Title Page
ABSTRACT
Contents
ABBREVIATIONS 15
BACKGROUND 16
1. Lifespan of Saccharomyces cerevisiae 17
2. rDNA stability of Saccharomyces cerevisiae 19
2.1. Ribosomal DNA 19
2.2. rDNA silencing 19
2.3. Msn2-Pnc1-Sir2 pathway 21
3. Hog1 MAP kinase 22
4. Previous study 24
5. Aim of this study 24
CHAPTER Ⅰ. Activation of Hog1 promotes rDNA stability and extends lifespan 26
1. Introduction 27
2. Results 30
2.1. Loss of Nbp2 or Ptc1 activates Hog1 30
2.2. Loss of Nbp2 or Ptc1 induces nuclear accumulation of Msn2 in a Hog1-dependent manner 32
2.3. Loss of Nbp2 or Ptc1 increased Pnc1 expression in a Hog1- and, Msn2/4- dependent manner 35
2.4. Loss of Nbp2 or Ptc1 enhances Sir2-mediated rDNA silencing in a Hog1 dependent manner 39
2.5. Loss of Nbp2 or Ptc1 promotes rDNA stability in a Hog1 dependent manner 42
2.6. Loss of Nbp2 extends RLS, whereas loss of Ptc1 does not 45
2.7. Osmotic stress and ER stress activate Hog1 49
2.8. Msn2 is activated under stress conditions 52
2.9. Pnc1 expression level is elevated under stress conditions 52
2.10. rDNA silencing is enhanced under stress conditions 56
2.11. rDNA stability is promoted under stress conditions 58
2.12. Sorbitol or DTT treatment extend RLS 60
3. Discussion 64
4. Materials and Methods 69
4.1. Yeast strains and growth media 69
4.2. Amplification of PCR fragment 69
4.3. Transformation of yeast cells 70
4.4. Western blot analysis 70
4.5. Measurement of protein phosphorylation 71
4.6. Quantification of GPD1, PNC1 and mURA3 transcript level 72
4.7. Fluorescence microscopy 72
4.8. rDNA silencing assay 73
4.9. rDNA recombination assay 73
4.10. RLS analysis 74
CHAPTER Ⅱ. Phosphorylation of Thr¹⁷⁴ and Tyr¹⁷⁶ of Hog1 contributes to the regulation of rDNA stability and longevity 80
1. Introduction 81
2. Results 85
2.1. Thr¹⁷⁴/Tyr¹⁷⁶ dual phosphorylation is essential for adaptation to osmotic stress but not for adaptation to ER stress or mutant viability 85
2.2. Thr¹⁷⁴/Tyr¹⁷⁶ dual phosphorylation is important to rDNA stability. 88
2.3. Thr¹⁷⁴/Tyr¹⁷⁶ dual phosphorylation is necessary to Hog1- mediated RLS extension 91
2.4. Mono-phosphorylatable Hog1 mutants affect adaptation to osmotic stress but not for adaptation to ER stress or mutant viability 94
2.5. Phosphorylation of Tyr¹⁷⁶ is not essential for the regulation of rDNA stability by loss of Nbp2 or Ptc1 97
2.6. Mono-phosphorylatable Hog1 mutants did not extend RLS in the absence of Nbp2 101
2.7. Phosphorylation of Thr¹⁷⁴ is not essential for the regulation of rDNA stability under ER stress conditions 105
2.8. Mono-phosphorylatable Hog1 mutants did not extend RLS under DTT-treated condition 109
2.9. Hog1³A responds similarly to wild-type Hog1[이미지참조] 113
2.10. Hog1³A does not affect Hog1-mediated rDNA stability and lifespan regulation[이미지참조] 113
3. Discussion 122
4. Materials and Methods 135
4.1. Yeast strains and growth media 135
4.2. Amplification of PCR fragment 135
4.3. Construction of plasmid 136
4.4. Transformation of yeast cells 136
4.5. Western blot analysis Measurement of protein phosphorylation 137
4.6. Quantification of GPD1, STL1, and PNC1 transcript level 138
4.7. Viability assay 139
4.8. rDNA recombination assay 139
4.9. RLS analysis 140
4.10. Fluorescence microscopy 140
CONCLUSION 146
REFERENCES 150
APPENDIX 159
국문초록 169
CHAPTER Ⅰ 14
Table 1. Strains used in this study 75
Table 2. Oligonucleotide primers used in this study 78
CHAPTER Ⅱ 14
Table 3. Strains used in this study 141
Table 4. Oligonucleotide primers used in this study 144
BACKGROUND 11
Figure 1. Lifespan of yeast and three factors that cause replicative aging 18
Figure 2. rDNA silencing in yeast and evolutionary conservation of Sir2 in aging process 20
Figure 3. Function of Hog1 and evolutionary conservation of the HOG pathway 23
CHAPTER Ⅰ 11
Figure 4. Loss of Nbp2 or Ptc1 activated Hog1 31
Figure 5. Loss of Nbp2 or Ptc1 induced nuclear accumulation of Msn2 in a Hog1-dependent manner 33
Figure 6. Loss of Nbp2 or Ptc1 increased Pnc1 expression in a Hog1- and, Msn2/4- dependent manner 37
Figure 7. Loss of Nbp2 or Ptc1 promoted rDNA silencing in a Sir2- and Hog-dependent manner 41
Figure 8. Loss of Nbp2 or Ptc1 reduced rDNA recombination in a Sir2- and Hog-dependent manner 43
Figure 9. Loss of Nbp2 extended RLS, whereas loss of Ptc1 did not 47
Figure 10. Osmotic stress and ER stress activated Hog1 51
Figure 11. Msn2 was activated under osmotic stress and ER stress conditions 53
Figure 12. Pnc1 expression was elevated under osmotic stress and ER stress conditions 55
Figure 13. rDNA silencing was enhanced under osmotic stress and ER stress conditions in a Sir2- and Hog1-dependent manner 57
Figure 14. rDNA recombination was reduced under osmotic stress and ER stress conditions in a Sir2- and Hog-dependent manner 59
Figure 15. Sorbitol and DTT extended RLS in a Hog1- and Msn2/4-dependent manner 63
CHAPTER Ⅱ 12
Figure 16. Comparison of protein sequences in yeast Hog1, nematodes PMK-1, human p38α, and yeast Slt2 82
Figure 17. Hog1T¹⁷⁴A,Y¹⁷⁶F did not affect mutant viability and ER stress adaptation[이미지참조] 87
Figure 18. Hog1T¹⁷⁴A,Y¹⁷⁶F did not increase PNC1 expression under ER stress conditions or by genetic mutations[이미지참조] 89
Figure 19. Hog1T¹⁷⁴A,Y¹⁷⁶F did not promote rDNA stability under ER stress conditions or by genetic mutations[이미지참조] 90
Figure 20. Hog1T¹⁷⁴A,Y¹⁷⁶F did not extend RLS under ER stress conditions or by genetic mutations[이미지참조] 93
Figure 21. Hog1T¹⁷⁴A and Hog1Y¹⁷⁶F had partial activity[이미지참조] 95
Figure 22. Hog1T¹⁷⁴A and Hog1Y¹⁷⁶F did not affect mutant viability and ER stress adaptation[이미지참조] 96
Figure 23. Hog1Y¹⁷⁶F promoted rDNA stability by loss of Nbp2 or Ptc1, whereas Hog1T¹⁷⁴A did not[이미지참조] 99
Figure 24. Neither Hog1T¹⁷⁴A nor Hog1Y¹⁷⁶F extended the RLS in the absence of Nbp2[이미지참조] 103
Figure 25. Hog1T¹⁷⁴A promoted rDNA stability under ER stress conditions, whereas Hog1Y¹⁷⁶F did not 107
Figure 26. Neither Hog1T¹⁷⁴A nor Hog1Y¹⁷⁶F extended the RLS under DTT-treated condition[이미지참조] 111
Figure 27. Hog13A did not affect Thr¹⁷⁴/Tyr¹⁷⁶ dual phosphorylation, stress adaptation and cell viability 115
Figure 28. Hog13A did not affect rDNA stability and lifespan regulation function of Hog1 121
Figure 29. Hog1Y¹⁷⁶F promoted rDNA stability by loss of Smi1, whereas Hog1T¹⁷⁴A did not[이미지참조] 125
Figure 30. Hog1 PM-tethering impairs rDNA stability and RLS only under stress conditions 134
CONCLUSION 13
Figure 31. The model for Hog1-mediated rDNA stability and lifespan regulation 149