The rapid emergence of multidrug resistant bacterial strains represents a major global healthcare issue. Amongst five known classes of membrane transporters, which play a huge role in multidrug efflux, primary-active ATP-binding cassette (ABC) transporters are ATP powered whilst secondary-active transporters utilize electrochemical ion gradients to drive substrate transport. Mechanistic insights into transport by these proteins can help with the design and development of novel therapeutic agents against multidrug resistance, and can increase our understanding of the physiological functions of these transporters. Although available crystal structures illustrate a common alternate access model for transport by ABC transporters, the mechanisms by which metabolic energy is coupled to the transport cycle is still elusive. This thesis presents a series of functional studies using whole cells as well as artificial phospholipid membranes to study the energetics of transport, and the influence of membrane phospholipids on substrate transport by the homodimeric Escherichia coli lipid A/multidrug ABC exporter MsbA. Current alternating access models for ABC exporters involve cycling between conformations with inward- and outward-facing substrate-binding sites in membrane domains (MDs) in response to engagement and hydrolysis of ATP at the nucleotide-binding domains (NBDs). Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. In this thesis, analogous substrate transport reactions are also studied for two other ABC exporters, the MsbA homologue LmrA and the human multidrug transporter ABCG2. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. It also raises questions about the role of NBDs in the transport process. Comparisons of drug transport and resistance in cells expressing MsbA-MD (truncated MsbA lacking the NBD) and full length MsbA (MsbA-WT) demonstrate increased transport efficiency of MsbA-WT compared to MsbA-MD. In addition, growth studies using E. coli WD2 cells, which are conditionally defective in MsbA’s essential activity in lipid A transport, show that lipid A transport can be restored by the expression of MsbA-WT but not MsbA-MD or ATP-hydrolysis impaired Walker A mutant (MsbA- ΔK382). Lastly, we also present biochemical experiments with proteoliposomes with a defined phospholipid composition, which suggest that cardiolipin is essential for the transport activity of MsbA. These techniques open the way to further explore lipid-proteins interactions and examine the physiological role(s) of MsbA. In conclusion, this thesis produces new insights in the mechanisms of transport and energy coupling in ABC exporters.

Leung Kim-chuen Andrew. / "August 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 168-200). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

No description available.

胞吐作用定義為囊運小泡將物質運輸到質膜或細胞外空間的轉運過程。其中關鍵的一步發生在同源SNARE 蛋白介導的膜融合之前，即將胞吐囊泡瞄向並靶定在適當的質膜位點。先前在酵母和哺乳動物中的研究表明，一個名為exocyst 的蛋白質複合體在這一關鍵步驟發揮作用。exocyst 蛋白複合體最早在酵母發現，之後這個複合體也在哺乳動物中被發現。這個複合體包含8 個不同的亞基：SEC3，SEC5，SEC6，SEC8，Sec10，Sec15，Exo70 和Exo84。Exocyst 同源蛋白也已在植物中發現。相比酵母和動物，exocyst 在植物體內的功能還鮮為人知，尤其是在胞吐運輸過程中的作用 。通過瞬時表達熒光蛋白標記的擬南芥同源的exocyst 蛋白Exo70：AtExo70E2 以及使用這個同源物的特異抗體，我們在擬南芥和煙草BY－2 懸浮培養細胞中發現了一種新的細胞器，並命名為exocyst 陽性細胞器（EXPO）。這種細胞器分別位於質膜或是細胞質中。由於它未能與任何傳統的細胞器標記物重合，或是被布雷菲爾德菌素A，渥曼青黴素和刀豆素A 影響，以及不能與FM4-64 重合，我們判斷這些細胞器不定位於常規的分泌或胞吞途徑中。對於快速冷凍樣本進行的免疫電子顯微鏡顯示EXPO 的雙膜性質，同時也發現了陽性標記的位於質膜外的單膜囊泡的存在。與此同時，在野生型細胞中也發現了同樣結構的細胞器。EXPO和自噬體非常相似， 都有兩層膜。然而，EXPO 不能被的自噬標記物（AtAtg8e）所標記。同時，在營養脅迫條件下，EXPO 的數量也沒有增加。因此，EXPO 代表著植物所特有的一種非常規分泌形式。 / 此外，通過在擬南芥原生質體內進行瞬時表達，我進一步證實在AtExo70E2 存在的條件下， 一些exocyst 成員可以被招募到EXPO 。AtExo70E2 的旁系同源物AtExo70A1 是在這方面物法取代AtExo70E2 的作用。蛋白蛋白相互作用分析證實了AtSec10 或AtSec6 與AtExo70E2 之間的相互作用。 AtExo70E2，而不是它在酵母或是動物中的同源蛋白，可以誘導EXPO 在動物細胞中的形成。反之，人或是酵母Exo70 同源蛋白都不能誘導EXPO 在植物細胞中的形成。這些結果表明AtExo70E2 在EXPO 形成過程中的特定的以及至關重要的作用。 / Exocytosis defines the process in which vesicles transport substances to the plasma membrane (PM)/extracellular space of the cell. One key step of exocytosis is the targeting and docking of the exocytic vesicles to the appropriate PM sites, which is prior to membrane fusion mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE). Previously studies have demonstrated that a protein complex called exocyst complex is involved in this key step in yeast and mammals. The exocyst complex, containing eight different subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, was first identified in yeast and subsequently in mammals. Exocyst homologs have also been found in plants. In comparison to its yeast and animal counterparts, little is known about the function of exocyst proteins in plants especially in the process of exocytosis. By using both antibodies specific for one of the orthlogs of exocyst protein: AtExo70E2 as well as transiently-expressed fluorescently-tagged constructs for this exocyst subunit, a novel organelle termed exocyst-positive organelle (EXPO) was identified in suspension cultured Arabidopsis and tobacco BY-2 cells. These organelles were located to both the plasma membrane and cytosol. Based on their failure to overlap with any conventional organelle markers or response to brefeldin A (BFA), wortmannin or concanamycin A (ConcA) treatments, as well as their inability to take up the endocytic dye FM4-64, these organelles were thus not lie on the conventional secretory or endocytic pathways of plant cells. Immunogold electron microscopy (EM) of cryofixed samples revealed the double membrane nature of EXPO and also produced labeling of large single-membrane bound vesicles outside of the PM. These structures were also identified in wild type cells. EXPO and autophagosomes are similar in that both have two boundary membranes. However, EXPO did not label positively with YFP-AtAtg8e, a standard marker for autophagosomes, nor did the number of EXPO increase when the cells were subjected to nutrient stress. Therefore, EXPO represents a form of unconventional secretion unique to plants. / Further studies demonstrated that a number of exocyst subunits can be positively recruited to EXPO in the presence of AtExo70E2 by performing transient expression in Arabidopsis protoplasts. The paralog AtExo70A1 is unable to substitute for AtExo70E2 in this regard. Protein-protein interaction assay have confirmed the interaction between AtExo70E2 and AtSec6 and AtSec10. AtExo70E2, but not its yeast counterpart, is also capable of inducing EXPO formation in animal cells. Inversely, neither human nor yeast Exo70 homologs are able to cause the formation of EXPO in Arabidopsis protoplasts. These results point to a specific and crucial role for AtExo70E2 in EXPO formation. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Ding, Yu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 101-118). / Abstracts also in Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xi / List of Abbreviations --- p.xiv / Chapter CHAPTER 1 --- p.1 / General Introduction --- p.1 / Chapter 1.1 --- The secretory system in eukaryotic cells --- p.2 / Chapter 1.2 --- Exocytosis and exocyst complex --- p.6 / Chapter 1.3 --- Project Objectives --- p.7 / Chapter CHAPTER 2 --- p.9 / Exocyst-positive organelles (EXPOs) mediate unconventional protein secretion in plant cells --- p.9 / Chapter 2.1 --- Abstract --- p.10 / Chapter 2.2 --- Introduction --- p.11 / Chapter 2.3 --- Materials and Methods --- p.12 / Chapter 2.4 --- Results --- p.20 / Chapter 2.4.1 --- Expression pattern of different AtExo70 paralogs with fluorescent tag in Arabidopsis protoplasts --- p.20 / Chapter 2.4.2 --- The organelles labeled by AtExo70E2 are distinct from well known endomembrane markers --- p.23 / Chapter 2.4.3 --- The AtExo70E2 positive organelles do not lie on the secretory or endocytic pathways --- p.27 / Chapter 2.4.4 --- Arabidopsis Exo70E2-specific antibodies confirm identity of AtExo70E2-positive organelles --- p.31 / Chapter 2.4.5 --- AtExo70E2 positive organelles are true and novel double membrane organelles --- p.33 / Chapter 2.4.6 --- EXPO are not autophagosomes but sequester cytosolic proteins to release them into the apoplast --- p.41 / Chapter 2.5 --- Discussion --- p.53 / Chapter 2.5.1 --- EXPO: novel organelles labeled by exocyst --- p.53 / Chapter 2.5.2 --- EXPO and autophagosome: same or not? --- p.55 / Chapter 2.5.3 --- EXPO: the evidence of unconventional secretion in plant cells --- p.56 / Chapter 2.6 --- Perspectives --- p.56 / Chapter CHATER 3 --- p.58 / AtExo70E2 is essential for exocyst subunit recruitment and for EXPO formation in both plants and animals --- p.58 / Chapter 3.1 --- Abstract --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Materials and Methods --- p.62 / Chapter 3.4 --- Results --- p.70 / Chapter 3.4.1 --- AtExo70E2 is required for the membrane recruitment of a number of exocyst subunits --- p.70 / Chapter 3.4.2 --- AtExo70E2 is required for the recruitment of some other, but not all, AtExo70 subunits --- p.74 / Chapter 3.4.3 --- AtExo70A1 is unable to recruit other exocyst subunits --- p.74 / Chapter 3.4.4 --- FRET and BiFC confirm interactions between AtExo70E2 and other exocyst subunits --- p.80 / Chapter 3.4.5 --- Arabidopsis Exo70E2 can also induce EXPO formation in animal cells --- p.84 / Chapter 3.4.6 --- Neither human nor yeast Exo70 can induce EXPO in plant protoplasts --- p.84 / Chapter 3.4.7 --- EXPO induced by AtExo70-GFP expression in HEK cells do not colocalize with standard organelle markers --- p.87 / Chapter 3.4.8 --- Electron microscopy confirms the presence of EXPO-like, double membrane structures in HEK cells after expression of AtExo70E2-GFP --- p.87 / Chapter 3.5 --- Discussion --- p.91 / Chapter 3.5.1 --- Plant exocyst and the discovery of EXPO --- p.91 / Chapter 3.5.2 --- AtExo70E2 is a key player in exocyst recruitment onto EXPO --- p.93 / Chapter 3.5.3 --- AtExo70E2 expression as a signal for EXPO formation --- p.96 / Chapter 3.6 --- Perspectives --- p.100 / References: --- p.101 / Chapter List of publications derived from this Ph.D. thesis research --- p.119

by Cheung Siu Tim. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (p. 155-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

Single-particle reconstruction technique is one of the major approaches to studying ribosome structure and membrane proteins. In this thesis, I report the use of time-resolved cryo-EM technique to study the structure of short-lived ribosome complexes and conventional cryo-EM technique to study the structure of ribosome complexes and membrane proteins. The thesis consists three parts. The first part is the development of time-resolved cryo-EM technique. I document the protocol for how to capture short-lived states of the molecules with time-resolved cryo-EM technique using microfluidic chip. Working closely with Dr. Lin’s lab at Columbia University Engineering Department, I designed and tested a well-controlled and effective microspraying-plunging method to prepare cryo-grids. I demonstrated the performance of this device by a 3-Å reconstruction from about 4000 particles collected on grids sprayed with apoferritin suspension. The second part is the application of time-resolved cryo-EM technique for studying short-lived ribosome complexes in bacteria translation processes on the time-scale of 10-1000 ms. I document three applications on bacterial translation processes. The initiation project is collaborated with Dr. Gonzalez’s lab at Chemistry Department, Columbia University. The termination and recycling projects are collaborated with Dr. Ehrenberg’s lab at Department of Cell and Molecular Biology, Uppsala University. I captured and solved short-lived ribosome intermediates complexes in these processes. The results demonstrate the power of time-resolved cryo-EM to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology. The last part is the application of conventional cryo-EM technique to study ribosome complexes and membrane proteins. This part includes five collaboration projects. Human GABA(B) receptor project is the collaboration with Dr. Fan at Department of Pharmacology, Columbia University. Cyclic nucleotide-gated (CNG) channels project is the collaboration with Dr. Yang at Department of Biological Sciences, Columbia University. The cryo-EM study of Ybit-70S ribosome complex and Cystic fibrosis transmembrane conductance regulator (CFTR) project are the collaboration with Dr. Hunt at Department of Biological Sciences, Columbia University. The cryo-EM study of native lipid bilayer in membrane protein transporter is the collaboration with Dr. Hendrickson at Department of Biochemistry and Molecular Biophysics, Columbia University and Dr. Guo at Department of Medicinal Chemistry, Virginia Commonwealth University.

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Heterogeneity in immunodominant outer membrane proteins has been proposed as a significant factor in the failure of an NTHi infection to induce immune protection against subsequent infections. This study has examined the vaccine potential of three outer membrane proteins in an attempt to identify conserved regions that could be targeted by an immune response after vaccination. The three proteins investigated were: TbpB, P5 and P48 (HI0164). The optimal route of immunisation in clearing a bolus inoculum of NTHi to the lung in the rat has been shown to be a combination of gut sensitisation with a respiratory boost and this regime was used in the present study. A panel of NTHi isolates was assessed to determine the frequency with which strains were able to bind transferrin and thus be targeted by a TbpBspecific immune response. A high proportion of strains was able to bind transferrin with similar frequencies in isolates associated with infection and those from normal throat swabs. A protocol was developed to purify nonlipidated recombinant TbpB from NTHi using a glutathione-Stransferase (GST)-rTbpB fusion protein and Glutathione-Sepharose affinity chromatography. Mucosal-directed immunisation with rTbpB significantly enhanced clearance of an NTHi challenge to the lung, however, whilst rTbpB-specific antibodies were cross-reactive on Western immunoblots, the cross-reactivity was variable in both transferrin binding inhibition assays and bactericidal activity. This suggested that the rTbpB-specific humoral response would be variable in the recognition of heterologous NTHi isolates. The secondary structure of P5 has been controversial with several reports suggesting that P5 was a fimbrin protein composed of coiled coils. In this present study the interstrain variation in P5 amongst isolates from diverse anatomical sites, as well as computer prediction methods and spectrophotometric analysis, generated a model of P5 based on the homologous E. coli protein, OmpA. This model suggested a B-barrel conformation with no evidence of coiled coils. Synthetic peptides corresponding to conserved regions of P5 that were thought to be surface exposed, as well as a region (H3) with some homology to a protective epitope in the P. aeruginosa protein, OprF, were then combined with a "promiscuous" T cell epitope from the measles virus F protein (MVF) and used for immunisation studies. Whilst variable protection was seen with the peptides, the MVF/H3 peptide was the most efficacious of the antigens assessed in this study in enhancing clearance of NTHi. This occurred in the absence of detectable peptide- or PS-specific antibody leading to the suggestion that cell mediated responses may have played an important role in enhancing clearance in this model. The highly conserved nature of the region in P5 represented by the H3 peptide suggests that further study should be focused on this peptide as a potential NTHi vaccine candidate. The last antigen, P48, is homologous to a A. pleuropneumoniae antigen, AopA, which has been proposed to have potential as a vaccine component against pleuropneumonia in pigs. Sequence analysis of the gene encoding P48 from several isolates showed that this protein was well conserved. Recombinant P48 was purified from a GST-rP48 fusion protein and used for immunisation, which also conferred significant protection. However, immunisation with rP48 was not as efficacious as immunisation with the MVF/H3 peptide. Whilst immunisation with rP48 induced high antibody titres, no bactericidal activity could be detected indicating that bactericidal antibody had not contributed to the observed clearance. In addition, the rP48- specific serum IgG was predominantly of the IgG2a isotype suggesting that Thl cell mediated responses had been induced by immunisation with rP48.

Expression of membrane-type matrix metalloproteinases (MT) and tissue inhibitors of matrix metalloproteinases (TIMP) was evaluated in sheep embryos and uterus during the pre- and peri-implantation periods. Embryos and uterine samples were surgically collected from ewes on days 9, 11, 13, and 15 of pregnancy (n=3 ewes/day) and of the estrous cycle (n=2 ewes/day). Total RNA was extracted and RT-PCR were performed using primers specifically designed from published human, mouse, and bovine complete cDNA sequences for MT-1, -2, -3, and -5, and TIMP-1, -2 and -3. Multiplex PCR were performed on uterine samples for each gene at optimal cycles and temperatures with 18S rRNA as the internal standard. For embryos, PCR were conducted for 40 cycles at optimal temperatures. MT-1, -2, -3, and -5 were observed in pregnant and nonpregnant uterus during all days of collection. No difference (P>0.10) was observed in MT-1 or -2 expression due to day of collection. However, pregnant uterus expressed more (P=0.096) MT-1 than nonpregnant uterus, whereas expression of MT-2 was greater (P<0.05) in nonpregnant compared to pregnant uterus. No differences (P>0.10) in MT-3 expression were observed due to pregnancy status, however Day 9 and 11 expressed more MT-3 than Day 15. Uterine MT-5 expression was not different (P>0.10) between pregnant and nonpregnant females, however Day 15 uterus expressed less (P<0.05) MT-5 then Day 11 and 13 uteri. TIMP-1 expression was greater (P<0.05) in pregnant compared to nonpregnant uterus, but did not differ (P>0.10) by day of collection. TIMP-2 did not differ (P>0.10) by pregnancy status or day of collection but the interaction was significant (P<0.05). TIMP-2 expression was greatest in Day 9 pregnant uterus and least in Day 9 nonpregnant uterus. No difference (P>0.10) was observed in expression of TIMP-3 due to day of collection or pregnancy status. Embryos expressed MT-3 and -5 during Days 9-15 of development, however, MT-1 and -2 were not detected. The presence of MT and TIMP in the endometrium suggests these proteins may play important roles in regulating extracellular matrix degradation and activating other matrix metalloproteinases for endometrial remodeling and preparation for implantation. Embryonic MT may participate in the processes of embryonic expansion, elongation and attachment. / Graduation date: 2002

In recent years, the general public has recognized the dangers of a high fat diet and are demanding meat with lower fat content. This demand has stimulated research in the growth and regulation of adipocytes. However, despite much effort, no adipocyte-specific plasma membrane markers from any species are available as an aid to accurately distinguish adipocytes from non-adipocytes. One potential candidate for such a marker in porcine adipocytes has been identified by Killefer and Hu (1990b). Characterization of the cDNA for this protein, designated porcine adipocyte membrane protein (PAMP), is presented here. Sequence for the 910 by clone is 80% similar to an internal region of a rat prostaglandin F[subscript 2��] receptor regulator protein (FPRP) described by Orlickey (1996). Western blot analysis suggests that the pig protein is a homotetramer held together with disulfide bonds which form very close to the transmembrane region making the tetramer extremely difficult to reduce to monomeric units. Oligonucleotide primers were designed to amplify a genomic fragment by the polymerase chain reaction (PCR) and for a reverse transcriptase PCR (RT-PCR) assay to study the expression of the mRNA. A 2114 bp genomic clone revealed one intron in the coding region. A serum-free primary cell culture system was used to study the expression of the mRNA. Although message was detected every day over a ten day period, it appeared to peak between 6 to 8 days after plating. The PAMP protein is clearly of the same family as the rat FPRP but its size and conformation are quite different so it is not clear what function it performs in porcine adipocytes. Further experiments should focus on attaining full length cDNA's, confirming the molecular conformation of the protein, and assessing its function in a serum-free primary cell culture system. / Graduation date: 1997