, MullerR, FunkM (1994) Regulatable promoters of Saccharomyces cerevisiae: comparison of transcriptional activity and their use for heterologous expression. Nucleic Acids Res 22: 5767–5768.

42. NathanDF, LindquistS (1995) Mutational analysis of Hsp90 function: interactions with a steroid receptor and a protein kinase. Mol Cell Biol 15: 3917–3925.

43. RoscoeBP, ThayerKM, ZeldovichKB, FushmanD, BolonDN (2013) Analyses of the Effects of All Ubiquitin Point Mutants on Yeast Growth Rate. J Mol Biol 425: 1363–77.

44. WayneN, BolonDN (2007) Dimerization of Hsp90 is required for in vivo function. Design and analysis of monomers and dimers. J Biol Chem 282: 35386–35395.

45. PicardD, KhursheedB, GarabedianMJ, FortinMG, LindquistS, et al. (1990) Reduced levels of hsp90 compromise steroid receptor action in vivo. Nature 348: 166–168.

46. DillKA (1990) Dominant forces in protein folding. Biochemistry 29: 7133–7155.

47. KingJL, JukesTH (1969) Non-Darwinian evolution. Science 164: 788–798.

48. SauerRT, MillaME, WaldburgerCD, BrownBM, SchildbachJF (1996) Sequence determinants of folding and stability for the P22 Arc repressor dimer. Faseb J 10: 42–48.

49. Toth-PetroczyA, TawfikDS (2011) Slow protein evolutionary rates are dictated by surface-core association. Proc Natl Acad Sci U S A 108: 11151–11156.

50. AliMM, RoeSM, VaughanCK, MeyerP, PanaretouB, et al. (2006) Crystal structure of an Hsp90-nucleotide-p23/Sba1 closed chaperone complex. Nature 440: 1013–1017.

51. KelloggEH, Leaver-FayA, BakerD (2011) Role of conformational sampling in computing mutation-induced changes in protein structure and stability. Proteins 79: 830–838.

52. DykhuizenDE, DeanAM (1990) Enzyme activity and fitness: Evolution in solution. Trends Ecol Evol 5: 257–262.

53. DykhuizenDE, DeanAM, HartlDL (1987) Metabolic flux and fitness. Genetics 115: 25–31.

54. Dykhuizen DE, Dean AM (2009) Experimental Evolution from the Bottom Up. In: Theodor, editor. Experimental Evolution: Concepts, Methods, and Applications of Selection Experiments.

55. MumbergD, MullerR, FunkM (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156: 119–122.

56. ZaretKS, ShermanF (1984) Mutationally altered 3′ ends of yeast CYC1 mRNA affect transcript stability and translational efficiency. J Mol Biol 177: 107–135.

57. LiMZ, ElledgeSJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4: 251–256.

58. HenikoffS, HenikoffJG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci U S A 89: 10915–10919.

59. Geiler-SamerotteKA, DionMF, BudnikBA, WangSM, HartlDL, et al. (2010) Misfolded proteins impose a dosage-dependent fitness cost and trigger a cytosolic unfolded protein response in yeast. Proc Natl Acad Sci U S A 108: 680–685.

60. FutcherB, CarbonJ (1986) Toxic effects of excess cloned centromeres. Mol Cell Biol 6: 2213–2222.

61. TschumperG, CarbonJ (1983) Copy number control by a yeast centromere. Gene 23: 221–232.

62. TschumperG, CarbonJ (1987) Saccharomyces cerevisiae mutants that tolerate centromere plasmids at high copy number. Proc Natl Acad Sci U S A 84: 7203–7207.