Covalently Dimerized SecA Is Functional in Protein Translocation

Abstract

The ATPase SecA provides the driving force for the transport of secretory proteins across the cytoplasmic membrane of Escherichia coli. SecA exists as a dimer in solution, but the exact oligomeric state of SecA during membrane binding and preprotein translocation is a topic of debate. To study the requirements of oligomeric changes in SecA during protein translocation, a non-dissociable SecA dimer was formed by oxidation of the carboxyl-terminal cysteines. The cross-linked SecA dimer interacts with the SecYEG complex with a similar stoichiometry as non-cross-linked SecA. Cross-linking reversibly disrupts the SecB binding site on SecA. However, in the absence of SecB, the activity of the disulfide-bonded SecA dimer is indistinguishable from wild-type SecA. Moreover, SecYEG binding stabilizes a cold sodium dodecylsulfate-resistant dimeric state of SecA. The results demonstrate that dissociation of the SecA dimer is not an essential feature of the protein translocation reaction.Translocase mediates the translocation of precursor proteins (preproteins) across the cytoplasmic membrane of Escherichia coli and other bacteria. The ATPase SecA (1) is the peripheral motor domain of the translocase. The SecYEG complex constitutes a membrane-embedded protein-conducting channel (reviewed in Ref. 2) and a high affinity binding site for SecA (3). When bound to SecYEG, SecA functions as a high affinity membrane receptor for the molecular chaperone SecB with associated preproteins (3). The movement of the preprotein through the protein-conducting channel is driven by multiple cycles of ATP binding and hydrolysis at SecA (4) and the proton motive force.In solution, SecA exist as a homodimer (5) that equilibrates with its monomeric form in a temperature-, salt-and protein concentration-dependent manner (6). Under physiological conditions, the dissociation constant (K D ) for the monomer-dimer equilibrium is ϳ0

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

“…This conclusion is consistent with the early biochemical study of Driessen (8) as well as the genetic observation from Kumamoto's group that duplication of the secA gene that resulted in a head-to-tail covalent dimer was functional in vivo (17). While we were writing up our work, a study similar to ours was published (6). While the approach and conclusion of that study were similar to ours, there are important differences between the two studies.…”

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

“…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 Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

“…Remarkably, structural determinations have so far revealed different oligomeric states of SecA, including the antiparallel dimer (6,7), the intertwined dimer (8), the parallel dimer (9), and the monomer (10). It is unresolved whether its working form is a monomer or a dimer (11)(12)(13)(14)(15). The translation level and the functionality of SecA are controlled in vivo by SecM, an unusual secretion monitor protein (16).…”