Investigating the stability of the SecA-SecYEG complex during protein translocation across the bacterial membrane.

During post-translational translocation in E. coli, polypeptide substrates are driven across the membrane through the SecYEG protein conducting channel using the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. Recent studies suggest that SecA is a highly dynamic enzyme, able to repeatedly bind and dissociate from SecYEG during substrate translocation, yet other studies indicate that these dynamics - here referred to as "SecA processivity" - are not a requirement for transport.Here, we employ a SecA mutant (PrlD23) which associates more tightly to membranes than wild-type SecA, in addition to a SecA-SecYEG crosslinked complex, to demonstrate that SecA-SecYEG binding and dissociation events are important for efficient transport of the periplasmic protein proPhoA. Strikingly however, we find that transport of the precursor of the outer membrane protein proOmpA does not depend on SecA processivity. By exchanging signal sequence and protein domains of similar size between PhoA and OmpA, we find that SecA processivity is not influenced by the sequence of the protein substrate. In contrast, using an extended proOmpA variant and a truncated derivative of proPhoA, we show that SecA processivity is affected by substrate length. These findings underscore the importance of the dynamic nature of SecA-SecYEG interactions as a function of the preprotein substrate, features which are not yet reported by other biophysical or in vivo methods.