Every cell is faced continuously with the task of transducing a vast number of external signals into appropriate intracellular responses. In mammalian cells, membrane bound receptors modulate the production of secondary messenger molecules, such as cyclic AMP (cAMP). Cell signaling through second messengers relies on the diffusion of such second messengers from sites of production to sites of intracellular action to modulate the activities of an intracellular effector molecule, which in turn modulates a cellular response. Protein kinase A is the primary sensor of intracellular concentrations of cAMP, and protein kinase A (PKA)-mediated phosphorylation events are regulated by fluctuations in cAMP concentrations. This thesis examines the mechanism by which protein kinase A functions within fish melanophore to regulate intracellular activities in response to cAMP. A novel in situ assay to monitor protein kinase A-dependent phosphorylation is described here. By using a fluorescent substrate, the activities of protein kinase A are characterized in living cells as a function of phosphorylation-dependent fluorescence decay. Physiologically manipulated cells were microinjected with the fluorescent substrate to correlate the activities of protein kinase A and the subsequent effects on melanosome distribution. Cytoplasmic compartmentalization of protein kinase A is one means to regulate the specificity of a multifunctional kinase. The physiologic effects of an overexpressed PKA anchoring protein, Ht31, demonstrated that protein kinase A anchoring is required to direct the movements of melanosomes. Additional studies demonstrated that protein kinase A redistributes with the stimulated movements of melanosomes and interacts with proteins from a motor-protein complex. These observations shed light on how a transduction complex, consisting of a group of proteins, which localize components within the cell and provide motor activity, is able to integrate signaling information that regulate melanosome movements. I propose a model and suggest that protein kinase A is a component of the transduction complex: the proposed PKA complex constitutes a molecular servomechanism comprised of a sensor, an effector, and a regulator that function together in feedback loop to direct the motor-protein dependent transport of melanosomes. / Graduation date: 2001

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

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在酵母和动物细胞中，内膜蛋白（EMP）隶属于进化上保守的九跨膜结构域（TMD）蛋白家族，此类蛋白的共同结构特征是有一个很长的N 末端，紧接着九个跨膜结构域后面连着暴露于胞质的C 末端短肽。在黏菌以及酵母中，EMP 蛋白被发现参与蛋白分泌功能以及细胞的贴壁生长。拟南芥基因组中有12 个EMP 编码基因，关于它们所编码蛋白质的定位以及功能甚少有研究报道。在此项研究中，借助于不同的生化以及细胞生物学手段，包括瞬时表达、共聚焦成像、电子显微镜分析、pull down 相互作用蛋白捕获以及质谱分析，我将主要研究拟南芥中EMP12 蛋白的亚细胞定位以及分选信号和蛋白靶定机理。通过研究我发现：1）在拟南芥植物中，内源性的EMP12 蛋白（通过EMP12 特异性抗体标记）和绿色荧光蛋白标记的GFP-EMP12 蛋白都定位于高尔基体；2）C 末端连接的GFP 导致 EMP12-GFP 融合蛋白错误地定位到后高尔基体细胞器，并最终被运送到液泡而降解；3）EMP12 蛋白的C 末端有两个分选信号：内质网输出信号（FV/Y）和一个新发现的高尔基体滞留信号（KXD/E），这两个分选信号分别和COPII 和COPI 囊泡相互作用从而实现其功能；4）把EMP12 的高尔基体滞留信号连接到其他后高尔基体定位的膜蛋白时可以滞留它们在高尔基体。EMP12 中发掘的内质网输出信号和高尔基体滞留信号在所有的植物EMP 蛋白家族中都是非常保守的，这也预测了植物EMP 蛋白家族都通过类似的分选途径而定位于高尔基体并且预示这些保守的分选信号对于植物EMP 蛋白家族的靶定是非常重要的。 / Endomembrane Proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N-terminus, nine transmembrane domains (TMD) and a short cytoplasmic tail (CT). In the slime mold and yeast, it has been reported that EMP family proteins are involved in protein secretion function and cell adhesion growth. The Arabidopsis genome contains 12 EMP members (EMP1 to EMP12) with little information about their protein subcellular localization and function. Here I studied the subcellular localization and targeting mechanisms of EMP12 in Arabidopsis through a combination of biochemical and cell biological approaches including transient expression, confocal observation, electron microscopy, pull down and mass spectrometry. I found that 1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; 2) GFP fusion at the C-terminus of EMP12 caused mis-localization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; 3) EMP12 CT contained dual sorting signals: an ER export motif (FV/Y) and a novel Golgi retention signal (KXD/E) that interacted with COPII and COPI subunits respectively to achieve their ER export or Golgi retention functions; 4) the Golgi-retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all the plant EMP isoforms, thus likely representing a general mechanism for EMP targeting in plant cells. / Detailed summary in vernacular field only. / Gao, Caiji. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 86-94). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Thesis/Assessment Committee --- p.I / Statement --- p.II / Abstract --- p.III / 摘要 --- p.V / Acknowledgements --- p.VI / Table of Contents --- p.VIII / List of Tables --- p.XI / List of Figures --- p.XII / List of Abbreviations --- p.XIV / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The Plant Secretory Pathway --- p.2 / Chapter 1.2 --- Sorting Signals for Membrane Protein Trafficking between ER and Golgi --- p.5 / Chapter 1.3 --- Endomembrane Proteins (EMPs) in Non-plant Species --- p.7 / Chapter 1.4 --- Endomembrane Proteins (EMPs) in Plants --- p.8 / Chapter 1.5 --- Objectives of this Research --- p.11 / Chapter Chapter 2 --- Materials and Experimental Procedures --- p.12 / Chapter 2.1 --- Plasmid Construction --- p.13 / Chapter 2.2 --- Plant Materials and Transient Expression in Protoplasts --- p.17 / Chapter 2.3 --- Immunofluorescence Study and Confocal Microscopy --- p.17 / Chapter 2.4 --- Electron Microscopy (EM) Study --- p.18 / Chapter 2.5 --- Antibodies, Protein Preparation and Western Blot Analysis --- p.21 / Chapter 2.6 --- In Vitro Binding Assay of COPI and COPII Coat Proteins to Sorting Motifs --- p.22 / Chapter 2.7 --- Mass Spectrometry (MS) Identification of Binding Proteins --- p.23 / Chapter 2.8 --- Topology Analysis and Protease Protection Assay --- p.23 / Chapter Chapter 3 --- Results --- p.25 / Chapter 3.1 --- At EMP12 is a Golgi-localized Multiple TMDs Protein --- p.26 / Chapter 3.1.1 --- Trypsin Digestion and Topology Analysis of EMP12 --- p.26 / Chapter 3.1.2 --- Golgi Localization of Endogenous EMP12 and GFP-EMP12 Fusion in Arabidopsis Plant --- p.29 / Chapter 3.1.3 --- Golgi Localization of GFP-EMP12 Fusion in Arabidopsis Protoplasts --- p.36 / Chapter 3.2 --- The Cytosolically Exposed C-terminal Region Contains Essential ER Export Signals for the Trafficking of EMP12 from the ER to the Golgi --- p.38 / Chapter 3.2.1 --- C-terminus is Essential for ER export of EMP12 --- p.38 / Chapter 3.2.2 --- Identification of FV/Y Residues as the ER Export Signals for EMP12 --- p.40 / Chapter 3.2.3 --- The ER Export Signals, FV/Y, Can Interact with COPII Subunit Sec24 --- p.46 / Chapter 3.3 --- The C-terminal Fused GFP-tag Causes Mislocalization of EMP12-GFP Fusion to TGN, PVC and Vacuole --- p.48 / Chapter 3.4 --- The EMP12 CT Contains a Novel KXD/E Motif for Golgi Retention --- p.51 / Chapter 3.4.1 --- Identification of the KXD/E Motif for Retaining the Mislocalized EMP12-GFP in the Golgi Apparatus --- p.51 / Chapter 3.4.2 --- The Mislocalized EMP12-GFP Fusions Go to Vacuole via PVC for Degradation --- p.58 / Chapter 3.4.3 --- Western Blot Analysis of Protoplasts Expressing various EMP12-GFP fusions --- p.61 / Chapter 3.5 --- The KXD/E Motif Shows Similar Golgi Retention Function for Other Plant EMP Homologues --- p.63 / Chapter 3.6 --- The KXD/E Motif Interacts with COPI Vesicle to Achieve its Golgi Retention Function --- p.65 / Chapter 3.7 --- The RNIKCD Functions as a Golgi Retention Motif for Post-Golgi Membrane Proteins --- p.69 / Chapter 3.7.1 --- The RNIKCD Can Retain the SCAMP1-GFP in the Golgi Apparatus --- p.69 / Chapter 3.7.2 --- The RNIKCD Motif Causes Partial Golgi Retention of TPK1-GFP --- p.71 / Chapter 3.8 --- Localization Patterns of Singly-expressed Various EMP12 Fusions and Their Mutants in Arabidopsis Protoplasts --- p.74 / Chapter Chapter 4 --- Discussions, Conclusions and Perspectives --- p.76 / Chapter 4.1 --- Discussions --- p.77 / Chapter 4.1.1 --- The Position of GFP Tag Affects the Proper Golgi Localization of EMP12 --- p.77 / Chapter 4.1.2 --- Multiple Sorting Signals and Proper COPII Vesicle Function are Involved in ER Export of EMP12 --- p.79 / Chapter 4.1.3 --- KXD/E Motif and COPI Vesicle Mediate Golgi Localization of EMP12 --- p.80 / Chapter 4.2 --- Conclusions and Working Model of EMP12 Trafficking --- p.83 / Chapter 4.3 --- Future Perspectives --- p.85 / References --- p.86 / List of Publications --- p.95

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

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The twin-arginine translocase allows the translocation of folded protein substrates across the cytoplasmic membrane of bacteria and archaea or the thylakoid membrane of plants. In Escherichia coli, its protein components TatA, TatB and TatC assemble dynamically upon interaction with protein substrates. Prior to export, the machinery performs a quality control check so that only correctly folded proteins are translocated. The first objective of this work was to derive and apply new methodologies based on the inherent qualities of the pathway. We developed a new bacterial two-hybrid system that capitalizes on the folding quality control mechanism of the Tat pathway. One protein (prey) is fused to Tat-specific signal peptide. A second (bait) protein is produced as a fusion to a reporter that produces a "signal" (growth or enzymatic activity) only when the bait-reporter fusion binds to the prey and the resulting complex is exported into the periplasm via the Tat pathway. As a second biotechnological application of the Tat pathway, we developed a phage display system that allows the protein of interest to fold within the cytoplasm prior export and display onto phage particles. This is in contrast to the conventional phage display system, in which displayed protein folds in the periplasm. We took advantage of this new system to screen a library of 2 x 10⁶ of fluorescent GFP variants containing a hexameric peptide insertion for ligand binding. Despite the diversity of the hexamer, we were not able to isolate single GFP variants that would bind with specificity to various ligands. This highlights the difficulty in engineering GFP variants that can bind to other proteins while retaining the ability to fluoresce. The second aspect of this research was to examine mechanistic aspects of the Tat pathway. TatB and TatC are responsible for the recognition of Tat signal peptides. Here, we established the importance of TatC as the crucial component of the Tat pathway for the interaction with the hallmark twin-arginine motif within Tat signal peptide. Substitution of the RR dipeptide with a KK sequence completely abolishes export. In a genetic screen using a ssTorA(KK)-GFP-SsrA as a reporter. We identified several amino acid substitutions within TatC that allowed the alteration of the substrate specificity of the pathway as indicated by the impairment of indigenous Tat substrates. Finally, we analyzed the conformational dynamics of TatA using GFP fusions and by incorporation of the chemically reactive, non-canonical amino acid azidohomoalanine.

The primary cilium is an organelle that serves as a signaling center of the cell and is involved in the hedgehog signaling, cAMP pathway, Wnt pathways, etc. Ciliary function relies on the transportation of molecules between the primary cilium and the cell, which is facilitated by intraflagellar transport (IFT). IFT88, one of the important IFT proteins in complex B, is known to play a role in the formation and maintenance of cilia in various types of organisms. The ciliary transition zone (TZ), which is part of the gating apparatus at the ciliary base, is home to a large number of ciliopathy molecules. Recent studies have identified important regulating elements for TZ gating in cilia. However, the architecture of the TZ region and its arrangement relative to intraflagellar transport (IFT) proteins remain largely unknown, hindering the mechanistic understanding of the regulation processes. One of the major challenges comes from the tiny volume at the ciliary base packed with numerous proteins, with the diameter of the TZ close to the diffraction limit of conventional microscopes. Using a series of stimulated emission depletion (STED) superresolution images mapped to electron microscopy images, we analyzed the structural organization of the ciliary base. Subdiffraction imaging of TZ components defines novel geometric distributions of RPGRIP1L, MKS1, CEP290, TCTN2 and TMEM67, shedding light on their roles in TZ structure, assembly, and function. We found TCTN2 at the outmost periphery of the TZ close to the ciliary membrane, with a 227±18 nm diameter. TMEM67 was adjacent to TCTN2, with a 205±20 nm diameter. RPGRIP1L was localized toward the axoneme at the same axial level as TCTN2 and TMEM67, with a 165±8 nm diameter. MKS1 was situated between TMEM67 and RPGRIP1L, with an 186±21 nm diameter. Surprisingly, CEP290 was localized at the proximal side of the TZ close to the distal end of the centrin-labeled basal body. The lateral width was unexpectedly close to the width of the basal body, distant from the potential Y-links region of the TZ. Moreover, IFT88 was intriguingly distributed in two distinct patterns, forming three puncta or a Y shape at the ciliary base found in human retinal pigment epithelial cells (RPE), human fibroblasts (HFF), mouse inner medullary collecting duct (IMCD) cells and mouse embryonic fibroblasts (MEFs). We hypothesize that the two distribution states of IFT88 correspond to the open and closed gating states of the TZ, where IFT particles aggregate to form three puncta when the gate is closed, and move to form the branches of the Y-shape pattern when the gate is open. Two reservoirs of IFT particles, correlating with phases of ciliary growth, were localized relative to the internal structure of the TZ. These subdiffraction images reveal unprecedented architectural details of the TZ, providing a basic structural framework for future functional studies. To visualize the dynamic movement of IFT particles within primary cilia, we further conducted superresolution live-cell imaging of IFT88 fused to EYFP in IMCD cells. Our findings, in particular, show IFT88 particles pass through the TZ at a reduced speed by approximately 50%, implying the gating mechanism is involved at this region to slow down IFT trafficking. Finally, we report the distinct transport pathways of IFT88 and Smo (Smoothened), an essential player to hedgehog signaling, to support our hypothesis that two proteins are transported in different mechanisms at the ciliary base, based on dual-color superresolution imaging.

Huntington's disease (HD) is caused by an expanded plyglutamine repeat in the huntingtin protein. In this study, I focused on the effect of the mutant huntingtin protein (mhtt) on the subcellular localization of glutamic acid decarboxylase (GAD), the enzyme responsible for synthesizing gama-aminobutyric acid (GABA). Subcellular distribution of GAD65 is significantly altered in two neuronal cell lines that express either the N-terminus or full length mhtt. GAD65 is predominantly associated with the Golgi membrane in cells expressing normal huntingtin (Htt). However, it diffuses in the cytosol of cells expressing mhtt. Palmitoylation of GAD65 is required for GAD65 trafficking, and I demonstrated the palmitoylation of GAD65 is reduced in the HD model. Overexpression of huntingtin-interacting protein 14 (HIP14), the enzyme that palmitoylates GAD65, rescues GAD65 palmitoylation and vesicle-associated trafficking. This data suggests that impairment of GAD65 palmitoylation by mhtt may alter its localization and lead to altered inhibitory neurotransmission in HD. / by Daniel Rush. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2012. Mode of access: World Wide Web.