Abstractβ-barrel membrane proteins in Gram-negative bacteria, mitochondria, and chloroplasts are assembled by highly conserved multi-protein complexes. The mechanism by which these molecular machines fold and insert their substrates is poorly understood. It has not been possible to dissect the folding and insertion pathway because the process has not been reproduced in a biochemical system. We purified the components that fold and insert E. coli outer membrane proteins and reconstituted β-barrel protein assembly in proteoliposomes using the enzymatic activity of a protein substrate to report on its folding state. The assembly of this protein occurred without an energy source but required a soluble chaperone in addition to the multi-protein assembly complex.The outer membranes of Gram-negative bacteria and the mitochondria and chloroplasts of higher eukaryotes contain proteins with β-barrel structure, which are assembled in their respective membranes by multi-protein machines (1-6). The folding and insertion of these β-barrels must be coordinated because they would have many unsatisfied hydrogen bonds in the membrane if they were inserted in an unfolded state, but, conversely, they would be "inside out" if they folded first in the aqueous environment and were subsequently inserted. In order to understand how β-barrel proteins assemble into membranes, we purified the proteins comprising the E. coli outer membrane protein (OMP) folding machinery and established a reconstituted system to monitor the activity of this machinery.The β-barrel assembly machine (Bam) in E. coli consists of an integral β-barrel protein, BamA (formerly YaeT), and four lipoproteins, BamB, C, D, and E (formerly YfgL, NlpB, YfiO, and SmpA, respectively). Only BamA and BamD are essential for cell survival, but deleting or depleting any member of the complex causes defects in OMP assembly (6-9). BamA has homologs in prokaryotes and eukaryotes and contains five periplasmic polypeptide transport associated (POTRA) domains, which scaffold the Bam lipoproteins (2-5,10). Unfolded OMPs are delivered to this complex following their synthesis in the cytoplasm and translocation across the inner membrane by the secretion machinery (SecYEG) ( Figure 1A) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript that are released from the Sec machine from aggregating and misfolding as they transit the periplasm. These chaperones are thought to transport OMPs in two parallel, but separate pathways-one that relies on SurA and one that involves both Skp and DegP (12,13). Most OMPs can be handled by either pathway, but SurA delivers the bulk of OMPs to the OM (14).The process of OMP assembly can be monitored in isolated mitochondria (15) and in vivo in E. coli (16). We sought to develop an in vitro system to study the function of the Bam proteins. Expressing all five bam genes in a single strain produced a mixture of complexes and sub-complexes that could not be easily separated. Previous lipoprotein and POTRA domain deletion experiments indicat...

β-barrel membrane proteins perform important functions in the outer membranes (OMs) of Gram-negative bacteria and of the mitochondria and chloroplasts of eukaryotes. The protein complexes that assemble these proteins in their respective membranes have been identified and shown to contain a component that has been conserved from bacteria to humans. β-barrel proteins are handled differently from α-helical membrane proteins in the cell in order to efficiently transport them to their final locations in unfolded but folding-competent states. The mechanism by which the assembly complex then binds, folds, and inserts β-barrels into the membrane is not well understood, but recent structural, biochemical, and genetic studies have begun to elucidate elements of how the complex provides a facilitated pathway for β-barrel assembly. Ultimately, studies of the mechanism of β-barrel assembly and comparison to the better-understood process of α-helical membrane protein assembly will reveal whether there are general principles that guide the folding and insertion of all membrane proteins.

The protein complex that assembles integral membrane β-barrel proteins in the outer membranes of Gram-negative bacteria is an attractive target in the development of new antibiotics. This complex, the β-barrel assembly machine (Bam), contains two essential proteins, BamA and BamD. We have identified a peptide that inhibits the assembly of β-barrel proteins in vitro by characterizing the interaction of BamD with an unfolded substrate protein. This peptide is a fragment of the substrate protein and contains a conserved amino acid sequence. We have demonstrated that mutations of this sequence in the full-length substrate protein impair the protein's assembly, implying that BamD's interaction with this sequence is an important part of the assembly mechanism. Finally, we have found that in vivo expression of a peptide containing this sequence causes growth defects and sensitizes Escherichia coli to antibiotics to which they are normally resistant. Therefore, inhibiting the binding of substrates to BamD is a viable strategy for developing new antibiotics directed against Gram-negative bacteria.β-barrel | outer membrane | protein folding | Bam complex | inhibition

The outer membrane (OM) of Gram-negative bacteria such as Escherichia coli contains lipoproteins and integral β-barrel proteins (outer-membrane proteins, OMPs) assembled into an asymmetrical lipid bilayer. Insertion of β-barrel proteins into the OM is mediated by a protein complex that contains the OMP BamA and four associated lipoproteins (BamBCDE). The mechanism by which the Bam complex catalyzes the assembly of OMPs is not known. We report here the isolation and characterization of a temperature-sensitive lethal mutation, bamAE373K, which alters the fifth polypeptide transport-associated domain and disrupts the interaction between the BamAB and BamCDE subcomplexes. Suppressor mutations that map to codon 197 in bamD restore Bam complex function to wildtype levels. However, these suppressors do not restore the interaction between BamA and BamD; rather, they bypass the requirement for stable holocomplex formation by activating BamD. These results imply that BamA and BamD interact directly with OMP substrates.Omp85 | membrane protein folding | conditional lethal | allele-specific

The Bam machine assembles β-barrel membrane proteins into the outer membranes of Gram-negative bacteria. The central component of the Bam complex, BamA, is a β-barrel that is conserved in prokaryotes and eukaryotes. We have previously reported an in vitro assay for studying the assembly of β-barrel proteins by the Bam complex and now apply this assay to identify the specific components that are required for BamA assembly. We establish that BamB and BamD, two lipoprotein components of the complex, bind to the unfolded BamA substrate and are sufficient to accelerate its assembly into the membrane.