轉運蛋白顆粒 TRAPP 是一個從酵母到哺乳類細胞高度保守的蛋白復合體，參與胞內囊泡運輸的定位融合。在酵母中，有三種形式的TRAPP 復合體存在，共用六個亞基（Bet3, Bet5, Trs20, Trs23, Trs31 and Trs33）。這六個亞基被稱為六亞基核心，是TRAPP復合體的結構基礎，並保守存在於哺乳類細胞中。三種TRAPP復合體分別作用於內質網到高爾基體的運輸，高爾基體內部和內涵體的蛋白轉運以及自噬過程。在哺乳類細胞中，關於TRAPP的作用和復合體的構成尚在研究中。本論文對哺乳類TRAPP（mTRAPP）復合體的構成，以及mTRAPP在 COPII 囊泡運輸過程中的作用進行研究。 / 在論文第一部分，我們在哺乳類細胞中發現了兩個不同的TRAPP復合體，它們分別包含亞基TRAPPC9和TRAPPC8，被確定為mTRAPPII和mTRAPPIII。酵母Trs20的同源體TRAPPC2作為銜接蛋白，分別連接TRAPPC9或TRAPPC8與六亞基核心，從而形成mTRAPPII和mTRAPPIII。D47Y是TRAPPC2的錯義突變體之一，被確認為X-連鎖遲發性脊柱骨骼發育不良（SEDT）的致病基因。與野生型TRAPPC2相比，發生突變的D47Y蛋白與TRAPPC9和TRAPPC8的親和力顯著降低，並且過表達時對破壞高爾基體的結構和改變TRAPPC9的定位顯示出更強的能力。這說明，D47Y能破壞TRAPP復合體的形成並引起早期分泌途徑的缺陷。 / 在論文第二部分，TRAPP被證明可以通過TRAPPC9與p150Glued相互作用。 p150[superscript Glued]作為dynactin的一個亞基，據報道，可以通過與COPII衣被蛋白綁定，使COPII囊泡與微管連接，以實現COPII囊泡從內質網到內質網高爾基體中間復合體（ERGIC）間的移動。在ERGIC附近，COPII衣被蛋白和TRAPP的相互作用可以介導COPII囊泡與其他COPII囊泡（同型拴系）或ERGIC（異型拴系）的拴系過程。我們發現TRAPPC9主要位於ERGIC和cis-Golgi， 並且TRAPPC9的存在減弱了p150[superscript Glued] 和COPII囊泡間的相互作用。這說明在ERGIC附近，TRAPPC9通過與COPII囊泡競爭來綁定p150[superscript Glued]，形成dynactin-TRAPP-COPII三聯復合體。這個復合體允許融合的囊泡（ERGIC）在向cis-Golgi移動的過程中與新來的囊泡發生拴系，從而使COPII囊泡的移動過程和拴系過程協調進行。 / 在論文最後一部分，TRAPPC9的N末端被確認為p150[superscript Glued]的綁定位點。TRAPPC9 N末端片段被發現定位於微管組織中心（MTOC）, 提示TRAPPC9可能具有與MTOC定位相關的功能。在智力遲緩病人中發現的TRAPPC9的兩個缺失突變體，依然能夠與p150[superscript Glued]相互作用，但是因為不能被招募到TRAPP復合體中，很可能不能實現野生型TRAPPC9在囊泡移動拴系過程中的協調作用。 / 總之，本論文證明了TRAPPC2在不同TRAPP復合體形成過程中的銜接作用，以及TRAPPC9在囊泡移動拴系過程中的協調作用。文中對突變蛋白的研究可以為TRAPP突變引起的遺傳學疾病提供生化解釋。 / TRAPP (transport protein particle), a conserved protein complex from yeast to mammals, is well known for its functions in vesicular transport. Three forms of TRAPP complexes found in yeast share six subunits (Bet3, Bet5, Trs20, Trs23, Trs31 and Trs33), referred as the six-subunit core, which is conserved in mammals. These three yeast TRAPP complexes act in ER to Golgi traffic, intra-Golgi and endosomal traffic and autophagy, respectively. But the functions and composition of TRAPP complex remain largely unknown in mammalian cells. This thesis investigated the functions of mammalian TRAPP (mTRAPP) subunits in the assembly of TRAPP complexes and the COPII (coat protein complex II) vesicle transport. / In the first part of this thesis, two different TRAPP complexes containing subunits TRAPPC9 and TRAPPC8 were identified in mammalian cells as mTRAPPII and mTRAPPIII respectively. TRAPPC2, the homologue of yeast TRAPP subunit Trs20, served as an adaptor that links TRAPPC9 or TRAPPC8 with the six-subunit core for the formation of these two mTRAPP complexes. D47Y is previously reported to be one of the missense mutations of TRAPPC2 that have been identified in patients suffering from X-linked spondyloepiphyseal dysplasia tarda (SEDT). We found that D47Y significantly reduced the affinity of TRAPPC2 with TRAPPC9 and TRAPPC8 and disrupted the Golgi structure and TRAPPC9 localization, suggesting that D47Y impairs the formation of TRAPP complexes and causes defect in the early secretory pathway. / In the second part of this thesis, TRAPP was demonstrated to interact with p150[superscript Glued] through TRAPPC9. It is previously reported that p150[superscript Glued], a subunit of dynactin, is responsible for targeting COPII vesicles to the microtubules for movement from ER (endoplasmic reticulum) to ERGIC (ER and Golgi intermediate compartment) by interacting with COPII coat proteins. At ERGIC, COPII vesicles tether with the target compartments: COPII vesicles (homotypic tethering) or ERGIC (heterotypic tethering), which is mediated by the interaction between COPII coat and TRAPP. In this study, we found that TRAPPC9 mainly localized at ERGIC and cis-Golgi and inhibited the interaction between p150[superscript Glued] and COPII vesicle, suggesting that TRAPPC9 competes with COPII vesicle to bind to p150[superscript Glued] to form the dynactin-TRAPP-COPII tripartite complex at the target compartment. This tripartite complex allows vesicles or ERGIC to tether with the incoming vesicles during the movement towards cis-Golgi. Therefore, TRAPPC9 mediates the interaction between TRAPP and dynactin to coordinate the COPII vesicle movement and tethering. / In the last part of this thesis, we found that the N terminal domain of TRAPPC9 was the p150[superscript Glued] binding site. The N terminal fragment was observed to localize at the microtubule organizing center (MTOC), suggesting that TRAPPC9 has native functions related to the MTOC. The two deletional mutations of TRAPPC9 identified for autosomal-recessive mental retardation, retained in interaction with p150[superscript Glued] but could not accomplish the coordination function of the wildtype TRAPPC9 in the COPII vesicle transport due to the failure of being recruited into TRAPP complex. / In summary, this thesis demonstrated the adaptor function of TRAPPC2 in the formation of different TRAPP complexes and the coordination function of TRAPPC9 in the COPII vesicle movement and tethering. Our study on the mutant proteins provides a biochemical explanation to the genetic diseases caused by TRAPP mutant proteins. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zong, Min. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 127-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 中文摘要 --- p.iii / Acknowledgements --- p.vi / List of Publications --- p.vii / List of Abbreviations --- p.viii / List of Figures --- p.xi / Chapter Chapter 1 --- General introduction --- p.1 / Chapter 1.1 --- Intracellular protein transport --- p.1 / Chapter 1.2 --- COPII vesicle is responsible for ER to Golgi transport --- p.2 / Chapter 1.2.1 --- COPII vesicle formation --- p.2 / Chapter 1.2.2 --- COPII vesicle transport pathway --- p.4 / Chapter 1.3 --- TRAPP complexes are involved in vesicular transport --- p.11 / Chapter 1.3.1 --- Yeast TRAPP --- p.12 / Chapter 1.3.2 --- Mammalian TRAPP --- p.14 / Chapter 1.4 --- Objectives --- p.19 / Chapter Chapter 2 --- Materials and methods --- p.20 / Chapter 2.1 --- Materials --- p.20 / Chapter 2.2 --- Cell culture --- p.20 / Chapter 2.3 --- Subcloning --- p.20 / Chapter 2.4 --- Transfection --- p.25 / Chapter 2.5 --- Western Blot --- p.26 / Chapter 2.6 --- Immunoflurescence --- p.28 / Chapter 2.7 --- Antibody development and purification --- p.30 / Chapter 2.8 --- Direct binding assay --- p.33 / Chapter 2.9 --- Statistics --- p.35 / Chapter Chapter 3 --- The Adaptor Function of TRAPPC2 for the formation of mTRAPP complexes Explains TRAPPC2-Associated SEDT --- p.36 / Chapter 3.1 --- Introduction --- p.36 / Chapter 3.1.1 --- The different compositions in Yeast TRAPP complexes result in different functions --- p.36 / Chapter 3.1.2 --- The studies on the compositions of mammalian TRAPP complexes --- p.37 / Chapter 3.1.3 --- The mutants of TRAPPC2 in spondyloepiphyseal dysplasia tarda (SEDT) --- p.39 / Chapter 3.2 --- Results --- p.41 / Chapter 3.2.1 --- TRAPPC2 interacts with TRAPPC9 --- p.41 / Chapter 3.2.2 --- TRAPPC2 interacts with TRAPPC8 --- p.42 / Chapter 3.2.3 --- A disease-causing mutant of TRAPPC2 fails to interact with either TRAPPC9 or TRAPPC8 --- p.48 / Chapter 3.2.4 --- Overexpression of D47Y mutant has the stronger ability to fragment the Golgi and disrupt TRAPPC9 localization than wildtype TRAPPC2 --- p.48 / Chapter 3.3 --- Discussion --- p.52 / Chapter Chapter 4 --- The TRAPPC9- p150[superscript Glued] interaction coordinates COPII Vesicle movement and tethering at the Target Membrane --- p.57 / Chapter 4.1 --- Introduction --- p.57 / Chapter 4.1.1 --- The role of microtubules in ER to Golgi transport --- p.57 / Chapter 4.1.2 --- p150[superscript Glued] is involved in the COPII vesicle transport --- p.58 / Chapter 4.1.3 --- TRAPP tethers COPII vesicles --- p.61 / Chapter 4.1.4 --- COPII coat proteins are involved in vesicle tethering and movement --- p.61 / Chapter 4.2 --- Results --- p.64 / Chapter 4.2.1 --- TRAPP interacts with p150[superscript Glued] --- p.64 / Chapter 4.2.2 --- Characterization of the functions of p150[superscript Glued] and TRAPPC9 in COPII vesicle transport --- p.74 / Chapter 4.2.3 --- TRAPPC9 competes with Sec23/24 to bind with p150[superscript Glued] --- p.87 / Chapter 4.3 --- Discussion --- p.95 / Chapter Chapter 5 --- Characterization of the p150[superscript Glued]-binding domain of TRAPPC9 --- p.100 / Chapter 5.1 --- Introduction --- p.100 / Chapter 5.2 --- Results --- p.103 / Chapter 5.2.1 --- Disease-associated mutations in TRAPPC9 interacts with p150[superscript Glued] --- p.103 / Chapter 5.2.2 --- The N terminal small fragment can interact with CT[superscript Glued] --- p.103 / Chapter 5.2.3 --- C9-2f competes with the full length TRAPPC9 to bind with CT[superscript Glued] --- p.104 / Chapter 5.2.4 --- C9-2f localizes at the MTOC --- p.105 / Chapter 5.2.5 --- C9-2f directly binds with CT[superscript Glued] --- p.111 / Chapter 5.3 --- Discussion --- p.115 / Chapter Chapter 6 --- General Discussion --- p.119 / Chapter 6.1 --- How does mTRAPP function in the COPII vesicle transport? --- p.119 / Chapter 6.2 --- How do the disease-causing TRAPP mutants impair the normal functions? --- p.121 / Chapter 6.3 --- The implicated functions of TRAPPC9 with the localization at MTOC --- p.124 / Chapter 6.4 --- Future studies --- p.124 / Chapter 6.5 --- Conclusion --- p.126 / References --- p.127 / Chapter Appendix A- --- chemicals and reagents --- p.139 / Chapter Appendix B- --- Antibody lists --- p.141 / Chapter Appendix C- --- DNA lists --- p.142 / Chapter Appendix D- --- Buffer recipes --- p.144
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328255 |
| Date | January 2012 |
| Contributors | Zong, Min., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xii, 145 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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