Cysteine-Directed Cross-Linking Demonstrates That Helix 3 of SecE Is Close to Helix 2 of SecY and Helix 3 of a Neighboring SecE

Abstract

Preprotein translocation in Escherichia coli is mediated by translocase, a multimeric membrane protein complex with SecA as the peripheral ATPase and SecYEG as the translocation pore. Unique cysteines were introduced into transmembrane segment (TMS) 2 of SecY and TMS 3 of SecE to probe possible sites of interaction between the integral membrane subunits. The SecY and SecE single-Cys mutants were cloned individually and in pairs into a secYEG expression vector and functionally overexpressed. Oxidation of the single-Cys pairs revealed periodic contacts between SecY and SecE that are confined to a specific alpha-helical face of TMS 2 and 3, respectively. A Cys at the opposite alpha-helical face of TMS 3 of SecE was found to interact with a neighboring SecE molecule. Formation of this SecE dimer did not affect the high-affinity binding of SecA to SecYEG and ATP hydrolysis, but blocked preprotein translocation and thus uncouples the SecA ATPase activity from translocation. Conditions that prevent membrane deinsertion of SecA markedly stimulated the interhelical contact between the SecE molecules. The latter demonstrates a SecA-mediated modulation of the protein translocation channel that is sensed by SecE.

Glycolipozyme MPIase is essential for topology inversion of SecG during preprotein translocation

“…In contrast, the overproduced SecYEG with SecG 60C was resistant to oxidation [SecYEG(60C) ++ ]. Conversely, when SecE 106C IMV were used in which SecE 106C had been expressed at the WT level in the secE null background, the activity did not change on oxidation (ΔSecE/SecE 106C), whereas translocation into SecYE(106C)G ++ IMV was affected by oxidation [SecYE(106C) G ++ ], which is consistent with a previous report (11). These results strongly suggest that the dimer interface of SecYEG differs between WT SecYEG and overproduced SecYEG.…”

“…3C, Right) as MPIase increased. Although the recovery of the SecE 106C monomer after oxidation of PL was less efficient, as reported (11), an MPIase-dependent decrease of the SecE dimer was clearly observed. These results confirm that MPIase modulates the dimer orientation of SecYEG.…”

“…We also demonstrated that MPIase modulates the dimer orientation of SecYEG, with this being the reason MPIase is essential for SecG inversion. The back-to-back structure, which is predominantly found in crystals (4,5) and SecYEG-overproducing conditions (11), and the front-to-front structure found on cryo-electron microscopy (8) may be caused by a lack of MPIase. However, SecYEG in WT cells or purified SecYEG supplemented with MPIase exhibited the side-by-side orientation, allowing five-to 10-fold more efficient preprotein translocation with SecG inversion.…”

“…However, the structures that have so far been determined are those of the resting or preactive states; the structure of the translocating channels is largely unknown. Biochemical studies also have revealed that E. coli SecYEG forms oligomers, such as a dimer and a tetramer (9)(10)(11)(12). In some crystals, SecYEG forms a dimer, of which the interface is either SecE (back-to-back) (4,6) or SecY (front-to-front) (8), with the back-to-back interface being predominantly formed (13).…”

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

“…12 Within the C5 loop, Arg357 has been shown to play a crucial role in SecY functioning, 24 and therefore we mutated 15 amino acids around this residue (Val353 to Asp367) into single cysteine residues. Cysteine-less SecYEG, which behaves identically to wild-type SecYEG, 25 was used as a control throughout this work. All 15 single cysteine SecY mutants were overexpressed to similar levels as cysteine-less SecY (data not shown), and except for the SecY(R357C) mutant, all were equally active for in vitro translocation of proOmpA (Figure 1(b), compare lane 6 to all other lanes).…”