Characterization of the SecA Insertion Cycle Through in vivo Photo-Crosslinking Utilizing Maltose-binding Protein as a Topology Probe

<p>The post-translational translocation of preproteins across the bacterial plasma membrane is a Sec-dependent mechanism in which the SecA ATPase motor protein drives protein substrates through the SecYEG channel complex. The precise nature of this transport mechanism remains unknown, however, a ratchet model has been proposed in which a 30kD carboxyl-terminal region of SecA undergoes ATP-driven membrane insertion and retraction cycles at SecYEG to promote the translocation of preproteins. This result was determined using protease probing of an<em> in vitro</em> protein translocation system, but it remains highly controversial.</p> <p>Recent <em>in vivo</em> photo-crosslinking results support the ratchet model, where it was found that SecA interacts both superficially with the cytosolic domains of SecY, and also within the channel interior and periplasmic domains of SecY when engaged with a substrate protein. However, the specific region(s) of SecA that undergoes substratedependent insertion was not determined, nor was the depth of insertion, and accessibility of SecA from the periplasm explored in this study.</p> <p>In order to address these questions, we have probed the periplasmic accessibility of SecA by <em>in vivo</em> photo-crosslinking to maltose-binding protein (MBP), a periplasmic protein. While this specific crosslinking methodology has been previously utilized to reveal specific protein-protein interactions, the high concentration of MBP in the periplasm (approaching millimolar levels) make it amenable for use as a general crosslinking reagent to detect periplasmic exposure of proteins on the trans side of the plasma membrane. A series of plasmid-containing MBP constructs were engineered by site-directed mutagenesis to introduce UV-dependent crosslinkers at different molecular environments on the MBP protein surface. These plasmid-containing strains were then utilized to test for MBP-SecA interaction by <em>in vivo</em> photo-crosslinking. For this purpose, cells were grown and induced for MBP over-expression, then UV treated and broken by sonication. SecA-containing membranes were isolated and probed by western blotting for MBP-SecA adducts. In addition, detergent-solubilized membranes have been eluted through a dextran affinity column, which specifically binds MBP, to attempt to enrich for the desired MBP-SecA adducts. Positive crosslinking results indicate that SecA becomes periplasmically accessible during protein transport. In addition, further limited proteolysis studies of the MBP-SecA chimeras, if detected, can be carried out with region-specific SecA antisera, which will allow us to map the region of SecA that inserts into the SecYEG channel complex and becomes periplasmically accessible during protein transport. In sum, these results should allow us to build a structure-function model of SecA-dependent protein transport through the SecYEG channel complex.</p>