The focus of this thesis is to characterise the interactions between GLUT4-related SNARE proteins syntaxin4, SNAP23 and VAMP2 and a regulatory protein, Munc18c. GLUT4 is the primary insulin-regulated glucose transporter and is presentin fat and muscle cells. GLUT4 is held in intracellular pools of vesicles until it is transported to the cell surface upon insulin stimulation. Insulin initiates a cellular signalling cascade via the insulin receptor on the cell membrane, which in turn stimulates GLUT4 vesicles to move to the cell surface where they fuse to the plasmamembrane via SNARE proteins. SNAREs are membrane-anchored proteins present on both vesicle and target membranes that form a tight complex which brings themembranes together for fusion. Fusion of vesicles to the target membrane releases the vesicular cargo.SNARE-mediated membrane fusion is a conserved mechanism that controls many other vesicle fusion processes such as neurotransmitter release and yeast vesicular trafficking. However, the regulation of the SNARE mechanism is not fully understood. SNAREs can interact with many other proteins that could act as regulatory factors,and studies have focused primarily on a group of effector proteins called Sec1p/Munc18 (SM) proteins. SM proteins were discovered and characterised because they bind to one type of SNARE protein, syntaxin. The SM protein that interacts with the GLUT4-related SNARE, syntaxin4, is Munc18c.The aim of this thesis was to investigate Munc18c interactions with SNARE proteins, principally syntaxin4, using biochemical techniques with purified recombinant proteins. This work was carried out in several stages including: 1) development of methods to produce and purify GLUT4-related SNARE proteins, SNARE complexes and Munc18c, 2) development of an assay to quantify Munc18c interactions with binding partners using surface plasmon resonance, 3) investigation into interactions between Munc18c and SNARE ternary complex, 4) characterising Munc18c interactions with syntaxin4, and 5) developing a method to produce selenomethionine-containing Munc18c in a baculovirus system to be used in structural studies. The methods and outcomes of these experiments are described inthis thesis. There were two major outcomes from this work. Firstly, Munc18c interacts with SNARE ternary complex, and secondly, Munc18c requires only the N-terminal 29residues of syntaxin4 for an interaction to occur. These results were determined using pulldown assays with purified proteins, as well as other chromatographic methods to show that protein complexes were formed. The steps taken to develop these binding assays are also discussed. Initial crystallisation conditions forMunc18c-HIS and a peptide consisting of syntaxin4 residues 1-20 have been identified using crystallisation screens. The interactions determined for Munc18c binding to Sx4 are in direct contrast to those of neuronal SM protein, Munc18a, and its interaction with neuronal SNARE proteins - Munc18a does not bind to its ternary complex and binds to the entire cytoplasmic domain of Sx1a. Rather, the Munc18c:Sx4 interactions are similar to that for the yeast SM protein, Sly1p, which can interact with both its SNARE ternary complex and with its syntaxin via the Nterminal residues. Another interesting outcome of this research was that syntaxin4 binds to metals (cobalt and nickel). This finding represents the first reported for a syntaxin interacting with metals. Preliminary results indicate that un-tagged syntaxin4 can bind to cobalt resin, and to nickel immobilised on a chip. This interesting and novel property of syntaxin4 binding was serendipitously discovered while investigating conditions for the Munc18c assay. Overall, I have shown that Munc18c, the SM protein involved in GLUT4 trafficking, interacts with SNARE proteins in a different manner to its mammalian counterpart inneurons, Munc18a, and is more like Sly1p, a yeast ER-Golgi SM protein. Munc18c interacts with SNARE complexes and only the N-terminal residues of syntaxin4.These interactions demonstrate that the regulatory mechanism for SNARE-mediated fusion is conserved between yeast and mammals. This finding has several implications for the role of Munc18c in the exocytosis of GLUT4-containing vesicles. Munc18c could act at several stages in the fusion process via syntaxin4 binding.These interactions could involve binding to other proteins (such as synip or tomosyn), conformational switching of syntaxin4 or interaction with metal ions to induce conformational changes in the proteins. Finally, these studies of GLUT4 exocytosis contribute to our understanding of glucose transport disorders such as Type 2 diabetes and could one day pave the way for the design of therapeutic agents.
| Identifer | oai:union.ndltd.org:ADTP/253652 |
| Creators | Latham, Catherine Frances Mary |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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