Rab27a and Munc18 as Molecular Determinants of Snare-Mediated Vesicle Docking, Priming, And Fusion In Glucose Regulatory Tissues

Abstract

Vesicle exocytosis involves number of essential protein families that cooperatively mediate physical attachment between the vesicle and the plasma membrane, including Rab GTPases, Sec1/Munc18 proteins, and SNARE proteins. We have examined two specific molecules in the regulated exocytotic pathway, Munc18c and Rab27a, at sites of SNARE-mediated vesicle fusion in tissues integral to the controlled maintenance of blood glucose, adipocytes and pancreatic β-cells. We have hypothesized that Munc18c regulates the progression of GLUT4 storage vesicle (GSV) trafficking and fusion via the insulin-induced alteration of its binding to the t-SNARE syntaxin4. Our results indicate that Munc18c interaction with a conformationally folded syntaxin4 acts as a negative regulator GSV docking. By comparison, syntaxin4 mutants that promote a SNARE complex-forming conformation demonstrate a shift in the state of Munc18c interaction that is sufficient for GSV docking, and is likely to mimic insulin activation of GSV trafficking, resulting in increased GLUT4 at the plasma membrane. Upstream of SM proteins, Rab GTPases and their effector proteins assist with the transport to and docking of vesicles at the plasma membrane, where they are believed to interact directly with the Munc18/syntaxin complex. We focused on identifying sites of Rab27a action in the insulin secretory pathways using membrane capacitance measurements of pancreatic β-cells isolated from Rab27a-null (ashen) mice. We demonstrate that Rab27a exerts dual roles in insulin granule exocytosis, facilitating the glucose-induced refilling of releasable granule pools while also limiting release from these pools. In summary, the investigations of this dissertation support a role for Munc18c and Rab27a in controlling both the timing and magnitude of vesicle docking and priming (i.e., SNARE complex formation) at the plasma membrane, thereby acting as crucial regulators in the regulated exocytotic pathways in glucose regulatory tissues.