腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)為細胞中調控能量代謝的關鍵蛋白，廣泛存在於真核生物中，當其受到腺苷酸活化時，會促進細胞內的分解代謝作用產生能量；而AMPK活性的異常與很多疾病有關，如第二型糖尿病、帕金森氏症、阿茲海默症、癌症。酵母菌中的Snf1 (Sucrose non-fermenting 1)為AMPK的同源蛋白，而Snf1也與細胞內的能量代謝有關。當環境中缺乏葡萄糖時，Snf1會被活化，進而促進基因表現或下游蛋白質的活化，使酵母菌能夠利用環境中其他碳源生長。在先前實驗中發現某酵母菌菌株的Snf1蛋白表現量與正常酵母菌相比明顯較低，因此本實驗想要探討造成該菌株Snf1蛋白表現量下降的原因。利用全基因體定序(Whole genome sequencing)及細胞培養穩定同位素胺基酸標記(Stable Isotope Labeling with Amino acids in Cell culture, SILAC)尋找可能造成Snf1蛋白下降的突變基因或蛋白質，希望能發現目前尚未被找出的Snf1蛋白表現量的調控機制。實驗結果發現，Snf1蛋白表現量較低的菌株其SNF1基因上具有兩個點突變，但經由質體表現該突變蛋白後，Snf1蛋白表現量並無顯著下降的現象。另外，也發現標記Snf1蛋白的TAP (Tandem affinity purification)上存在著一個點突變，目前推測該突變可能是造成Snf1蛋白下降的原因。另一方面，SILAC的實驗結果發現大量表現Hsp104蛋白可能可以回復Snf1蛋白的表現量。因此，我們推論TAP上的點突變可能會去影響Snf1蛋白的結構，進而造成Snf1蛋白降解。

AMPK plays an important role in energy homeostasis, and it is also named as “energy sensor of the cell”. AMPK is highly conserved in eukaryotes, it stimulates catabolism to generate energy in cells upon activation. Abnormality in AMPK regulation may relate to many diseases, such as type 2 diabetes, neurodegenerative diseases, and cancer. The yeast homolog of AMPK Snf1 is required for maintaining energy homeostasis and stress resistance. The SNF1 protein kinase is consist of α, β, and γ subunits, when glucose limitation occurs, α subunit—Snf1 is activated by phosphorylation of T210, and then promotes gene expression to utilize other carbon sources. The phosphorylation level of Snf1 can be altered when mutations occur in SNF1. In this study, I investigated the underlining mechanisms that lead to the reduction of the Snf1 protein level. I used the whole genome sequencing and SILAC (Stable Isotope Labeling with Amino acids in Cell culture) analysis to search the possible gene mutation(s) or protein which may cause the diminishment of the Snf1 protein level. Surprisingly, I identified two specific mutations resided at the C terminus of SNF1 in the Snf1 lowly expressed strain, but my further analysis indicated that these two point mutations within the SNF1 did not induce significant reduction in the Snf1 protein level. I further discovered that the TAP which tagged at the C-terminal of SNF1 carried a novel mutation and I demonstrated that this mutation on TAP is likely responsible for the reduced Snf1 protein level. In addition, SILAC analysis showed that overexpression of Hsp104 may restore the Snf1-TAP protein level. Thus I propose that the newly identified variant of the Snf1-TAP may be structurally altered and lead to an instability and degradation.

國立臺灣大學碩士學位論文口試委員會審定書 i
謝誌 ii
摘要 iii
ABSTRACT iv
CONTENTS vi
LIST OF FIGURES viii
LIST OF TABLES ix
CHAPTER I　INTRODUCTION 1
1.1 Yeast as a model organism 1
1.2 AMP-activated protein kinase (AMPK) 3
1.2.1 Energy homeostasis 3
1.2.2 Structure and regulation of AMPK 4
1.3 SNF1 protein kinase 6
1.3.1 Structure of SNF1 protein kinase 6
1.3.2 Regulation of SNF1 protein kinase 9
1.4 Three prime untranslated region (3’-UTR) 12
1.4.1 Composition of 3’-UTR 13
1.4.2 Regulation of polyadenylation 15
1.4.3 Regulation of mRNA stability 15
1.4.4 Regulation of translation efficiency 16
1.5 Tandem affinity purification (TAP) 17
CHAPTER II　RESEARCH OBJECTIVE 20
CHAPTER III　MATERIALS AND METHODS 21
3.1 Materials 21
3.1.1 Yeast strains 21
3.1.2 Plasmids 24
3.1.3 Medium 25
3.1.3.1 YPD medium 25
3.1.3.2 G418 and HPH plates 26
3.1.3.3 Amino acids dropout medium 27
3.1.3.4 5-FOA plate 28
3.1.3.5 LB medium 29
3.1.3.6 Lysis buffer for cell extraction of western blotting 29
3.2 Methods 30
3.2.1 Cell extraction for western blot analysis 30
3.2.2 Western blot analysis 31
3.2.3 Site-directed mutagenesis 32
3.2.4 Construction of pRS315- SNF1-3’-UTR--3xHA 33
3.2.5 Construction of RDKY3615 HXT13 strain 34
3.2.6 Overexpression of pBG1805-yeast ORF-HA 34
CHAPTER IV　RESULTS 36
4.1 Snf1 protein level was dramatically decreased in the specific strain 36
4.2 Search for the mechanisms that lead to the reduced Snf1 protein level in the Tzu strain 37
4.2.1 The genomic approach 39
4.2.1.1 Search for the mutation(s) in the genome of the Tzu strain 39
4.2.1.2 Different SNF1-TAP fragments influence Snf1 protein level 41
4.2.1.3 The specific mutation(s) on SNF1 expressed by plasmid 43
4.2.1.4 The relationship between SNF1 and its 3’-untranslated region 45
4.2.1.5 The effect of different C-terminal tagging on SNF1 48
4.2.1.6 Use various antibodies to determine Snf1 protein level 49
4.2.2 The proteomic approach 52
4.2.2.1 Search candidate(s) which protein level decreased in the Tzu strain 52
4.2.2.2 Screen several candidates which may rescue Snf1 protein level 55
4.2.2.3 Snf1 might be rescued by the overexpression of PYC1, PFK1, HSP104, and VAS1 58
CHAPTER V　DISCUSSION 62
5.1 The specific mutations on SNF1 62
5.2 The relationship between Snf1 and its C-terminal tagging 63
5.3 Hsp104 may play a role in Snf1 stability 64
5.4 Future perspective 65
CHAPTER VI　CONCLUSION 68
CHAPTER VII　REFERENCES 70
APPENDIX 77

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