SecA Dimer Cross-Linked at Its Subunit Interface Is Functional for Protein Translocation

Abstract

SecA facilitates protein transport across the eubacterial plasma membrane by its association with cargo proteins and the SecYEG translocon, followed by ATP-driven conformational changes that promote protein translocation in a stepwise manner. Whether SecA functions as a monomer or a dimer during this process has been the subject of considerable controversy. Here we utilize cysteine-directed mutagenesis along with the crystal structure of the SecA dimer to create a cross-linked dimer at its subunit interface, which was normally active for in vitro protein translocation.

In Vivo Membrane Topology of Escherichia coli SecA ATPase Reveals Extensive Periplasmic Exposure of Multiple Functionally Important Domains Clustering on One Face of SecA

“…Our results were represented on the highly homologous B. subtilis SecA dimer structure (8), although we caution the reader that it is uncertain how SecA structure changes upon SecYEG binding and membrane insertion (23, 43, 44). It has been reported previously that membrane-bound SecA functions as a dimer (34,(45)(46)(47), although this result has been disputed in favor of a monomer action model and remains controversial (48,49). The MBP labeling pattern was highlighted on both SecA protomers, because we were unable to distinguish whether any subunit labeling asymmetry was obtained in our study.…”

Additional In Vitro and In Vivo Evidence for SecA Functioning as Dimers in the Membrane: Dissociation into Monomers Is Not Essential for Protein Translocation in Escherichia coli

“…Contradictory results have been reported for a similar SecA monomer, SecA⌬11, in which no in vivo or in vitro activities were detected (14), indicating that the SecA dimer is the functional unit of protein translocation (13,14,25,27). Interestingly, however, a recombinant SecA protein (amino acids 9 to 861, lacking both the N and C termini) from E. coli was recently crystallized as an antiparallel dimer at 2 Å resolution, revealing that neither the N nor the C terminus, as previously reported (15), is related to dimerization (25).…”

“…Or et al (24) showed that a disulfide-cross-linked SecA dimer is inactive, suggesting that dissociation of SecA dimers into monomers is required during protein translocation. However, a similar cross-linked SecA-Cys637/801 dimer retained its in vitro activity (13). In addition, a nondissociable SecA dimer, crossed-linked at its C terminus, is functional in protein translocation in vitro, demonstrating that dissociation of the SecA dimer is not an essential step in preprotein translocation (6).…”

The bacterial Sec-translocase: structure and mechanism

“…However, a recent single-molecule study suggested that the dimeric organization of SecA is maintained when actively engaged in preprotein translocation [93]. Covalent stabilization of the SecA dimer by crosslinking did not interfere with the protein translocation activity [93,103,108,109]. In this respect, the T. maritima SecYEG-SecA crystal structure shows a monomer of SecA bound to SecYEG, but this structure may not reflect the physiological oligomeric state of SecA as the crystallization process was accompanied with detergents and high salt, both of which have been shown to negate interactions in a SecA dimer interface [93,106].…”