Thesis (Ph. D.)--University of Oklahoma. / Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 105-122).

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. [93]-106.

Thesis (Ph. D. (Biochemistry, Microbiology & Biotechnology))--Rhodes University, 2004.

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. [93]-106.

Quiescence, a transitory period of non-growth, is a ubiquitous aspect that is present in all organisms. In addition to being present in all forms of life, quiescence is a feature that has been observed in cells that are important for human health, including stem cells in mammals and antibiotic tolerant cells in bacteria. In bacteria, quiescence per se has recently been suggested to underlie the transient tolerance to a wide range of antibiotics. Furthermore, most microbial life exists in a quiescent state. Despite their prevalence and importance, relatively little is known about the physiology of quiescent bacteria. One aspect of bacterial quiescence that has been repeatedly observed is their lowered metabolic activity compared to actively growing bacteria. How do cells that grow and divide enter into a temporary state of non-growth? In particular, how are the energy-intensive processes that are required for growing cells regulated during a non-growing state? The main subject of this thesis is to investigate how protein synthesis, the most energy-intensive process in growing bacterial cells, is regulated during entry into a quiescent phenotype (stationary phase). I first investigate how protein synthesis is regulated using a single cell method that fluorescently tags nascent polypeptide chains. In chapter 3, I show that during entry into stationary phase, protein synthesis is downregulated heterogeneously with one group of cells having comparatively low protein synthesis, resulting in a population that is approximately bimodal. I further show that this bimodality is dependent on a signaling system (PrkC and its partner phosphatase PrpC) that senses cell wall metabolism. I connect signaling from this system to the expression of an enzyme (SasA) that produces a group of nucleotides that are major regulators of growth in bacteria ((pp)pGpp). Lastly, I show that the bimodality is dependent on the three enzymes that synthesize (pp)pGpp. In chapter 4, I explore in detail how the bimodality in protein synthesis is generated. This heterogeneity requires the production of (pp)pGpp by three synthases: SasA, SasB, RelA. I first show that these enzymes differentially affect this bimodality: RelA and SasB are necessary to generate the sub-population exhibiting low protein synthesis, whereas SasA is necessary to generate cells exhibiting comparatively higher protein synthesis. The RelA product (pppGpp) allosterically activates SasB, and I find that the SasA product (pGpp) competitively inhibits this activation. I provide in vivo evidence that this antagonistic interaction mediates the observed heterogeneity in protein synthesis. This chapter, therefore, identifies the mechanism underlying the generation of phenotypic heterogeneity in the central physiological process of protein synthesis. In chapter 5, I next turn to understand the biochemical mechanism by which cells with comparatively low levels of protein synthesis down-regulate this process. I first show that ppGpp is sufficient to inhibit protein synthesis in vivo. I then show that ppGpp inhibits protein synthesis by inhibiting translation initiation directly by binding to the essential GTPase, Initiation Factor 2 (IF2). In collaboration with Ruben Gonzalez’s lab, we also show that ppGpp prevents the allosteric activation of IF2. Finally, I demonstrate that the observed attenuation of protein synthesis during the entry into quiescence is a consequence of the direct interaction of (pp)pGpp and IF2.

No description available.

Vesicular trafficking, the movement of vesicles between organelles and the plasma membrane for secretion, consists of multiple highly regulated processes. Many protein families function as specificity and regulatory determinants to ensure correct vesicle targeting and timing of trafficking events. The SNARE proteins dock and fuse vesicles to their target membranes. Sec1/Munc18 (SM) proteins regulate membrane fusion through interactions with the SNAREs—SM proteins have been shown to act as both inhibitors and stimulators of SNARE assembly and membrane fusion. However, the details of these SM protein functions are not understood. Constructing a model of SM protein function has been challenging due to the various modes of interactions reported between SM proteins and their SNAREs. SM proteins interact with their cognate SNAREs and SNARE complexes through several distinct modes. The most conserved mode is an interaction with the syntaxin N-peptide; other modes of binding, such as the syntaxin closed conformation, are hypothesized to be specific for specialized cell types. In order to elucidate the general function of SM proteins, I investigated the function of the endosomal SM protein Vps45p by analyzing its interactions with its cognate syntaxin Tlg2p and its role in SNARE assembly. I had two main hypotheses: that the Tlg2p N-peptide does not solely mediate the interaction between Vps45p and Tlg2p; and that Vps45p functions to stimulate SNARE complex assembly. I systematically mapped the interaction between Vps45p and Tlg2p using various Tlg2p truncations containing the different domains of Tlg2p and discovered a second binding site on Tlg2p that corresponds to the closed conformation. The neuronal SM-syntaxin pair interacts in a similar manner, indicating that this interaction mode is conserved. To characterize the closed conformation binding mode further, and determine its relationship to the N-peptide binding mode, I developed a quantitative fluorescent electrophoretic mobility shift assay. Results indicate that these two sites do not bind simultaneously and that the N-peptide binding modulates the closed conformation affinity. Furthermore, I monitored the effect of Vps45p on SNARE complex assembly using size exclusion chromatography. Under the conditions tested, Vps45p did not appear to stimulate SNARE complex assembly. The work presented here addresses several puzzling issues in the field and significantly contributes to the construction of a new mechanistic model for SM protein function. In this new model, the SM protein is recruited to the membrane by its interaction with the syntaxin N-peptide. The SM protein then binds the syntaxin closed conformation thus inhibiting SNARE complex assembly. Upon dissociation of the SM protein from the closed conformation, an event perhaps regulated by the SM protein, syntaxin opens and interacts with the other SNAREs to form a SNARE complex. Fusion ensues, stimulated by the SM protein.

Hoi-Yeung Li. / "May 2000." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (p. 151-168). / 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.