Vesicle fusion is a biological process that is central to cellular function. In eukaryotic cells, the partnership between soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) and Sec1p/Munc18 (SM) proteins drives most intracellular fusion steps. According to the ‘‘zipper’’ model, SNARE proteins assemble into stable membrane-bridging complexes that bring membranes in juxtaposition, providing the necessary energy for overcoming the repulsive forces between two membranes.
Previous studies (mostly using Munc18a from neurons) have clearly confirmed that Munc18 proteins are important components of the vesicle fusion process. However, conflicting models have been proposed for how these different SM proteins regulate fusion, paradoxically suggesting that they can either promote or inhibit SNARE assembly and membrane fusion in vivo and in vitro. A detailed review of the literature suggested that the confounding results showing either a positive or a negative role for SM proteins could be at least partly the result of the use of different Sx constructs with deficiencies in key N and C-terminal sequences (either being absent or inhibited by fusion tags), combined with the wide variety of protein-protein interaction techniques that have been used to dissect the mechanism of Munc18 regulation of SNARE complex formation. These include immuno-precipitations (IPs), in vitro pull-down assays, fluorescence assays, energetics of protein-protein interaction using ITC (isothermal titration calorimetry); kinetics using SPR (Surface Plasmon Resonance) and liposome reconstitutions. The inconsistencies of results obtained from different methods have impacted significantly on developing a cohesive understanding of the role of Munc18/SNARE complexes in vesicle transport.
The research presented in this thesis focuses on characterizing the protein-protein interactions involved in insulin regulated GLUT4 vesicle exocytosis, specifically the Sec1/Munc18 (SM) protein Munc18c and SNARE proteins Sx4, SNAP23, and VAMP2. Importantly, for the first time the role played by Sx4 C–terminal membrane anchoring is addressed, and reveals a key role for this structure in allowing Munc18c to regulate SNARE complex assembly.
Research on Munc18c has been hampered by difficulties in producing recombinant Munc18c in sufficient quantities for protein-protein interaction assays. I therefore successfully established and optimized a protocol to produce correctly folded and fully functional Munc18c protein in a bacterial expression system. This allowed characterization of the Munc18c/Sx4 interaction both biochemically and structurally.
To date, research on Sx4, an integral membrane protein has only focused on its soluble C-terminally truncated form. Due to the deletion of the transmembrane domain of Sx4, I hypothesized that the soluble form may have a relatively more flexible C-terminus compared with the native form and this may affect its interactions. C-terminal anchoring of Sx4 may be critical to the ability of Munc18 proteins to regulate the conformational changes necessary for the assembly of the SNARE complex and vesicle fusion. To enable investigation of the role of Sx4 C-terminus anchoring, an artificial construct was engineered combining Sx4 (residues 1-275) with a T4-Lysozyme fusion at its C-terminus. The data obtained using this construct showed that T4L does not affect Sx4 protein binding to either Munc18c or SNARE partner proteins. My results however, showed that the Sx4 Cterminal anchoring may be critical in the ability of Sx4 to interact with its cognate SNARE proteins in the presence of Munc18c. A range of biochemical approaches was used to investigate the role of the Sx4 C-terminal region. Sx4 lacking its C-terminal transmembrane domain was found to be deficient in SNARE complex formation in the presence of Munc18c. Kinetic studies using fluorescence anisotropy confirmed that the role of Munc18c was affected if the Sx4 C-terminus was not stabilised by T4L. The results presented support a positive role for Munc18c in SNARE complex formation for GLUT4 vesicle fusion.
I then established a protocol to produce recombinant full-length Sx4 (residues 1-298) that included the transmembrane domain. These two recombinant proteins (Sx4T4L and Sx4-TMD) were then used for functional and structural studies. I was able to generate small crystals for both the Munc18c/Sx4-T4L and Munc18c/Sx41-298TMD complexes. Although structures have not yet been forthcoming the work provides a solid base to begin crystal optimization towards future diffraction quality crystals. For membrane protein crystallization, training was provided at the Scripps Research Institute, using innovative lipidic cubic phase crystallization methods.
Overall I was able to show that tethering or anchoring or stabilising the Sx4 transmembrane domain is important for Munc18c to be able to regulate SNARE complex assembly. Collectively my research provides new insight into the mechanism of Munc18c for SNARE complex formation and provides optimised methods to produce and purify recombinant Munc18c and the full-length integral membrane protein Sx4 for structural and functional studies.