The Escherichia coli vitamin B12 transporter BtuCD-F is a type II importer belonging to the ABC transporter superfamily. Available data suggest both exporters and type I importers in the ABC superfamily employ similar transport mechanism in which the transmembrane (TMDs) are open to cytoplasm in the resting state, and ATP binding induces a major conformational change resulting in opening of the TMDs instead to the periplasm. However, the crystal structures of BtuCD from E. coli and recent EPR spectroscopy studies indicate that this type II importer employs a substantially different mechanism in which the TMDs are open to the periplasm in the resting state and to the cytoplasm after ATP binding. We have developed robust methods to study the conformation and transport mechanism of BtuCD-F reconstituted into lipid bilayers using single molecule fluorescence resonance energy transfer (smFRET) measurements. Fluorescent probes have been introduced at a variety of diagnostic sites, enabling smFRET to be used to measure distance changes in different conformational states as well as to observe the transitions between these states in real time. These data suggest that thermal fluctuations enable the transporter to explore different functional conformational states in the absence of ATP or other ligands. They also suggest that the ATP-bound state is indeed open to the cytoplasm and ATP binding/hydrolysis increases the rate of transition between open and closed states. Efforts are currently underway to observe the transport of vitamin B12 through a single BtuCD-F oligomer in real-time.

by Tse Yu Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 133-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The Plant secretory pathway --- p.2 / An overview on the secretory pathway --- p.2 / Vesicular pathways and transport vesicles --- p.4 / Chapter 2. --- Vacuolar sorting receptors --- p.6 / BP-80 and its homologues --- p.6 / RMR proteins --- p.7 / Chapter 3. --- Prevacuolar compartments --- p.8 / PVCs in mammalian and yeast cells --- p.8 / PVCs for seed protein storage vacuoles --- p.9 / PVCs for lytic vacuoles --- p.11 / Chapter Chapter 2 --- Development of Transgenic Tobacco BY-2 Cell Lines Expressing Fluorescent Markers for Golgi and Prevacuolar Compartments --- p.15 / Chapter 1. --- Introduction --- p.16 / Chapter 1.1 --- Fluorescent proteins are useful tools in studying protein trafficking and subcellular localization in living cells --- p.16 / Chapter 1.2 --- Tobacco BY-2 cells --- p.18 / Chapter 1.3 --- Plant prevacuolar compartments --- p.19 / Chapter 2. --- Materials and Methods --- p.21 / Chapter 2.1 --- Construction of RFP-BP-80 and RFP-α-TIP reporters --- p.21 / Chapter 2.2 --- Construction of YFP-BP-80 and YFP-α-TIP reporters --- p.27 / Chapter 2.3 --- Construction of YFP markers for Golgi organelles --- p.32 / Chapter 2.4 --- Agrobacterium electroporation --- p.33 / Chapter 2.5 --- Transformation of tobacco BY-2 cells --- p.34 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing RFP markers --- p.35 / Chapter 2.8 --- Production of anti-BP-80 CT antibody --- p.43 / Chapter 2.9 --- Chemicals --- p.45 / Chapter 2.10 --- Primers --- p.45 / Chapter 2.11 --- Bacterial strain --- p.46 / Chapter 3. --- Results --- p.47 / Chapter 3.1 --- Generation and characterization of transgenic BY-2 cell lines expressing RFP reporters --- p.47 / Chapter 3.2 --- Generation and preliminary characterization of transgenic BY-2 cell lines expressing YFP reporters --- p.55 / Chapter 3.3 --- Confocal detection ofYFP reporters in transgenic cell lines --- p.64 / Chapter 3.4 --- Characterization of anti-BP-80 CT antibody --- p.66 / Chapter 4. --- Discussion --- p.68 / Chapter Chapter 3 --- Dynamic of Plant Prevacuolar Compartments in Transgenic Tobacco BY-2 Cells --- p.72 / Chapter 1. --- Introduction --- p.73 / Chapter 1.1 --- The plant secretory pathway --- p.73 / Chapter 1.2 --- Organelle markers in plant secretory pathway --- p.74 / Chapter 1.3 --- Markers for Lytic PVCs --- p.75 / Chapter 2. --- Materials and Methods --- p.77 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.77 / Chapter 2.2 --- FM4-64 uptake study --- p.79 / Chapter 2.3 --- Brefeldin A treatment --- p.79 / Chapter 2.4 --- Wortmannin treatment --- p.80 / Chapter 2.5 --- Movement study of YFP-marked PVC --- p.82 / Chapter 3. --- Results --- p.83 / Chapter 3.1 --- Different internal organelles were labeled by two different YFP reporters --- p.83 / Chapter 3.2 --- The YFP-BP-80 reporter localized with endogenous VSR proteins --- p.86 / Chapter 3.3 --- Brefeldin A enlarged PVC organelles --- p.89 / Chapter 3.4 --- Identity of PVC-derived BFA-induced compartments --- p.99 / Chapter 3.5 --- Wortmannin induced PVCs to form small vacuoles --- p.102 / Chapter 3.6 --- PVCs are mobile organelles in living cells --- p.112 / Chapter 4. --- Discussion --- p.114 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.123 / Chapter 1. --- Summary --- p.124 / The hypothesis --- p.124 / Development of three transgenic cell lines --- p.125 / Distinct organelles were marked by two different YFP reporters --- p.126 / The YFP-BP-80 reporter defined the lytic PVCs --- p.126 / Response of YFP-marked PVCs to Brefeldin A treatment --- p.127 / Response of YFP-marked PVCs to Wortmannin treatment --- p.127 / PVCs are mobile organelles in living cells --- p.129 / Chapter 2. --- Future perspectives --- p.130 / References --- p.133

Further in vivo and in vitro studies using the truncated OsRMR1 proteins from the culture media of transgenic BY-2 cells demonstrated that OsRMR1 functioned as a receptor in transporting vicilin-like storage proteins via specific interaction with their vacuolar sorting determinants. Taken together, the OsRMR1 is a sorting receptor for the PSV pathway that defines a novel organelle as PVC for PSV in rice. / Receptor-mediated protein sorting is one of the mechanisms for transporting soluble proteins to the protein storage vacuoles (PSVs) in plant cells. Members of vacuolar sorting receptor (VSR) family proteins and receptor homology region-transmembrane domain-RING-H2 (RMR) family proteins have been shown to function in mediating the transport of storage proteins to PSVs in plants. However, no prevacuolar compartment (PVC) for the PSV pathway has been identified. In this study, I used a rice RMR protein (OsRMR1) as a probe to study the PSV pathway in rice. Using confocal immunofluorescent and immunogold electron microscopy (EM) with specific OsRMR1 antibodies, I have identified a novel organelle as a PVC for the PSV pathway, because OsRMR1 antibodies labeled the Golgi apparatus, trans-Golgi network (TGN) and the novel organelle in both rice cultured cells and developing rice seeds, as well as the protein body Type II (PBII) in developing rice seeds. This novel organelle is morphologically distinct from the lytic PVC or multivesicular body (MVB). / Shen, Yun. / "May 2008." / Adviser: Liwen Jiang. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1428. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 124-139). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Thesis (Ph.D.)--McMaster University, 2006. / Supervisor: Michael J. O'Donnell. Includes bibliographical references (leaves 279-310).

Thesis (M. Phil.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 130-155). Also available on the Internet.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 162-172).

Ph.D.

Ph.D.

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 102-113).