Date of Graduation

Summer 2019

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Kyoungtae Kim

Keywords

Vps1, Tlg2, endosome, TGN, membrane fusion, proteoliposome reconstitution, simultaneous fusion assay, lipid mixing, content mixing

Subject Categories

Biochemistry | Molecular Biology

Abstract

Intracellular membrane fusion events can be reconstituted by exploiting isolated organelles from cellular hosts or artificial membranes made of purified phospholipid components. Artificial construction of membranes provides two significant advantages. First, cellular isolation of the endosome-derived vesicles and TGN (trans-Golgi Network) compartments needed for the fusion assay would be extremely challenging. Second, reconstituting the membranes provides the added benefit of controlling size and lipid compositions to functionally mimic the individual membrane architectures and introduce only the purified proteins that are under investigation. For these reasons, I have developed the first simultaneous lipid and content mixing fusion assays that measures the efficacy of endosome-to-TGN fusion and its promoted fusion capability with the dynamin-like protein Vps1 for the purposes of recapitulating the fusion. To quantify lipid mixing between the donor and recipient membrane fluorescent lipids (Rhodamine-PE and NBD-PE) were used, while content mixing was assessed by analyzing the increase of FRET between Cy5- streptavidin and PhycoE-biotin.

Copyright

© Jared Christopher Smothers

Open Access

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