Breaking the exocyst

The vesicle trafficking machinery is essential for the regulation of cell polarity. A large protein complex, the exocyst, has an utmost important role in the polarised trafficking processes. <br/><br/>Most studies of the exocyst machinery and functions have been made by suppression of genes or deletion of a single subunit which may prevent the holocomplex formation. However, loss of even a single subunit causes further disintegration of subcomplexes. To prevent this and limit disassembly to only the holocomplex, a new approach is required. Disruption of the holocomplex formation, while maintaining intact subcomplexes, could enable us to study the role of exocyst subcomplexes.<br/><br/>These challenges and limitations lead to the ultimate goal of this study, breaking the exocyst. By breaking the exocyst, I aim to prevent the holocomplex formation and preserve both subcomplexes. In order to achieve that, I have determined the region that contributes to subcomplex interactions, which I hypothesized could be deleted or mutated to break the interaction between subcomplexes.<br/><br/>With this scheme, I initially applied structural and sequence analysis approaches to the human exocyst subunits and their orthologs. The analysis suggested potential variants that may prevent the formation of the exocyst octameric holocomplex.<br/><br/>I confirmed the interactions between exocyst breaker variants and their partner exocyst subunits by pull-down assays. I used direct and inverted approaches, swapping the bait and prey, and also confirmed expression of both bait and prey by pull-down from the flow-through. The results suggest two exocyst breaker variants that prevent the holocomplex formation while maintaining the stability of the subcomplexes.<br/><br/>The exocyst breaker variants are potentially useful for the exocyst mechanisms and regulation studies in the future. The variants can be used as a control to determine the requirement of the exocyst holocomplex for vesicle tethering processes. It can also reveal in part the mechanism for exocyst specificity and selectivity for vesicle tethering in cells. In conclusion, these exocyst breaker variants are potentially useful for understanding the in vivo mechanisms and regulation of the complex.

Name dc:type.qualificationname

Master of Science

Level dc:type.qualificationlevel

Master's Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2020

Author dc:creator

• Sopipong, Watanyoo

Advisor dc:contributor.advisor

• Murray, David

• Exocyst

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/22b917d6-7ed2-4d60-91d8-f551c7eec5ac

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/22b917d6-7ed2-4d60-91d8-f551c7eec5ac