Year of Award

2014

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Other Degree Name/Area of Focus

Integrative Microbiology and Biochemistry, Cellular and Molecular Biology

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

Jesse Hay

Commitee Members

Mark Grimes, Michele McGuirl, Stephen Sprang, Erica Woodahl

Keywords

COPII, ER/Golgi, p115, SNARE, tether, trafficking

Publisher

University of Montana

Abstract

The endomembrane system consists of several connected yet distinct compartments, constantly exchanging material via the flow of membrane-enclosed vesicles. Protein transport between the endoplasmic reticulum (ER) and the Golgi is one of the most fundamental trafficking pathways in eukaryotic cells, as well as the first step in the secretory pathway. The fusion between vesicles and target membranes requires the orchestration of tethers, Rabs, SM proteins and SNAREs. Tethering factors regulate the targeting of membrane-enclosed vesicles and enhance the efficiency as well as specificity of membrane fusion. P115, a Golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, p115's mechanism of action is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that p115's function is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from COPII vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNARE assembly. Our results suggest a model in which p115 forms a mixed p115/SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at pre-fusion sites, and is subsequently ejected as SNARE complex formation and fusion proceed.

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© Copyright 2014 Ting Wang