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Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1984 M44 / Master of Science

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Cellular membranes were once considered static and passive structures, but are now appreciated as a fluidic and dynamic assembly of macromolecules that play an active role in cellular function. Membrane composition has been proposed to play a critical role in modulating protein function by affecting everything from post-translational modifications to conformation, but the physiologic relevance of the relationship between protein and membrane has been difficult to establish. For example, membrane-associated proteins such as Flotillin-1 (Flot1) have been implicated to scaffold proteins into cholesterol-rich membranes, as well as play a role in a wide array of functions such as endocytosis and axon pathfinding; however, genetic elimination of Flot1 expression had little to no reported consequence, leaving to question the physiologic importance of scaffolding proteins to membrane microdomains. Using genetic and biochemical approaches, I sought to understand how the immediate lipid environment can influence the function of a transmembrane protein, and how this might impact brain function. Specifically, I examined how a cholesterol-rich environment can affect the function of the cell surface neurotransmitter transporter for dopamine, the dopamine transporter (DAT), and how this interaction may influence the ability of an organism to respond to the psychostimulant amphetamine (AMPH). Although neurotransmitter transporters (NTTs) such as DAT and the serotonin transporter (SERT), have been predicted to reside in membrane rafts, it has been difficult to establish the role of microdomain localization in transporter function. DAT localizes to the plasma membrane, where it modulates the strength and duration of neurotransmission by clearing dopamine (DA) from the perisynaptic space. Defects in DAT have been implicated in a range of psychiatric and neurological disorders, from schizophrenia to Parkinson’s disease. Additionally, as a target of psychostimulants, such as AMPH and cocaine (COC), the role of DAT in addiction is of societal interest. Given that Flot1 was required for scaffolding heterologously expressed DAT to cholesterol-rich membranes in cell-based systems, and was selectively necessary for the non-exocytic release of DA through DAT in response to AMPH, I sought to test the hypothesis that the Flot1-mediated membrane localization of DAT was significant for the ability of mice to respond to AMPH. To this end, I created a series of genetic models to determine how the presence of Flot1 impacts DAT function in the brain. I found that Flot1 is not only important for scaffolding DAT into cholesterol-rich membranes, but that the ability of DAT to partition into these membranes was necessary for DAergic neurons, DAT, and ultimately mice, to respond to AMPH. Given that the other parameters of DA neuron function, as well as the ability of the animals to respond to COC was unaffected by DAT partitioning, my findings demonstrate that AMPH and COC exert different mechanisms of action in vivo. Moreover, I found that the cholesterol-rich membrane environment promoted a conformation of DAT that was favorable for reverse transport of DA through DAT, namely increasing the ability of its N-terminus to bind to the phospholipid, PIP2. This dissertation provides the first glimpse into not only how membrane localization can affect protein conformation and function but also the physiologic relevance of these Flot1-dependent membrane microdomains in brain.

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The insulin-dependent uptake of glucose by adipose and muscle tissues is accomplished through the regulated vesicle trafficking of the GLUT4 glucose transporter to the plasma membrane. The distal trafficking events comprising the tethering, docking and fusion of GLUT4 vesicles with the plasma membrane are poorly defined, but represent vital steps in this pathway. This dissertation encompasses a series of complementary studies that have provided new insights into how these events are regulated in the adipocyte. The Sec1p homologue Munc18c, is believed to play a central role in the docking of GLUT4 vesicles by controlling SNARE complex assembly. Munc18c was shown to bind the t-SNARE Syntaxin4 and form a stable complex in vivo. Protein binding studies demonstrated that Munc18c interacts with Syntaxin4 via an evolutionarily conserved N-terminal binding mode and the formation of the Munc18c/Syntaxin4 hetero-dimer was shown to promote SNARE complex assembly. In contrast to previous reports, I propose that Munc18c is positive regulator of SNARE assembly and vesicle docking. The exocyst complex is thought to promote the tethering of exocytic GLUT4 vesicles with the plasma membrane. Yeast two-hybrid screens revealed interactions between the exocyst subunits Sec6 and Exo70 and the SNARE-associated proteins Munc18c and Snapin, respectively. Snapin was subsequently shown to have a novel role in GLUT4 trafficking. These interactions suggest Munc18c and Snapin provide a course for cross-talk between the exocyst complex and the SNAREs to stimulate GLUT4 vesicle tethering and docking. In addition to its interactions with Munc18c and Snapin, the exocyst was also found to interact with the GTP-bound form of RalA, a small GTPase regulated by insulin. RalA was almost exclusively localised to the plasma membrane of the adipocyte and a novel role for the RalA/exocyst interaction in GLUT4 trafficking was demonstrated. Specifically, overexpression of a GTP-deficient RalA mutant significantly inhibited insulin-stimulated GLUT4 appearance on the plasma membrane. In addition to its role in GLUT4 trafficking, a novel role for RalA was demonstrated in insulin release from pancreatic -cells, indicating that RalA may represent a universal component of regulated exocytosis. It is becoming increasingly apparent that vesicle trafficking events from yeast to mammals rely on similar protein complexes which communicate through multiple protein interactions, ensuring vesicle transport is highly coupled. Similarly, the Munc18c studies demonstrate that while mammalian cells have evolved to fulfil specialised functions throughout the body, some proteins appear to have retained the biochemical properties of their ancestors, emphasing the importance of this family of proteins throughout eukaryotic vesicle transport. In contrast, proteins such as RalA have evolved only in higher eukaryotes and appear to play a universal role in vesicle transport despite vast differences in the specialised functioning of mammalian cells.

Thesis (Ph.D.)--York University, 2008. Graduate Programme in Mathematics and Statistics. / Typescript. Includes bibliographical references (leaves 166-175). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR46017