李芳仁臺灣大學：分子醫學研究所詹琍婷Jang, Li-TingLi-TingJang2007-11-262018-07-092007-11-262018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/51366壹、酵母菌核醣核酸結合蛋白Rbp1p之功能性探討 在酵母菌中，一個可以與核醣核酸結合的蛋白質Rbp1p最初被鑑定為一個負生長調控者。Rbp1p經由加速一個可以轉譯做出粒線體蛋白質Por1p的核醣核酸的降解速度來減少POR1核醣核酸的量。最近，在細胞中的一種細胞質聚集顆粒已經被確定了，這種顆粒被稱為P-bodies，包含有調控核醣核酸降解的蛋白質和核醣核酸本身，核醣核酸的降解也會在此處發生。這裡，我們顯示Rbp1p可以坐落在P-bodies。 Rbp1p的氮端、第一個可以與核醣核酸結合的序列和碳端皆對於Rbp1p坐落到P-bodies很重要。我們也證明氮端和第一個可以與核醣核酸結合的序列對於Rbp1p坐落到P-bodies是必要的，但是不足夠的。Rbp1p的碳端對於自我的結合作用扮演著重要角色。Dhh1p是可以坐落到P-bodies的。我們結果顯示在細胞內Rbp1p和Dhh1p能交互作用，但是這交互作用並不影響Rbp1p是否可以坐落到P-bodies。利用螢光染色來揭示Rbp1p在以下兩種突變株rbp1Δ xrn1Δ ccr4Δ和rbp1Δ xrn1Δ pan2Δ 的位置，結果顯示Rbp1p在xrn1剔除細胞株中坐落到P-bodies是不需要核醣核酸進行去線嘌呤化。我們也顯示Rbp1p在xrn1剔除細胞株中坐落到P-bodies是不需要POR1核醣核酸。我們觀察在不同的環境壓力下，P-bodies和Rbp1p之間的關係。我們的數據顯示在葡萄糖剝奪，滲透壓力和生長階段晚期的幾種壓力之下，Rbp1p 和Dhh1p會坐落在相同的位置。由於細胞經由cycloheximide的處理，Dhh1p 和Dcp2p會從P-bodies脫離，但是Rbp1p在細胞的分佈並沒有明顯的差別。雖然Rbp1p和P-bodies有著密切的關係，但是，Rbp1p對於P-bodies形成並不重要。我們猜測Rbp1p是構成P-bodies的種子之一，只不過並非是絕對需要的也不是唯一的。Rbp1p仍然可以出現在不包含核醣核酸的P-bodies。在我們的蛋白質雙向雜交研究過程中，有17種可以與Rbp1p相互作用的蛋白質被鑑定。 蛋白質與蛋白質相互作用對於執行各種各樣的生物機能扮演了決定性的角色。連接這些蛋白質的相互作用，將能對於Rbp1p的功能性有更深層的瞭解。 貳、酵母菌高基氏體結合蛋白Vps74p之功能性探討 在酵母菌的G1短期內發生頂端發芽生長，此時細胞膜和細胞壁沉積在正在芽化生長的頂端。Vps74p (YDR372C的產物)最近被報告可以改變cdc34-2突變株停止在頂端發展階段裡時被拉長的小芽形態。在這裡，我們顯示Vps74p在第十九號絲胺酸上的磷酸化是依靠生長階段的模式。而且Vps74p被磷酸化是需要cdc28p的存在。丙胺酸和天門冬胺酸分別代替第十九號的絲胺酸可以直接影響cdc34-2突變株被拉長的小芽形態。丙胺酸的取代可以直接終止Vps74p在頂端發展上的職能。相反的，天門冬胺酸的取代可以直接刺激細胞形成拉長的小芽。Vps74p坐落在高基氏體是不需倚賴氮端被磷酸化，但是需要碳端的胺基酸序列。我們也發現Vps74p會影響Gas1p送往細胞膜的的分佈，另外，剔除Vps74p碳端的胺基酸序列會影響細胞壁的完整，但氮端胺基酸序列的剔除並不會影響細胞壁的完整性。總而言之，我們推斷第十九號絲胺酸的磷酸化可以影響Vps74p在小芽頂端生長的調節功能，但並不是參與在細胞壁的完整性方面。Section I. Functional Characterization of RNA binding protein Rbp1p in Saccharomyces cerevisiae The Saccharomyces cerevisiae RNA-binding protein Rbp1p was initially identified as a negative growth regulator. Overexpression of Rbp1p can decrease the level of mitochondrial Por1p mRNA by enhancing its degradation. Recently, one kind of cytoplasmic foci had been defined, referred to processing bodies (P-bodies), wherein mRNA decay factors are concentrated and where mRNA decay can occur. Here, we show that Rbp1p is found in the mRNA processing bodies. Rbp1p localizes to P-bodies in an xrn1 strain and the N-terminus, C-terminus, and RRM1 domain are involved in this recruitment. We also demonstrate that the N-terminal and RRM1 domains of Rbp1p are necessary but not sufficient for its localization in P bodies. The C-terminus of Rbp1p is involved in oligomers formation. Dhh1p can be localized to P-bodies, and we show that in vivo Rbp1p interacts with Dhh1p. However, this interaction does not affect the recruitment of Rbp1p to P-bodies. Immunostaining also reveal that HA-Rbp1p located to P-bodies in rbp1Δxrn1Δccr4Δ and rbp1Δxrn1Δpan2Δ mutants, suggest that proteins involved in deadenylation are not required for the recruitment of Rbp1p to P-bodies. We also demonstrate that Rbp1p is not required for localizing POR1 mRNA to P bodies in an xrn1Δ strain. We have addressed how the relationship between P-bodies and Rbp1p responds to stress. Our data show that Rbp1p colocalizes with Dhh1p under several stress conditions including glucose deprivation, osmotic stress, and late growth stage. In response to cycloheximide treatment, Dhh1p and Dcp2p are dissociated from P-bodies, but there are no significant differences in Rbp1p localization. Despite the close relationship between Rbp1p and P-bodies, Rbp1p is not essential to P-bodies formation. Based on our data, we propose that Rbp1p is a redundant protein of P-body seed and remains in such foci even when mRNAs are no longer available. In our study, seventeen full-length Rbp1p interacting proteins have identified. Considering that protein-protein interactions play crucial roles in the execution of various biological functions, cumulative connection of these binary interactions would contribute considerably to the functional interpretation of Rbp1p. Section II. Functional Characterization of Golgi associated protein Vps74p in Saccharomyces cerevisiae In Saccharomyces cerevisiae, apical bud growth occurs for a brief period in G1 when the deposition of membrane and cell wall is restricted to the tip of the growing bud. Vps74p (product of YDR372C) was recently reported to alter the elongated bud morphology of cdc34-2 cells arrested in the apical growth phase. Here, we show that Vps74p is phosphorylated on serine-19 in a growth-phase-dependent manner. Cdc28 kinase is required for Vps74p phosphorylation. Both alanine and aspartate substitutions in serine-19 directly affect elongated bud morphology of cdc34-2 cells. The alanine substitution abolished Vps74p functional activity on apical growth. In contrast, the aspartate substitution stimulates cellular elongated buds formation. Localization of Vps74p at the Golgi is not dependent on the N-terminal phosphorylation, but on its C-terminal domain. Vps74p is involved in the transportation of GPI-anchored protein Gas1. In addition, deletion of C-terminus, but not N-terminus, of Vps74p affects cell wall integrity. Together, we infer that the distinct functional outcome from Ser-19 phosphorylation modulates Vps74p activity in apical growth, but not in cell wall integrity.中文摘要 1 Abstract 3 Section I: Functional characterization of Rbp1p 5 Introduction 6 Results 17 I Determinants of Rbp1p Localization on Specific Cytoplasmic mRNA-processing Foci, P-bodies 17 II To identify the localization of Rbp1p varies under different cellular condition 27 III To identify and characterize the structure and function of Rbp1p-interacting proteins 32 Discussion 37 Perspective 46 Section II: Functional characterization of Vps74p 48 Introduction 49 Results 53 Discussion 64 Materials and methods 67 Tables 78 Table 1 Yeast strains used in this thesis 78 Table 2. Primers used in this study 79 Table 3. A brief summary of plasmids used in this thesis 85 Table 4. Putative Rbp1p interacting proteins 86 Figures 87 Fig. 1. Rbp1p localizes to cytoplasmic foci similar to the P-bodies in an xrn1Δ mutant strain. 87 Fig. 2. Endogenous Rbp1p co-localizes with Dhh1p in the xrn1Δ mutant. 88 Fig. 3. N-terminus, C-terminus, and RRM1 domain of Rbp1p are involved in the recruitment of Rbp1p to P-bodies in an xrn1Δ mutant. 89 Fig. 4. Localization of Rbp1p and its mutants in an rbp1Δ mutant strain. 91 Fig. 5. Expression of Rbp1p recruits Rbp1p-dN, Rbp1p-rrm1, and Rbp1p-CT to P-bodies in an rbp1Δ/xrn1Δ mutant. 92 Fig. 6. Rbp1p interacts with itself and Rbp1p-dN in an RNA-independent manner. 93 Fig. 7. Rbp1p interacts with Dhh1p in an RNA-independent manner. 94 Fig. 8. Rbp1p interacts with Dhh1p in an rbp1Δ mutant in an RNA-independent manner in vivo. 95 Fig. 9. Xrn1p-GFP can be pulled with GST-Rbp1p in an rbp1Δ mutant in vivo. 96 Fig. 10. Dhh1p, Ccr4p and Pan2p are not required for the recruitment of Rbp1p to P-bodies in an xrn1Δ mutant. 97 Fig. 11. Sedimentation centrifugation analyses of GST-Rbp1p. 98 Fig. 12. RNA-binding ability of Rbp1p and its mutated or deleted mutants in vivo. 99 Fig. 13. Rbp1p is not required for the recruitment of POR1 mRNA to P-bodies in an xrn1Δ mutant. 100 Fig. 14. DsRed-Rbp1p is co-localized with Dhh1p-GFP in an rbp1Δ/xrn1Δ mutant. 102 Fig. 15. Growth phenotypes. 103 Fig. 16. Over-expression of HA-Rbp1p in either xrn1 or ccr4 mutant cells had no effect on the steady state level of POR1 mRNA. 104 Fig. 17. Xrn1p is involved in Rbp1p-mediated POR1 mRNA decay. 105 Fig. 18. The localization of endogenous Rbp1p, Dhh1p, and Dcp2p in response to the stage of cell growth. 106 Fig. 19. The co-localization of Rbp1p and Dhh1p increase with the stage of cell growth. 107 Fig. 20. Rbp1p co-localizes with Dhh1p under glucose deprivation. 108 Fig. 21. Rbp1p co-localizes with Dhh1p under osmotic stress. 109 Fig. 22. DsRed-Rbp1p does not co-localize with Dhh1p-GFP under H2O2-induced oxidative stress. 110 Fig. 23. Distribution of Rbp1p and Dhh1p in heat shock stress. 111 Fig. 24. Inhibition of translation elongation disrupts P-bodies but not granules containing Rbp1p. 112 Fig. 25. Inhibition of translation elongation could not dissociate Rbp1p from P-bodies in stationary phase. 113 Fig. 26. 2-D electrophoresis of GST-Rbp1p protein complexes. 114 Fig. 27. Immunoblot of the threonine phosphorylation GST-Rbp1p protein. 115 Fig. 28. 2D electrophoresis of GST-Rbp1p protein treated with alkaline phosphatase. 116 Fig. 29 Specific immunoreactivity and sensitivity of antibody against Hrp1p, Psp1p, Rpt2p, and Scj1p. 117 Fig. 30. Subcellular localization and subcellular distribution of Psp1p. 118 Fig. 31. Rbp1p localizes to P-bodies is not dependent on Psp1p and Rrp1p. 119 Fig. 32 Endogenous GFP-tagged Nrp1p and Pub1p can interact with the over-expressed GST-Rbp1p. 120 Fig. 33. Nrp1p partly co-localizes with Rbp1p. 121 Fig. 34. A small amount of Pub1p can locate to P-bodies in xrn1 mutant. 122 Fig. 35. Localization of Scj1p detected by indirect immunofluorescence and subcellular distribution of endogenous Scj1p-3HA. 123 Fig. 36. Subcellular localization and subcellular distribution of Hrp1p. 124 Fig. 37. Rbp1p interacts with Por1p in vivo. 125 Fig. 38. Subcellular distribution of endogenous Rpt2p. 126 Fig. 39. Rbp1p could be located to bud tip and daughter cell in sho1 mutant. 127 Fig. 40. These binary interactions were cumulative connections. 128 Fig. 41. Vps74p is a phospho-protein. 129 FIG 42. Phosphorylation of Vps74p does not regulated by cell cycle. 130 Fig. 43. Vps74p is phosphorylated in a growth-phase-dependent manner 131 Fig. 44. Expression of phosphorylated Vps74p is regulated by Cdc28p. 132 Fig. 45. Vps74 is critical for cells involved at least in apical projection between late G1 and before mitotic anaphase. 133 Fig. 46. Vps74p participates in apical growth. 134 Fig. 47. Phosphorylation of Ser-19 in Vps74p functions for apical growth. 135 Fig. 48. Determines of Vps74p localized to the Golgi apparatus. 136 Fig. 49. Subcellular distribution of Vps74p. 137 Fig. 50. Vps74p is not involved in known exocytosis and endocytosis. 138 Fig. 51. Vps74p is involved in transportation of GPI-anchored protein Gas1p. 139 Fig. 52. Vps74p is involved in the cell wall integrity. 140 Fig. 53. Vps74p is not involved in bud site selection. 141 Fig. 54. C-terminal GFP-fused Vps74p, Vps74-GFP appears diffusion in cytoplasm. 142 Fig. 55. Vps74p is not involved in the function of mitochondria. 143 Fig. 56. Arf1p facilitates elongated bud formation in cdc34-2 mutant. 144 References 1457392358 bytesapplication/pdfen-US酵母菌核醣核酸結合蛋白核醣核酸的降解高基氏體結合蛋白頂端發芽生長Saccharomyces cerevisiae RNA-binding proteinP-bodiesmRNA decayGolgi associated proteinapical growthotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51366/1/ntu-95-F88448002-1.pdf