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Regulation of SNARE-dependent fusion in an in vitro system

Aran-Ponte, Veronica (2009) Regulation of SNARE-dependent fusion in an in vitro system. PhD thesis, University of Glasgow.

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Abstract

Glucose homeostasis depends on the ability of insulin to stimulate glucose uptake into both muscle and adipose tissue by promoting the translocation of glucose transporters (GLUT4) from intracellular sites to the plasma membrane (PM). In individuals with Type 2 diabetes the ability of insulin to stimulate glucose transport is impaired. The incidence of Type 2 diabetes is increasing worldwide, highlighting the need to understand the molecular basis of insulin-stimulated glucose uptake. GLUT4 translocation is a specialised example of vesicular trafficking. Within the context of vesicle trafficking, all eukaryotic cells contain a common set of conserved components responsible for the execution of membrane fusion. Central to this machinery are members of the SNARE (soluble NSF attachment protein receptor) family of proteins. The process of SNARE-mediated membrane fusion needs to be tightly regulated and the SNARE proteins are partially responsible for the specificity in communication between eukaryotic subcellular organelles. Other proteins such as the Sec1p/ Munc18 (SM) proteins were shown to be essential for SNARE-mediated membrane fusion. Several methods were used to test the ability of SNARE proteins to drive membrane fusion, and one of the most important methods described to date is the in vitro fusion assay used in this study. The first topic addressed in this thesis was related to the molecular interactions between the regulatory SM protein Munc18c and the SNARE proteins VAMP2 and syntaxin 4. The use of pull-down assays revealed the novel fact that Munc18c interacts not only with the t-SNARE syntaxin 4 but also with the v-SNARE VAMP2 via its SNARE motif. The SM:v-SNARE interaction was disrupted by the presence of syntaxin 4 revealing that these two SNARE proteins compete for binding to Munc18c. Next, the role of Munc18c in membrane fusion driven by four different versions of syntaxin 4 plus SNAP23 and VAMP2 liposomes, was investigated using the well-characterised in vitro fusion assay. Results suggested that Munc18c negatively regulates SNARE- mediated membrane fusion by inhibiting the formation of SNARE complexes. Interestingly, deletion of the first 36 amino acids of syntaxin 4 was not sufficient to suppress Munc18c negative regulation of fusion indicating that this inhibition might involve other interactions apart from the short N-terminal peptide of syntaxin 4. Finally, the role of phosphorylation in SNARE complex formation was assessed using several techniques such as site-directed mutagenesis, pull-down assays and radiolabelling studies. Data obtained revealed that both syntaxin 4 and Munc18c become phosphorylated in vitro by a recombinant cytoplasmic insulin receptor kinase (CIRK). Munc18c phosphorylated by CIRK was unable to bind syntaxin 4 in vitro. Furthermore, phosphomimetic mutations were also introduced on both proteins and pull-down assays indicated that phosphorylated Munc18c is unable to interact with both syntaxin 4 and VAMP2, whereas phosphomimetic mutations in syntaxin 4 did not affect the interaction with its cognate SNARE proteins and Munc18c. These results were very useful to further understand and confirm the importance of phosphorylation in SNARE complex formation. Collectively, these data suggest that Munc18c acts through different modes of interaction with its cognate SNARE proteins, and support a model in which Munc18c negatively regulates SNARE complex formation. However, this regulation might also be dependent on other factors such as phosphorylation upon insulin signalling.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: in vitro fusion assay, membrane trafficking
Subjects: Q Science > QR Microbiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Gould, Prof. Gwyn
Date of Award: 2009
Depositing User: Dr Veronica Aran-Ponte
Unique ID: glathesis:2009-1030
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Aug 2009
Last Modified: 10 Dec 2012 13:31
URI: http://theses.gla.ac.uk/id/eprint/1030

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