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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李芳仁(Fang-Jen Lee) | |
| dc.contributor.author | Pei-Juan Cai | en |
| dc.contributor.author | 蔡佩娟 | zh_TW |
| dc.date.accessioned | 2021-07-10T21:40:19Z | - |
| dc.date.available | 2021-07-10T21:40:19Z | - |
| dc.date.copyright | 2020-09-10 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-13 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76914 | - |
| dc.description.abstract | 高爾基體蛋白Imh1透過由鳥嘌呤核苷酸交換因子(GEF)Syt1所活化的第一線嘌呤核苷二磷酸核醣化相似因子 (Arl1)被吸引到反式高基氏體網路 (trans-Golgi network)上。由於實驗室先前發表的文章中提到Imh1可去抑制Ypt6缺發所造成的反向運輸缺陷，暗示著Imh1在反向運輸中的重要性。然而其詳細的功能目前訂不是很清楚。在結構上，Imh1可分為三個部分，包含胺基端、中間捲曲螺旋(coiled-coil)的結構以及羧基端。因此在本篇的研究中，我們希望去了解Imh1各個部分對於其功能的重要性 首先，我們發現到Imh1在內質網壓力下的SNARE蛋白的運輸中扮演重要的腳色，並進一步找到特別是其胺基端的磷酸化對於其功能的執行是重要的。此外，除了胺基端磷酸化很重要外，我們發現其中間的螺旋結構有助於去穩定Imh1的雙聚體的結構去幫助SNARE 蛋白的運輸。接著，我們利用一個會增強Imh1去形成二聚體能力的突變體Imh1-M4. 我們發現這個會增強Imh1形成二聚體能力的突變使得Imh1仍可在缺乏GEF或膜的彎曲程度有缺陷的情況下被吸引到反式高基氏體網路上。然而，這樣的突變並無法完全的彌補當Imh1與Arl1或膜上脂質的結合有問題時的Imh1功能。總結本篇，我們發現Imh1各個部分都擁有它特別的特性去協助Imh1整體的功能，包括胺基端被推測具有接取囊泡的功能以及中間的捲曲螺旋結構參與穩定其二聚體的形成，而其羧機端則是與Arl1和膜上脂質的交互作用有關。每個部分都有獨特的功能並需要彼此的合作來維持Imh1的功能。 | zh_TW |
| dc.description.abstract | Imh1 is a golgin protein recruited by active Arl1 through the induction of Syt1, an Arl1 guanine nucleotide exchange factor (GEF). It has been proposed to play a role in retrograde transport. Our previous study showed that Imh1 acts as a high-copy suppressor of retrograde transport defect in ypt6Δ. However, the precise function and regulation of Imh1 in yeast is less known. The structure of Imh1 is divided into three parts, including the N-terminal region, three coiled-coil domains and the C-terminal GRIP domain. In this study, we try to verify the contribution of each region on the function of Imh1. We first found that Imh1 is required for SNARE protein transport in response to Unfolded-Protein-Response (UPR). Moreover, this function is determined by the ER stress-driven phosphorylation of Imh1 N-terminus (Ser25 and Thr27). We also revealed that the Imh1 coiled-coil region is required for sustaining Imh1 dimerization and therefore supporting the SNARE transport. By using a forced dimerization mutant of Imh1 (Imh1-M4), we found that it can bypass the defect of dysfunctional GEF (sty1Δ cells) and the damage of sensing membrane curvature (drs2Δ cells). However, Imh1-M4 can only partially suppressed the defect in its Arl1 interaction and lipid-binding ability. Collectively, we demonstrate the importance of each region on Imh1 function, including N-terminus for vesicle capturing, coiled-coil region for stabilizing dimer structure and C-terminus for Arl1 interaction and lipid membrane association. All of these regions possess unique roles and need to orchestrate properly to support the Imh1 functions. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-10T21:40:19Z (GMT). No. of bitstreams: 1 U0001-1008202012020600.pdf: 4834873 bytes, checksum: ecb64d1d5a20c9acd67309e9bb8e900d (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | 口試委員審定書 i 致謝 ii 中文摘要 iii Abstract iv Contents v Introduction 1 ADP-ribosylation Factors 1 ADP-ribosylation Factor-like proteins (Arls) 2 Golgin tethering protein 4 Unfolded protein response (UPR) 7 Material and Methods 9 Tables 19 Table 1. Yeast strains used in this study 19 Table 2. Plasmid used in this study 21 Table 3. Antibodies used in this study 24 Results 25 Part I. The functional characterization of Imh1 25 Imh1 is required for SNARE proteins recycling under ER stress 25 The N-terminus phosphorylation of Imh1 is important for its function under ER stress 26 The phosphorylation of Imh1 N-terminus under TM-induced ER stress is independent from Ire1 and Env7 28 The coiled-coil region of Imh1 is important for its function in SNARE proteins transport under ER stress 30 Imh1-M4 mutation enhances the dimerization ability of Imh1 31 Imh1-M4 enlarges the punctate of itself and the punctate of Arl1 32 The dynamic regulation of Imh1 dimerization is required to exert its function 32 Imh1-M4 protects Arl1 from Gcs1-catalyzed GTP hydrolysis of Arl1 33 Imh1-M4 can bypass the defect in syt1 deletion cells and the membrane curvature defect 34 The activation of Imh1-M4 still depends on Arl3-Arl1 cascade 36 Imh1-M4 suppresses the Arl1-interaction defect and lipid-interaction defect in Imh1 36 Part II. To characterize the interaction between Arl3 and Arl1 39 Arl3Q78L interacts with Arl1T32N in vivo 39 The interaction between Arl3Q78L and Arl1 is enhanced when there is more inactive Arl1 existing in the cells 40 Discussion 42 Imh1 N-terminal phosphorylation may affect the vesicle capturing ability of Imh1 42 Imh1 dimerization 43 Factors determine the Golgi association of Imh1 44 Figures 46 Figure 1. Imh1 is required for Snc1 and Tlg1 recycling under ER stress 46 Figure 2. Imh1 is phosphorylated in response to TM-induced ER stress. 47 Figure 3. The phosphorylation of S25 and T27 residues in Imh1 N-terminus is required for Snc1 and Tlg1 recycling upon ER stress. 49 Figure 4. The phosphorylation of both S25 and T27 at Imh1 N-terminus is required for Snc1 and Tlg1 recycling upon ER stress. 51 Figure 5. The function of Imh1 phosphorylation is independent from suppressing Snc1 and Tlg1 mislocalization in ypt6 deletion cells. 52 Figure 6. The function of Imh1 phosphorylation is independent from suppressing GARP complex mislocalization in ypt6 deletion cells. 53 Figure 7. The function of Imh1 phosphorylation is independent from suppressing high temperature sensitivity of ypt6 deletion defect. 54 Figure 8. Imh1 phosphorylation is independent from Ire1-signaling. 55 Figure 9 . Env7 is irresponsible for Imh1 phosphorylation. 56 Figure 10. Truncating the coiled-coil region of Imh1 does not affect its Golgi localization. 57 Figure 11. The coiled-coil region of Imh1 is required for SNAREs recycling in response to ER stress. 59 Figure 12. The Imh-M4 mutant enhances Imh1 dimer formation. 61 Figure 13. Imh1-M4 mutation enlarges Imh1 Golgi punctate. 62 Figure 14. Imh1-M4 caused the abnormal enlargement of Arl1 punctate. 64 Figure 15. Imh1-M4 fails to rescue the mislocalization of Snc1 in imh1 deletion cells under tunicamycin treatment. 65 Figure 16. Imh1-M4 fails to suppress mislocalization of Snc1, Tlg1, Vps53, and Vps52 in ypr6 deletion cells. 67 Figure 17. M4 mutation sustained Imh1 and Arl1 localization in ypt6 deletion cells. 68 Figure 18. Imh1-M4 fails to suppress high temperature sensitivity in ypr6 deletion cells. 69 Figure 19. Imh1-M4 competes with Gcs1 to sustained Arl1 localization. 70 Figure 20. The Imh1-M4 mutant can bypass the defect in syt1 deletion cells and the membrane curvature defect. 71 Figure 21. The activation of Imh1-M4 depends on Arl3-Arl1 cascade. 72 Figure 22. The interaction with Arl1 and lipid binding of Imh1 are both required for its Golgi recruitment. 73 Figure 23. Imh1-M4 partially suppresses the Arl1-interaction and lipid-interaction defect of Imh1. 75 Figure 24 A working model shows the function of Imh1 different region. 76 Figure 25 Arl1T32N interacts with Arl3Q78L in vivo. 77 Figure 26 Arl1T32N interacts with Arl3Q78L in syt1 deletion cells in vivo. 78 Figure 27. The interaction between Arl3Q78L and Arl1 is enhanced under glucose deprivation. 79 Figure 28. The interaction between Arl3Q78L and Arl1 is enhanced in syt1 deletion cells in vivo.. 80 Figure 29 A hypothesized model shows the possible role of Arl3 in regulating the activation of Arl1. 81 Reference 82 | |
| dc.language.iso | en | |
| dc.subject | Ypt6 | zh_TW |
| dc.subject | 反式高基氏體網路 | zh_TW |
| dc.subject | SNARE蛋白 | zh_TW |
| dc.subject | 內質網壓力 | zh_TW |
| dc.subject | 高爾基體蛋白 | zh_TW |
| dc.subject | GARP | zh_TW |
| dc.subject | 第一線嘌呤核苷二磷酸核醣化相似因子 | zh_TW |
| dc.subject | Arl1 | en |
| dc.subject | Trans-Golgi network | en |
| dc.subject | GARP | en |
| dc.subject | Ypt6 | en |
| dc.subject | SNAREs | en |
| dc.subject | ER stress | en |
| dc.subject | Imh1 | en |
| dc.title | 第三線嘌呤核苷二磷酸核醣化相似因子調控第一線嘌呤核苷二磷酸核醣化相似因子及其下游高爾基體蛋白Imh1在酵母菌中之功能探討 | zh_TW |
| dc.title | Functional Characterization of Arl3-regulated Arl1 and its effector Golgin Imh1 in Saccharomyces cerevisiae | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林敬哲(Jing-Jer Lin),鄧述諄(Shu-Chun Teng),陳瑞華(Ruey-Hwa Chen),王昭雯(Chao-Wen Wang) | |
| dc.subject.keyword | 第一線嘌呤核苷二磷酸核醣化相似因子,高爾基體蛋白,內質網壓力,SNARE蛋白,反式高基氏體網路,Ypt6,GARP, | zh_TW |
| dc.subject.keyword | Arl1,Imh1,ER stress,SNAREs,Ypt6,GARP,Trans-Golgi network, | en |
| dc.relation.page | 85 | |
| dc.identifier.doi | 10.6342/NTU202002780 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2020-08-14 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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