Investigation of the Relative Activity of Monomer and Dimer SecYEG Channels

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The general secretory (Sec) pathway is the principal protein transport pathway in bacteria. Two key components of this system are the protein complex SecYEG, which forms a protein-conducting channel within the plasma membrane, and the ATPase motor protein SecA, which drives the protein translocation process. Whether the SecYEG complex functions as a monomer or requires dimerization with a neighboring SecYEG complex for activity remains unclear. To address this important question, we have made use of both photobleaching and total internal reflection fluorescence (TIRF) microscopy to assess the fluorescently labeled SecY oligomeric state, as well as an in vitro protein translocation assay to assess Sec-dependent protein transport activity. The key component of our system is the SecYEG proteoliposome, which is a lipid vesicle reconstituted solely with SecYEG protein. In this study, we reconstituted proteoliposomes at different lipid to SecYEG protein ratios to produce distinct vesicle populations. Next, we utilized TIRF microscopy to determine the percent of singly or doubly-reconstituted proteoliposomes existing within a given population based on the ratio of single or double photobleaching events. Finally, we optimized and performed an in vitro protein transport assay to assess the overall protein translocation activity of a given population. Findings from these assays suggest that the distinct proteoliposome populations have very dissimilar translocation behavior. This difference in behavior is more extensive than that expected of simply different translocation rates. Therefore the data shows that the two different proteoliposome populations are fundamentally different, revealing a need to better understand liposome reconstitution dynamics in the Sec system field.

    Item Description
    Name(s)
    Thesis advisor: Oliver, Donald B.
    Date
    April 15, 2019
    Extent
    92 pages
    Language
    eng
    Genre
    Physical Form
    electronic
    Rights and Use
    In Copyright – Non-Commercial Use Permitted
    Digital Collection
    PID
    ir:1905