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# The role of Munc-18c in GLUT4 vesicle fusion

Boney, Kimberley
(2013)
*The role of Munc-18c in GLUT4 vesicle fusion.*
PhD thesis, University of Glasgow.

Full text available as:

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|  | PDF   [Download (4MB)](https://theses.gla.ac.uk/4300/1/2013boneyphd.pdf) |

Printed Thesis Information: <https://eleanor.lib.gla.ac.uk/record=b2981601>

## Abstract

Under normal physiological conditions, insulin is released from the pancreas in response to an increase in blood-sugar concentration. The insulin signalling pathway culminates with the presentation of the glucose transporter GLUT4 on the plasma membrane of muscle and adipose tissue, leading to the uptake of glucose into these tissues and the subsequent lowering of blood-glucose concentration to basal levels. This system is faulty in patients with Type 2 diabetes.

SNARE proteins have been identified as important regulators of membrane fusion in vivo. Formation of SNARE complexes has been shown to provide the energy required for two opposing membranes to fuse. The SNARE complex consisting of Syntaxin 4, SNAP-23 and VAMP 2 has been implicated in the fusion of GLUT4 Storage Vesicles (GSVs) with the plasma membrane of adipocytes in response to insulin, and thus unravelling the interactions involved in complex formation will allow a greater understanding into the translocation of GLUT4 in response to insulin.

In this thesis I developed an in vitro fusion assay, which confirmed that the SNARE complex consisting of Syntaxin 4, SNAP-23 and VAMP 2 is able to sustain fusion of two vesicle populations. This assay was utilised further to investigate the role of the SM protein Munc-18c on SNARE-mediated membrane fusion. Using this method, Munc-18c was shown to have both positive and negative regulatory roles, depending on experimental conditions. Site-directed mutagenesis of the SM protein was also used in an attempt to dissect the interactions involved in binding of the SM protein to the assembled SNARE complex.

Finally, I developed a second in vitro fusion assay which utilised isolated plasma membrane fractions from 3T3-L1 adipocytes. This assay was used to investigate the effect of insulin on the plasma membrane proteins found in these cells. Analysis of the fractions showed that insulin increased the rate of SNARE-mediated membrane fusion; however the levels of the t-SNAREs were unaltered in response to insulin, indicating that the hormone functions to alter protein structure or function, but not amount.

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| Item Type: | Thesis (PhD) |
| Qualification Level: | Doctoral |
| Keywords: | Membrane, fusion, vesicle, GLUT4, Syntaxin 4, SNAP-23, VAMP 2, Munc-18c, diabetes, insulin, membrane, fusion assay, 3T3-L1, liposome, FRET |
| Subjects: | [Q Science](https://theses.gla.ac.uk/view/subjects/Q.html) > [QH Natural history](https://theses.gla.ac.uk/view/subjects/QH.html) > [QH301 Biology](https://theses.gla.ac.uk/view/subjects/QH301.html) [Q Science](https://theses.gla.ac.uk/view/subjects/Q.html) > [QH Natural history](https://theses.gla.ac.uk/view/subjects/QH.html) > [QH345 Biochemistry](https://theses.gla.ac.uk/view/subjects/QH345.html) |
| Colleges/Schools: | [College of Medical Veterinary and Life Sciences](https://theses.gla.ac.uk/view/divisions/20000000/) > [School of Molecular Biosciences](https://theses.gla.ac.uk/view/divisions/25400000/) |
| Supervisor's Name: | Gould, Prof. Gywn |
| Date of Award: | May 2013 |
| Depositing User: | [Kimberley Boney](https://theses.gla.ac.uk/cgi/users/home?screen=User::View&userid=4383) |
| Unique ID: | glathesis:2013-4300 |
| Copyright: | Copyright of this thesis is held by the author. |
| Date Deposited: | 30 May 2013 10:31 |
| Last Modified: | 30 May 2013 10:31 |
| URI: | <https://theses.gla.ac.uk/id/eprint/4300> |

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