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

Abstract

SecA is an essential component in the Sec-dependent protein translocation pathway and, together with ATP, provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. Previous studies established that SecA undergoes monomer-dimer equilibrium in solution. However, the oligomeric state of functional SecA during the protein translocation process is controversial. In this study, we provide additional evidence that SecA functions as a dimer in the membrane by (i) demonstration of the capability of the presumably monomeric SecA derivative to be cross-linked as dimers in vitro and in vivo, (ii) complementation of the growth of a secA(Ts) mutant with another nonfunctional SecA or (iii) in vivo complementation and in vitro function of a genetically tandem SecA dimer that does not dissociate into monomers, and (iv) formation of similar ring-like structures by the tandem SecA dimer and SecA in the presence of lipid bilayers. We conclude that SecA functions as a dimer in the membrane and dissociation into monomers is not necessary during protein translocation.

Defining the Escherichia coli SecA Dimer Interface Residues through In Vivo Site-Specific Photo-Cross-Linking

“…At 100 mM KCl, the SecA (no His) dimer is only approximately 10-fold tighter than that of SecA ⌬2-11 (Table 1). This finding is consistent with previous studies, in which SecA ⌬2-11/N95 was found to exist primarily in the dimeric state in low-ionic-strength buffers (43,44).…”

“…In addition, SecA F263X and F10X produce a cross-linked dimer band with a faster electrophoretic mobility than other variants. The aberrant SDS-PAGE mobilities may result from changes in conformation dependent on the location of cross-linked residues (34), as observed in previous chemical cross-linking experiments (17,28,44).…”

“…Moreover, formaldehyde cross-linking, which links amino acids in very close proximity, also showed dimeric SecA ⌬2-11/N95 in vivo (44). This suggests that experiments such as those evaluating specific cysteine-cysteine cross-linking that indicate that SecA ⌬2-11/N95 is monomeric must be interpreted cautiously (17).…”

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].…”

Reexamination of the Role of the Amino Terminus of SecA in Promoting Its Dimerization and Functional State

“…Both in vivo and in vitro protein cross-linking techniques have been utilized to assess the fraction of the SecA dimer in soluble and membrane-bound states. Unfortunately, the studies have produced equivocal results (17,23,24,32) that are of limited value; on the one hand, cross-linking traps proteins in the oligomeric state and thus distorts a natural equilibrium, while on the other hand, suboptimal cross-linking conditions (e.g., nonideal cross-linker chemistry, insufficient cross-linker concentration, or nonphysiological buffer or salt conditions) can lead to underrepresentation of the true oligomeric population. Genetic techniques have been utilized to engineer SecA dimers that were linked in a head-to-tail fashion, and such modified proteins were found to be functional both in vivo and in vitro (22,32).…”