Differential use of the signal recognition particle translocase targeting pathway for inner membrane protein assembly in Escherichia coli

Abstract

Assembly of several inner membrane proteins-leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep-has been studied in vitro and in Escherichia coli strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the E. coli signal recognition particle (SRP), or SecE, an essential core component of the E. coli preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.

Is Ffh required for export of secretory proteins?

“…The extent to which precursor proteins accumulated in the absence of Ffh, relative to accumulation in the presence of Ffh, increased as the hydrophobicity of signal sequences decreased. Recent evidence [19] suggested that preproteins with more hydrophobic signal sequences would be more a¡ected by depleting Ffh. Interestingly, these results suggest that the role of SRP is not restricted to the transport of precursor proteins with very hydrophobic signal sequences or membrane proteins, but it is also responsible for the transport of proteins that have relatively weak hydrophobic signal sequences.…”

Basic Amino Acids in a Distinct Subset of Signal Peptides Promote Interaction with the Signal Recognition Particle

“…In E. coli, maltose binding protein (MBP) and OmpA are normally targeted to the IM by SecB, but increasing the net hydrophobicity of their signal peptides reroutes both proteins into the SRP pathway (18). Furthermore, the biogenesis of M13 procoat protein, a small IMP whose insertion normally does not require any targeting factor, becomes SRP-dependent when it contains an unusually hydrophobic signal peptide (19). Likewise, yeast SRP binds preferentially to signal peptides that have a high hydrophobicity index (20).…”

Ordered membrane insertion of an archaeal opsin in vivo

“…In Escherichia coli, polytopic membrane protein insertion seems to require the secretory translocase (8)(9)(10) as well as other proteins needed for insertion in eukaryotes, such as the signal recognition particle (10,11). In addition, insertion of E. coli polytopic membrane proteins in vitro occurs cotranslationally (12)(13)(14). Thus, the mechanism of polytopic membrane protein insertion in E. coli and other prokaryotes may be universally relevant.…”