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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 李芳仁 | |
| dc.contributor.author | Yu-Chun Tseng | en |
| dc.contributor.author | 曾鈺純 | zh_TW |
| dc.date.accessioned | 2021-06-16T17:39:37Z | - |
| dc.date.available | 2017-09-18 | |
| dc.date.copyright | 2012-09-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64302 | - |
| dc.description.abstract | 腺嘌呤核苷二磷酸核醣化因子(ADP-ribosylation factors)家族中的小鳥糞嘌呤核苷三磷酸結合蛋白主要在胞膜蛋白運輸的調節與細胞骨架的動態平衡中扮演著很重要的角色。腺嘌呤核苷二磷酸核醣化因子可進一步被活化，藉由鳥糞嘌呤核苷酸交換因子(GEF)來協助他從鳥糞嘌呤核苷二磷酸結合的形式轉換為鳥糞嘌呤核苷三磷酸結合的形式。在之前的報告指出鳥糞嘌呤核苷酸交換因子利用蛋白間的交互作用或是形成寡聚體進行活化，進而參與在調節本身的活性或是改變細胞內座落的位置。近來在我們實驗室的研究中也發現鳥糞嘌呤核苷酸交換因子Syt1p也可幫助第一腺嘌呤核苷二磷酸核糖化相似因子(Arl1p)的活化，進而募集下游的Imh1p到反式高基氏體上，且共同參與在Ypt6p調節液胞型態的能力。在此篇研究中，我們利用活體外結合試驗發現Syt1p能夠利用SEC7區域與pleckstrin同源結構域之間的其中一個羧基端片段(Syt1pC1)來進行分子間的結合。在細胞內若將此羧基端片段去除後確實會影響Syt1p的自相互作用，表示Syt1p確實能夠利用此羧基端片段相互結合形成二聚體(dimer)或寡聚體(oligomer)。而此種不能自相互作用的Syt1p並不影響到下游的Arl1p和Imh1p在細胞中的位置，但是在去除SYT1及YPT6基因的酵母菌中卻會影響到Ypt6p調節液胞型態的能力，且在剔除YPT6基因的細胞中對於溫度的敏感性也有些微的影響。這些發現都暗示著Syt1p形成二聚體(dimer)可能也參與在本身催化鳥糞嘌呤核苷酸交換的活性以外的重要功能。此外Syt1p可藉由胺基端片段的磷酸化進而調控二聚體(dimer)的形成，而這些磷酸化的位置可調節鳥糞嘌呤核苷酸交換因子的活性。所以我們認為Syt1p透過一羧基端片段(Syt1pC1)形成二聚體並參與在Ypt6p調節液胞型態的路徑之中。另一方面，Syt1p的磷酸化幫助形成二聚體，進而活化第一腺嘌呤核苷二磷酸核糖化相似因子並募集下游的Imh1p。 | zh_TW |
| dc.description.abstract | ADP-ribosylation factor (ARF) family proteins are small G proteins required for the regulation of membrane traffic and cytoskeletal dynamics. They are activated by guanine nucleotide exchange factor (GEF) that can stimulate the exchange of GDP for GTP. Previous reports show that GEF activation through protein–protein interactions or oligomerization, and its regulation can affect GEF activity or alter intracellular localization. Recently, our lab has reported that Syt1p function as a GEF that activates the Arl1p to recruit Imh1p to the late Golgi, which is subsequently involved in synthetic enhancement of Ypt6p-dependent vacuole fragmentation. Here, we identify Syt1p intermolecular interaction region located between SEC7 and PH domain (Syt1pC1) by in vitro pull down. Syt1p∆C1 defined the mutant here affects Syt1p self-interaction in vivo, which indicates that Syt1p can form dimers or oligomers through the C1 region. This mutant Syt1p∆C1 does not affect Arl1p and Imh1p localization. However, Syt1p∆C1 affects Ypt6p-dependent vacuolar fragmentation in syt1∆ypt6∆ strain and causes the inability to complement the temperature sensitivity of ypt6∆. These finding indicates that Syt1p homodimer has another essential functions rather than regulating its GEF activity. Besides, Syt1p forms dimer through its N-terminal phosphorylation, and the phosphorylation sites are required for the GEF activity. Thus, we suggest that Syt1pC1 is involved in the synthetic enhancement of Ypt6p-dependent pathway, and Syt1p dimer formation may be modulated by phosphorylation , which regulates Arl1p activation and Imh1p recruitment. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-16T17:39:37Z (GMT). No. of bitstreams: 1 ntu-101-R99448003-1.pdf: 1850399 bytes, checksum: a4b500e42e4f40aa32582d8be4619ddf (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | Contents 3
中文摘要 5 Abstract 6 Abbreviation 8 Introduction 9 ADP-ribosylation factors (ARFs) 9 Guanine nucleotide exchange factors (GEF) 13 ARF-like proteins (ARLs) 14 Regulation of GEF 17 Syt1p function in Arl3p-Arl1p-Imh1p pathway 19 Materials and Methods 23 Strains and Media 23 Construct and Primers 23 Polymerase chain reaction 24 Western blotting 24 Yeast extracts preparation 25 Expression and purification of recombinant proteins 26 6× His-Tagged Proteins 27 GST-tagged proteins 28 In vitro GST pull-down assay 28 In vivo pull-down assay 29 Fluorescent microscopy 30 Gel Filtration analysis 31 Cross-Linking of Syt1p 32 Fluorescence Microscopy 33 Results 34 I. Identification of Syt1p self-interaction binding site 34 Syt1p can interact with itself. 34 The C1 region of Syt1p can interact with C-terminus in vitro pull down assays 34 Syt1p∆C1 affects Syt1p self-interaction in vivo. 35 II. The Syt1p protein is present in soluble high-molecular-mass complexes. 37 III. Biological functions of the dimerization-deficient ∆C1 mutant of Syt1p 39 Syt1p∆C1 does not affect Arl1p and Imh1p localization 39 Syt1p∆C1 affects Ypt6p-dependent vacuolar fragmentation in syt1∆ypt6∆ strain. 40 Over-expression Syt1p∆C1 causes the inviability to complement the temperature growth defect of ypt6∆. 40 IV. Phosphorylative Syt1p mutations enhance Syt1p dimer formation 42 Syt1p self-interaction is affected through Syt1p Ser416417 and Thr277 phosphorylation. 42 Discussion 44 Dual forces are required for the dimer formation of Syt1p 44 The auto-inhibition of ARF-GEF regulation 47 Figures 49 Figure 1. Syt1p can interact with itself. 49 Figure 2. The C1 region of Syt1p is required for Syt1p self-interaction. 51 Figure 3. Syt1p∆C1 affects Syt1p self-interaction in vivo. 52 Figure 4. Gel filtration analysis of Syt1p protein. 53 Figure 5. Oligomerization of HA-Syt1p, HA-Syt1p∆C1 and HA-Syt1pC. 54 Figure 6.Syt1p∆C1 does not affect Arl1p localization in arl1syt1∆ strain 55 Figure 7. Syt1p∆C1 does not affect Imh1p localization in syt1∆ strain 56 Figure 8. Syt1p∆C1 affects Ypt6p-dependent vacuolar fragmentation in syt1ypt6∆ strain. 58 Figure 9. Over-expression Syt1p∆C1 does not suppress temperature growth defect of ypt6∆. 59 Figure 10. Syt1p self-interaction is affected through Syt1p Ser416417 phosphorylation. 60 Figure 11. Syt1p self-interaction is affected through Syt1p Thr277 phosphorylation. 61 Figure 12. Model of Syt1p self-interaction regulation in the Ypt6p-depentent pathway 62 Tables 63 Table 1. Yeast strains used in this study 63 Table 2. Constructs used in this study 64 Table 3. Antibodies used in this study 67 References 68 | |
| dc.language.iso | en | |
| dc.subject | 高基氏體 | zh_TW |
| dc.subject | 鳥糞嘌呤核苷 | zh_TW |
| dc.subject | 囊泡運輸 | zh_TW |
| dc.subject | 三磷酸結合蛋白 | zh_TW |
| dc.subject | 腺嘌呤核苷 | zh_TW |
| dc.subject | 二磷酸核醣化因子 | zh_TW |
| dc.subject | 小鳥糞嘌呤核苷 | zh_TW |
| dc.subject | 酸交換因子 | zh_TW |
| dc.subject | Golgi complex | en |
| dc.subject | Sec7 | en |
| dc.subject | GEF | en |
| dc.subject | vesicular trafficking | en |
| dc.subject | GTPase | en |
| dc.subject | ADP-ribosylation factor | en |
| dc.title | 探討第一腺嘌呤核苷二磷酸核糖化相似因子之鳥糞嘌呤核苷酸交換因子的自相互作用的功能特性 | zh_TW |
| dc.title | Functional characterization of self-interaction of an Arl1p GEF- Syt1p | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳瑞華,鄧述諄,王昭雯 | |
| dc.subject.keyword | 高基氏體,腺嘌呤核苷,二磷酸核醣化因子,小鳥糞嘌呤核苷,三磷酸結合蛋白,囊泡運輸,鳥糞嘌呤核苷,酸交換因子, | zh_TW |
| dc.subject.keyword | Golgi complex,ADP-ribosylation factor,GTPase,vesicular trafficking,GEF,Sec7, | en |
| dc.relation.page | 70 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2012-08-15 | |
| 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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