Investigating the SecY plug movement at the SecYEG translocation channel

Abstract

Protein translocation occurs across the energy-conserving bacterial membrane at the SecYEG channel. The crystal structure of the channel has revealed a possible mechanism for gating and opening. This study evaluates the plug hypothesis using cysteine crosslink experiments in combination with various allelic forms of the Sec complex. The results demonstrate that the SecY plug domain moves away from the center of the channel toward SecE during polypeptide translocation, and further show that the translocation-enhancing prlA3 mutation and SecG subunit change the properties of channel gating. Locking the plug in the open state preactivates the Sec complex, and a super-active translocase can be created when combined with the prlA4 mutation located in the pore of the channel. Dimerization of the Sec complex, which is essential for translocase activity, relocates the plug toward the open position. We propose that oligomerization may result in SecYEG cooperative interactions important to prime the translocon function.

Structural Determinants of Lateral Gate Opening in the Protein Translocon

“…A destabilization of the plug in functional dimers of the channel had already been predicted by simulation, modeling, and experiment (10,28,66). One early step in opening the channel, the insertion of the signal sequence, has been proposed to cause the lateral gate to open slightly, which then frees the plug (2).…”

Identification of Two Interaction Sites in SecY that Are Important for the Functional Interaction with SecA

“…The central role of Arg357 in SecY functioning is further illustrated by the recent findings that mutations in that region interfere with SecYEG oligomerization and movement of the plug domain. 30 The SecA interaction site we identified by peptide scanning (TMS4c: Phe170-Gln177) constitutes the cytoplasmic end of TMS4 from SecY. Instead of being an authentic SecA-SecY interaction site, the observed interaction could represent binding of SecA to a pre-protein.…”

The action of cardiolipin on the bacterial translocon

“…The membrane bound and active version of SecYEG is dimeric (11)(12)(13)(14), but only one copy is necessary to create the channel (15). We show that CL, and to a lesser degree PG, promotes the formation of SecYEG dimers and a high-affinity binding surface for SecA.…”

“…The experiments suggest a high-affinity CL binding site on SecYEG that, once occupied, stabilizes the SecYEG dimeric form and creates a high-affinity binding platform for SecA and a conferral of activated ATPase activity. Therefore, we tested if each of the positive effects of CL on SecA (i.e., binding and activation) were the consequence of SecYEG dimerization, in an analysis exploiting the covalently linked SecYEG dimer (11) termed SecYYE 2 G 2 .…”