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.
| Identifer | oai:union.ndltd.org:ADTP/235497 |
| Date | January 2007 |
| Creators | Lopez, Jamie Antonio, School of Medicine, UNSW |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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