P. Fekkes

The Sec system

Proteins designated to be secreted by Escherichia coli are synthesized with an amino-terminal signal peptide and associate as nascent chains with the export-specific chaperone SecB. Translocation occurs at a multisubunit membrane-bound enzyme termed translocase, which consists of a peripheral preprotein-binding site and an ATPase domain termed SecA, a core heterotrimeric integral membrane protein complex with SecY, SecE and SecG as subunits, and an accessory integral membrane protein complex containing SecD and SecF. Major new insights have been gained into the cascade of preprotein targeting events and the enzymatic mechanism or preprotein translocation. It has become clear that preproteins are translocated in a stepwise fashion involving large nucleotide-induced conformational changes of the molecular motor SecA that propels the translocation reaction.

The molecular chaperone SecB is released from the carboxy-terminus of SecA during initiation of precursor protein translocation.

The chaperone SecB keeps precursor proteins in a translocation‐competent state and targets them to SecA at the translocation sites in the cytoplasmic membrane of Escherichia coli. SecA is thought to recognize SecB via its carboxy‐terminus. To determine the minimal requirement for a SecB‐binding site, fusion proteins were created between glutathione‐S‐transferase and different parts of the carboxy‐terminus of SecA and analysed for SecB binding. A strikingly short amino acid sequence corresponding to only the most distal 22 aminoacyl residues of SecA suffices for the authentic binding of SecB or the SecB–precursor protein complex. SecAN880, a deletion mutant that lacks this highly conserved domain, still supports precursor protein translocation but is unable to bind SecB. Heterodimers of wild‐type SecA and SecAN880 are defective in SecB binding, demonstrating that both carboxy‐termini of the SecA dimer are needed to form a genuine SecB‐binding site. SecB is released from the translocase at a very early stage in protein translocation when the membrane‐bound SecA binds ATP to initiate translocation. It is concluded that the SecB‐binding site on SecA is confined to the extreme carboxy‐terminus of the SecA dimer, and that SecB is released from this site at the onset of translocation.

Preprotein transfer to the Escherichia coli translocase requires the co‐operative binding of SecB and the signal sequence to SecA

In Escherichia coli, precursor proteins are targeted to the membrane‐bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy‐terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA‐dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non‐functional signal sequence. Δ8proOmpA is not translocated across wild‐type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.

PrlA4 prevents the rejection of signal sequence defective preproteins by stabilizing the SecA–SecY interaction during the initiation of translocation

In Escherichia coli, precursor proteins are translocated across the cytoplasmic membrane by translocase. This multisubunit enzyme consists of a preprotein‐binding and ATPase domain, SecA, and the SecYEG complex as the integral membrane domain. PrlA4 is a mutant of SecY that enables the translocation of preproteins with a defective, or missing, signal sequence. Inner membranes of the prlA4 strain efficiently translocate Δ8proOmpA, a proOmpA derivative with a non‐functional signal sequence. Owing to the signal sequence mutation, Δ8proOmpA binds to the translocase with a lowered affinity and the recognition is not restored by the prlA4 SecY. At the ATP‐dependent initiation of translocation, the binding affinity of SecA for SecYEG is lowered causing the premature loss of bound preproteins from the translocase. The prlA4 membranes, however, bind SecA with a much higher affinity than the wild‐type, and during initiation, the SecA and preprotein remain bound at the translocation site allowing an improved efficiency of translocation. It is concluded that the prlA4 strain prevents the rejection of defective preproteins from the export pathway by stabilizing SecA at the SecYEG complex.

SecA is an intrinsic subunit of the Escherichia coli preprotein translocase and exposes its carboxyl terminus to the periplasm

SecA is the dissociable ATPase subunit of the Escherichia coli preprotein translocase, and cycles in a nucleotide‐modulated manner between the cytosol and the membrane. Overproduction of the integral subunits of the translocase,the SecY, SecE and SecG polypeptides, results in an increased level of membrane‐bound SecA. This fraction of SecA is firmly associated with the membrane as it is resistant to extraction with the chaotropic agent urea, and appears to be anchored by SecYEG rather than by lipids. Topology analysis of this membrane‐associated form of SecA indicates that it exposes a carboxy‐terminal domain to the periplasmic face of the membrane.