Determination of the Oligomeric State of SecY using in vivo photocrosslinking

SecYEG is a universally conserved heterotrimeric membrane protein complex that is part of the Sec system. The Sec system participates in protein trafficking across or integration into the bacterial cytoplasmic membrane. The oligomeric state and conformation of SecYEG during active protein transport is an ambiguous and well-debated topic in this field with evidence favoring either front-to-front or back-to-back dimers as well as the monomer as the active species. In this study, we set out to determine the presence of the front-to-front SecYEG dimer in live E. coli cells. We utilized site-specific in vivo photocrosslinking of selected SecY residues at the dimer interface followed by western blotting to determine the oligomeric state of SecYEG. We found that SecY mutants S111, N151, F154, and A324 showed strong UV-dependent bands at approximately 75 kDa consistent with the in vivo existence of the front-to-front SecYEG dimer. Furthermore, we assessed the SecYEG dimer state during active protein translocation by utilizing two plasmids that conditionally overproduce a translocation intermediate that jams most translocons within the cell, thus mimicking the active translocation state. One contains an arabinose-inducible promoter that produces a chimera between the outer membrane protein OmpA and GFP, and another contains a maltose-inducible promoter that produces a chimera between the periplasmic maltose-binding protein and beta-galactosidase. We found that during such arrested protein translocation, a SecYEG dimer is present. This suggests that a functional oligomeric state of the SecYEG channel in its active state is a dimer in the front-to-front orientation. Overall, this study will help to elucidate the mechanism of the Sec system in bacteria such as E. coli.

    Item Description
    Name(s)
    Thesis advisor: Oliver, Donald B.
    Date
    April 15, 2015
    Extent
    91 pages
    Language
    eng
    Genre
    Physical Form
    electronic
    Rights and Use
    In Copyright – Non-Commercial Use Permitted
    Restrictions on Use
    Access limited to Wesleyan Community only. Please contact wesscholar@wesleyan.edu for more information.
    Digital Collection
    PID
    ir:2039