Abstract
Many integral membrane proteins form oligomeric complexes, but the assembly of these structures is poorly understood. Here, we show that the assembly of OmpC, a trimeric porin that resides in the Escherichia coli outer membrane (OM), can be reconstituted in vitro. Although we observed the insertion of both urea-denatured and in vitro-synthesized OmpC into pure lipid vesicles at physiological pH, the protein assembled only into dead-end dimers. In contrast, in vitro-synthesized OmpC was inserted into proteoliposomes that contained the barrel assembly machinery (Bam) complex, a conserved heterooligomer that catalyzes protein integration into the bacterial OM, and folded into heat-stable trimers by passing through a short-lived dimeric intermediate. Interestingly, complete OmpC assembly was also dependent on the addition of lipopolysaccharide (LPS), a glycolipid located exclusively in the OM. Our results strongly suggest that trimeric porins form through a stepwise process that requires the integration of the protein into the OM in an assembly-competent state. Furthermore, our results provide surprising evidence that interaction with LPS is required not only for trimerization but also for the productive insertion of individual subunits into the lipid bilayer. IMPORTANCE Porins are a widespread family of homotrimers that represent a substantial fraction of the total protein located in the OM of many Proteobacteria. These proteins facilitate the nonspecific diffusion of small molecules across the outer membrane and strongly influence the susceptibility of bacteria to clinically used antibiotics. The assembly of porins and the mechanism by which they are integrated into the outer membrane, however, are poorly understood. Here, we show that assembly can be completely reconstituted in vitro and requires only phospholipid vesicles containing the Bam complex, a molecular chaperone, and LPS. Furthermore, by showing that LPS binding is required for membrane insertion, our results demonstrate that a native lipid promotes a specific stage of porin biogenesis.