Abstract
The bacterial Sec-dependent system is the major protein-biogenic pathway for protein secretion across the cytoplasmic membrane or insertion of integral membrane proteins into the phospholipid bilayer. The mechanism of SecA-driven protein transport across the SecYEG channel complex has remained controversial with conflicting claims from biochemical and structural studies regarding the depth and extent of SecA insertion into SecYEG during ongoing protein transport. Here we utilized site-specific in vivo photo-crosslinking to thoroughly map SecY regions that are in contact with SecA during its insertion cycle. An arabinose-inducible, rapidly folding OmpA-GFP chimera was utilized to jam the SecYEG channels with an arrested substrate protein to "freeze" them in their SecA-inserted state. Examination of 117 sites distributed throughout SecY indicated that SecA not only interacts extensively with the cytosolic regions of SecY as shown previously, but it also interacts with most of the transmembrane helices and periplasmic regions of SecY, with a clustering of interaction sights around the lateral gate and pore ring regions. Our observations support previous reports of SecA membrane insertion during in vitro protein transport as well as those documenting the membrane penetration properties of this protein. They suggest that one or more SecA regions transiently integrate into the heart of the SecY channel complex to span the membrane to promote the protein transport cycle. These findings indicate that high-resolution structural information about the membrane-inserted state of SecA is still lacking and will be critical for elucidating the bacterial protein transport mechanism.
Highlights
The bacterial Sec-dependent system is the major protein biogenic pathway promoting the secretion of proteins across the cytoplasmic membrane or the insertion of integral membrane proteins into the phospholipid bilayer
A number of models of SecA action have been proposed subsequently including (i) a second generation power stroke model whereby the SecA two-helix finger subdomain acts as an ATPdependent reciprocating piston to drive protein transport at the mouth of the SecY channel; this model was proposed based on the position of the two-helix finger within a co-crystal of SecA bound to SecYEG in an intermediate state of ATP hydrolysis [16, 17], (ii) the Brownian ratchet model that relies on two-way communication between SecA and SecY to coordinate channel opening and ATP hydrolysis events, thereby biasing Brownian motion of the substrate in one direction [18], and (iii)
We have previously utilized site-directed in vivo photocrosslinking to characterize the SecY-interactive face of SecA protein and to validate the in vivo relevance of the Thermotoga maritima SecA-SecYEG crystal structure for the E. coli model system [16, 36]
Summary
Duke University School of Medicine, Durham, NC 27710. 3 To whom correspondence should be addressed. The discovery that a protease-protected 30-kDa region of SecA appeared to undergo ATP-driven cycles of membrane insertion and retraction coupled to in vitro protein transport led to the original SecA power stroke model [14]. Integral membrane SecA was found to reveal multiple periplasmically-exposed regions under these conditions with its CTL4 region accessible to trypsinolysis [31, 33,34,35] In such studies it is impossible to distinguish whether the observed labeling pattern was due to the existence of authentic periplasmic regions of SecA or whether such labeling occurred utilizing the SecY channel to gain access to internal portions of SecA protein. Our results are described below, and they provide convincing evidence of extensive integration of SecA into the SecYEG channel complex, consistent with the original power stroke model as described by Economou and Wickner [14]
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