The Bacterial ATPase SecA Functions as a Monomer in Protein Translocation

Abstract

The ATPase SecA drives the post-translational translocation of proteins through the SecY channel in the bacterial inner membrane. SecA is a dimer that can dissociate into monomers under certain conditions. To address the functional importance of the monomeric state, we generated an Escherichia coli SecA mutant that is almost completely monomeric (>99%), consistent with predictions from the crystal structure of Bacillus subtilis SecA. In vitro, the monomeric derivative retained significant activity in various assays, and in vivo, it sustained 85% of the growth rate of wild type cells and reduced the accumulation of precursor proteins in the cytoplasm. Disulfide cross-linking in intact cells showed that mutant SecA is monomeric and that even its parental dimeric form is dissociated. Our results suggest that SecA functions as a monomer during protein translocation in vivo.Many bacterial proteins are transported post-translationally across the inner membrane by the Sec machinery, which consists of two essential components (1-4). One is the SecY complex, which forms a conserved heterotrimeric protein-conducting channel in the inner membrane (5, 6). The other is SecA, a cytoplasmic ATPase, which "pushes" substrate polypeptide chains through the SecY channel (7). SecA interacts not only with the SecY channel (8) but also with acidic phospholipids (9 -11) and with both the signal sequence and the mature part of a substrate protein (12). It also binds the chaperone SecB, which ushers some precursor proteins to SecA (8,13,14). When associated with the SecY complex, SecA undergoes repeated cycles of ATP-dependent conformational changes, which are linked to the movement of successive segments of a polypeptide chain through the channel (15,16). However the mechanism employed by SecA to translocate substrates polypeptide chains through the SecY channel remains largely unknown.An important issue concerning the function of SecA is its oligomeric state during translocation. SecA is a dimer in solution (17, 18), and previous work argued that this is its functional state (19). An x-ray structure of Bacillus subtilis SecA also indicates the existence of a dimer (7). However, recent evidence raises the possibility that SecA might actually function as a monomer; in solution, SecA dimers are in rapid equilibrium with monomers (20,21). Although the equilibrium favors dimers, it is shifted almost completely toward monomers in the presence of membranes containing acidic phospholipids or upon binding to the SecY complex (21). A synthetic signal peptide had a similar effect, although this result is controversial (22). A monomeric derivative of SecA containing six point mutations retained some in vitro translocation activity (21), but the low level of translocation precluded any firm conclusion. In addition, the previous results do not exclude models in which SecA cycles between monomeric and oligomeric states during the translocation of a polypeptide chain (22,23). Most importantly, the functional oligomeric state of SecA in vivo remains t...

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.…”

A cradle for new proteins: trigger factor at the ribosome

“…In contrast to prior assumptions, SecA is active in substrate association as a monomer, at least in the membrane-and translocon-interacting state. Substrate translocation through the translocon is driven by the SecA ATPase [51]. In the periplasm, an additional arsenal of chaperones welcomes the protein.…”

Probing the Affinity of SecA for Signal Peptide in Different Environments

“…Monomeric SecA in an open conformation has also been crystallized (20), and has a fold different from that of the proposed closed dimeric form from Bacillus subtilis (14) or Mycobacterium tuberculosis (21). Recently, a monomeric mutant of SecA has been found to be active in vivo, indicating that monomers of SecA must be active at some point during the translocation (22); however, the oligomeric state preferred for interactions with signal peptide in aqueous solution and membranes is unclear.…”