Graduation Year

2017

Document Type

Honors Thesis

Degree Name

Bachelor of Arts

Department

Natural Sciences

Program or Major

Biotechnology and Molecular Biology

Second Program or Major

Biology with Concentration in Neuroscience and Behavior

Faculty Advisor

Laura Marcotte

Abstract

The exocyst complex is a multi-subunit tethering complex that is used in the process of exocytosis. There are eight subunits in the complex, and these subunits interact with each other as well as proteins outside of the complex to facilitate membrane fusion. Prior research has shown that the exocyst subunit Sec6 and Sec1 from the Sec1/Munc18 (SM) family, a known regulator of membrane fusion, interact with each other. In this experiment, the goal was to crosslink the two proteins and in turn work to identify the interacting amino acid residues that are responsible for the proteins’ interaction. To do this, Sec1 and Sec6 were purified individually. Binding assays were then performed on the purified proteins to confirm that they folded properly and would interact. Crosslinking experiments were performed and resulted in the appearance of multiple new bands on the gel indicating that crosslinking had occurred. The proteins present in these bands will be analyzed by mass spectrometry to determine the interacting residues of the two protein subunits. Mutational and structural analysis will be performed in the future to confirm that the amino acids identified by mass spectrometry analysis are truly interacting. If the interacting residues are mutated, then the interactions between the two proteins should be weakened or even eliminated. This research is important in furthering our understanding of the exocyst complex and how it interacts with other proteins to facilitate the complex process of exocytosis.

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