No description available.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity. / AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.

No description available.

In eukaryotic cells, proteins continuously flux through the organelles of the secretory pathway in an essential cellular process called protein secretion. This dynamic process originates at the endoplasmic reticulum (ER), where translating ribosomes push linear peptides into the ER membrane and lumen. ER chaperones assist in folding nascent peptides into three-dimensional conformations and proteins are concentrated into membrane-encapsulated vesicles bound for the Golgi apparatus. ER to Golgi transport is mediated by a set of cytosolic coat proteins called COPII. The COPII coat polymerizes into a lattice on the ER membrane that is able to bend the membrane around secretory cargo and bud off a spherical vesicle. Protein secretion is subject to rapid changes as a cell responds to its environment and requirements for viability alter. In addition to accommodating short-term demands, such as translational up-regulation, evolved complexity of secretory proteins over time, has also required that secretory components adapt. In both cases changes in secretory demands require that the COPII proteins have an inherent flexibility to navigate these changes without disrupting secretory flux. In this work I have examined a family of quintessential secretory cargo, p24 proteins, that challenge protein secretion. This family of proteins forms a hetero-tetrameric complex that cycles between the ER and the Golgi and mediates transport of glycosylphosphatidylinositol-anchored proteins (GPI-APs). Here I present evidence that suggests, when present in vesicles, both p24 proteins and their GPI-AP cargo present a challenge to vesicle formation. I posit that three attributes of these proteins present a local barrier to membrane bending: Lumenal asymmetric distribution across the membrane, high cellular abundance and affinity for ceramide rich membranes. I have also elucidated mechanisms that the coat has evolved to accommodate troublesome cargo such as p24 proteins, which enhance structural scaffolding and increase average vesicle size. Finally I present preliminary findings indicating that p24s also contribute to ER homeostasis by preventing aberrant incorporation of proteins into vesicles. Comprehensively, these findings have shed light on the role of p24 proteins in vesicles. Traditionally thought to be canonical ER cargo receptors, these proteins also appear capable of contributing to the composition of the vesicles in which they reside, and impacting trafficking efficiency in two ways: First by directly mediating transport of GPI-APs and second by uniformly packing vesicles to avoid wasteful secretion. My work has contributed to a growing notion in the field that secretory cargo are not inert passengers but active participants in vesicle mediated secretion.

蛋白質分泌及胞吐作用是指蛋白質在內質網(ER)中合成後前往質膜(PM) ， 隨後， 被分泌到達細胞外的過程。 而細胞分泌路徑是指蛋白質途經數個含有膜包被的細胞器後運送到细胞之外。 這些細胞器，包括內質網， 高爾基體， 反式高爾基網絡(TGN) 及質膜。 分泌蛋白分泌出细胞之外後， 在细胞外基質中進行其功能。 / 為達到這項研究的目的，我們結合了細胞、分子和生物化學上的方法， 來對蛋白質運輸路徑及參與蛋白質分泌的細胞器進行研究。首先，通過MALDI-MS/MS對煙草懸浮BY-2細胞中的原態分泌性蛋白質進行分析。第二，把已識別的分泌蛋白包括陽離子過氧化物酶同工酶40K（40K）和N1過氧化物酶（N1），透過轉基因細胞的GFP融合表達方式、及應用特異性抗體於免疫螢光和膠體金免疫電鏡上的測定來對其特性作進一步分析。 第三，總合以上的研究，煙草懸浮BY-2細胞的典型蛋白質分泌路徑次序為質網 - 高爾基體 - 反式高爾基網絡 - 質膜。 / Protein secretion or exocytosis is the process by which proteins synthesized in the endoplasmic reticulum (ER) travel to the plasma membrane (PM) for their subsequent secretion outside of the cell. The secretory pathway responsible for protein secretion contains several membrane-bounded organelles such as the ER, Golgi apparatus, trans-Golgi Network (TGN), and PM. The secreted proteins move outside of the cell and perform their functions in the extracellular matrix. / The general objective of this study was to examine the transport pathways and organelles involved in protein secretion in plant cells using a combination of cellular, molecular and biochemical approaches. First, major native secreted proteins in suspension cultures of tobacco BY-2 culture cells were identified via MALDI-MS/MS analysis. Second, the identified secreted proteins, cationic peroxidase Isozyme 40K (40K) and peroxidase N1 (N1), were further characterized by examining the GFP fusion expression of transgenic cell lines and by generating specific antibodies in immunofluorescent and immunogold electron microscope (EM) studies. Third, throughout all of these studies, a typical ER-Golgi-TGN-PM pathway was mapped for protein secretion in tobacco BY-2 cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lam, Chun Kok. / "December 2012." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 79-84). / Abstracts also in Chinese. / Thesis /Assessment Committee --- p.i / Statement --- p.i / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.v / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1. --- Secreted protein --- p.1 / Chapter 1.2. --- Secretory Pathway --- p.1 / Chapter 1.3. --- Protein secretion --- p.2 / Chapter 1.4. --- Plant Peroxidases --- p.3 / Chapter 1.5. --- Project Objective --- p.4 / Chapter 1.6. --- Significance --- p.4 / Chapter Chapter 2 --- Materials and Methods --- p.6 / Chapter 2.1. --- Mass spectrometry analysis --- p.6 / Chapter 2.2. --- Generation of 40K/N1-GFP construct --- p.7 / Chapter 2.2.1. --- For transient expression --- p.7 / Chapter 2.2.2. --- For stable expressing constructs --- p.7 / Chapter 2.3. --- Transient expression of 40K/N1-GFP --- p.7 / Chapter 2.4. --- Generation of transgenic cell lines --- p.8 / Chapter 2.5. --- Fluorescence microscopic screening --- p.9 / Chapter 2.6. --- Generation and characterization of antibodies specific for 40K/N1 peroxidase --- p.9 / Chapter 2.7. --- Confocal immunofluorescence studies --- p.10 / Chapter 2.8. --- (TIRF) Total internal reflection fluorescence microscopy --- p.11 / Chapter 2.9. --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.11 / Chapter 2.10. --- Drug Treatment --- p.12 / Chapter 2.10.1. --- Dexamethasone (dex) --- p.12 / Chapter 2.10.2. --- Brefeldin A (BFA)/Concanamycin A (ConcA) --- p.12 / Chapter 2.11. --- Salt treatment (plasmolysis) --- p.13 / Chapter 2.12. --- EM (electron microscopy) study --- p.13 / Chapter Chapter 3 --- Results --- p.14 / Chapter 3.1. --- Protein secretion from tobacco BY2 cells --- p.14 / Chapter 3.2. --- Western blot analysis --- p.15 / Chapter 3.3. --- Protein expression in tobacco plant tissues --- p.15 / Chapter 3.4. --- EM labeling on the wild type BY-2 cells --- p.16 / Chapter 3.5. --- Localization in the tobacco root tip apoplast --- p.17 / Chapter 3.6. --- 40K/N1 peroxidase transient/stable cell line expression --- p.18 / Chapter 3.7. --- Time course study of 40K/N1 peroxidase-GFP cell line expression after induction --- p.18 / Chapter 3.8. --- Plasmolysis (salt treatment analysis) --- p.20 / Chapter 3.9. --- Brefeldin A (BFA) and concanamycin A (ConcA): Trafficking through the Golgi and TGN --- p.20 / Chapter 3.10. --- Examining exocytosis by total internal reflectance fluorescence (TIRF) --- p.22 / Chapter 3.11. --- Immunolabeling study --- p.23 / Chapter 3.12. --- EM study on transgenic 40K & N1 peroxidase-GFP cell lines --- p.24 / Chapter Chapter 4 --- Discussion --- p.26 / Chapter 4.1. --- Trafficking from the ER to the extracellular matrix --- p.26 / Chapter 4.2. --- Secretion through PM by exocytosis --- p.28 / Chapter 4.3. --- Time required for the secretory pathway --- p.29 / Chapter 4.4. --- Similarities of 40K and N1 --- p.30 / Chapter 4.5. --- Future perspectives --- p.30 / References --- p.79

The methylotrophic yeast. Pichia pastoris, is a powerful, adaptable, and inexpensive recombinant expression system commonly used to secrete heterologous protein. Although P. pastoris is a popular host organism, secretion inefficiency continues to be a major hurdle in its ability to produce high levels of foreign protein. Optimization of cis- and trans-acting factors has greatly enhanced the secretory capabilities of P. pastoris, however protein-specific engineering of a host organism is costly and not always effective. P. pastoris' secretion inefficiency is commonly due to trans-acting factors. Strains of S. cerevisiae have been engineered, through random genomic mutation, that are capable of overcoming these /ram-acting factors to secrete high levels of foreign protein. The Lin-Cereghino laboratory at University of the Pacific has developed a screen to identify mutations in P. pastoris capable of circumventing secretion obstacles. The P. pastoris genome was randomly disrupted through restriction enzyme-mediated integration of an antibiotic resistance marker. Supersecretion mutants were identified by their ability to secrete β-galactosidase, a reporter enzyme not natively secreted by P. pastoris. Sixteen β-galactosidase secretion (bgs) mutants were initially isolated by the Lin-Cereghino lab. This research focused on characterizing one of the resultant bgs mutants, ///. Initial sequencing and alignment studies identified the predicted LI1p sequence to be homologous to S. cerevisiae protein kinase C (PKC). Considering the role of PKC in the Cell Wall Integrity pathway of S. cerevisiae. the cell wall and secretory organelles of III were closely examined using transmission electron microscopy. Additionally, a qualitative alkaline phosphatase assay was used to evaluate the cell wall integrity of ///. Finally, the secretory phenotype of 111 was examined using a group of structurally and functionally diverse reporter proteins. In characterizing the bgs mutant, III, this research contributes to an understanding of cellular components that limit protein secretion in the yeast, P. pastoris.

Pseudomonas aeruginosa is a Gram-negative bacterium implicated in several clinical contexts. In its association with immunocompromised hosts including cystic fibrosis patients, P. aeruginosa is able to exploit the host immune response to acquire key factors essential to its adaptation. As such, key virulence factors including the Type III Secretion System (T3SS), initially essential in acute infection, is reduced in its significance in chronic colonization. On the contrary, other phenotypes are essential for the altered priorities in chronic colonization. The signals of the host immune response initiating the phenotypic switch from the expression of acute virulence factors to chronic virulence factors have not been well defined. Additionally, the function of the type VI secretion system (T6SS), a protein secretion apparatus, in chronic infection has been well established. Clinical isolates obtained from acute and chronic P. aeruginosa infections suggested selective regulation of the T6SS, namely up regulation of the H3-T6SS in chronic infection. We used murine models of infection to understand the in vivo transcriptional regulation of the T6SS of PAO1. Itaconate, an anti-inflammatory metabolite generated by the host, selectively upregulated transcription of a H3-T6SS-associated locus, vgrG3. Here we present evidence to show how the host immune response, namely metabolic changes in response to infection may be exploited to support the organism’s adaptation to the lung microenvironment. In the evaluation of such a phenotypic response notable in chronic infections, the Type VI Secretion System (T6SS) of P. aeruginosa is selectively regulated by a host-specific metabolic product, itaconate. While P. aeruginosa contains genetic clusters for three (H1-, H2-, and H3-T6SS) evolutionarily distinct T6SSs, we found the H3-T6SS to be up-regulated significantly (p<0.05) in the presence of this anti-inflammatory signal. Characterization of this response reveals that itaconate induces metabolic stress in P. aeruginosa. In an acute pneumoniae mouse model, deletion of the H3-T6SS locus results in increased colonization of the murine lung. Analysis of bronchoalveolar lavage fluid from wild type and H3-T6SS null-infected mice reveals alterations in metabolic pathways including purine metabolism, carbon metabolism, and arginine biosynthesis. Overall our work outlines the H3-T6SS as a phenotypic response to metabolic stress induced by the host immune response, serving to mediate pathways essential in pathogenesis. Further understanding of such phenotypes as the T6SS implicated in chronic infection is essential in treatment interventions in the clinic.

Exocyst is a protein complex composed of eight subunits, evolutionarily conserved in yeasts, animals, and plants. The main function of exocyst is to mediate the tethering of secretory vesicles to the plasma membrane. However, the involvement of exocyst in some other processes, especially in autophagy, has been recently discovered. Plant exocyst is specific because most of its subunits have multiple paralogs. The most diversified subunit is EXO70, which is encoded by 23 paralogous genes in Arabidopsis thaliana. In this thesis, I dealt with subunit AtEXO70E2 (AT5G61010), which has been localized to double-membrane compartments considerably reminiscent of autophagosomes. These compartments were named EXPOs (for exocyst-positive organelles) and described as a component of unconventional protein secretion pathways. There are also hints that EXO70E2 could play a role in autophagic processes. However, details of this relationship remained unexplored. For my experiments, I used stably transformed lines of A. thaliana and transiently transformed leaves of Nicotiana benthamiana. I performed numerous colocalization experiments, applied various pharmacological treatments to the studied lines, and analyzed a mutant line in the EXO70E2 gene. According to my observations, protein EXO70E2 is expressed especially...