Publication Date

5-2019

Advisor(s)

Donald Oliver

Department

Molecular Biology and Biochemistry

Abstract

The ubiquitous general Secretory Pathway (Sec pathway) is the most widely used protein transport pathway across the plasma membrane of bacteria. Key components include the SecA ATPase motor protein and the hetero-trimeric SecYEG channel complex. SecA binds both protein substrates and SecYEG, and promotes the stepwise transport of proteins across the channel by a largely unknown mechanism. While SecA has a highly conserved domain structure and protomer fold there is a multitude of quaternary states based on x-ray structures that has led to the proposal that the dimer may simply function as a storage form within the cell. We have examined the physiological form of the various SecA dimers by engineering a sitespecific crosslinker into potential dimer interfaces and performed in vivo photocrosslinking. Our results show that a single discrete dimer species is present within the cell, and this species was also captured during arrested protein transport through the SecYEG channel. This work strengthens models that posit, at least in part, a SecA dimer-driven translocation mechanism.

The mechanism of SecA-driven protein transport across the SecYEG channel complex has remained controversial. We undertook a novel approach, namely site-directed in vivo photo-crosslinking in order to map the SecA interaction sites throughout the SecY protein in a more physiological manner. Our SecA membrane topology map shows extensive integration of SecA into the SecYEG channel complex during arrested protein translocation, where SecA contacts most SecY transmembrane helices and periplasmic domains. This work helps to elucidate the SecA membrane insertion step that drives ongoing protein transport at the SecYEG channel complex.

Similar to the controversy surrounding the SecA oligomeric state, we have also addressed the functional oligomeric state of SecYEG protein utilizing an identical approach. However at the conclusion of this study, we were unable to resolve the question of whether the SecYEG monomer was active in promoting protein transport. In order to address this latter question we are currently using a combination of both single molecule and ensemble biophysical and biochemical approaches. In particular, by varying the SecYEG protein-to-lipid ratio (LPR), we have made different proteoliposome populations that contain either mostly SecYEG monomers or dimers as assessed by stepwise photobleaching of fluorescently labeled SecYEG under TIRF microscopy. The proteoliposome population that contains ~2 copies of SecYEG were subjected to crosslinking in order to verify its dimeric state. We have also developed a sensitive in vitro protein translocation system utilizing a fluorescently labeled protein substrate in order to overcome the low protein transport efficiency for proteoliposomes containing reduced SecYEG content. We measured the protein transport rate and maximal amplitude of these different proteoliposome populations. Our initial data hypothesize that SecYEG may monomerizes during initial step of protein transport and later in the process two copies of SecYEG comes closer to form the dimer in order to facilitate the further movement of substrate in association with SecA. However, further studies will be required to elucidate the functional oligomeric state of SecYEG during pre-protein translocation and validate the above-mentioned hypothesis.

Available for download on Monday, June 01, 2020

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