Dataset Information

Bam complex-mediated assembly of bacterial outer membrane proteins synthesized in an in vitro translation system.

ABSTRACT: Bacterial outer membrane proteins (OMPs) contain a unique "? barrel" segment that is inserted into the membrane by the barrel assembly machinery (Bam) complex by an unknown mechanism. OMP assembly has been reconstituted in vitro, but assembly reactions have involved the use of urea-denatured protein purified from inclusion bodies. Here we show that the E. coli Bam complex catalyzes the efficient assembly of OMPs synthesized de novo in a coupled in vitro transcription/translation system. Interestingly, the in vitro translated forms of the OMPs we analyzed were assembled more rapidly and were effectively engaged by fewer periplasmic chaperones than their urea-denatured counterparts. Taken together, our results strongly suggest that the mode of production influences the conformational states sampled by OMPs and thereby affects their recognition by both chaperones and the Bam complex. Besides providing insights into OMP biogenesis, our work describes a novel, streamlined method to reconstitute OMP assembly in vitro.

SUBMITTER: Hussain S

PROVIDER: S-EPMC7067875 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Bam complex-mediated assembly of bacterial outer membrane proteins synthesized in an in vitro translation system.

Hussain Sunyia S Peterson Janine H JH Bernstein Harris D HD

Scientific reports 20200312 1

Bacterial outer membrane proteins (OMPs) contain a unique "β barrel" segment that is inserted into the membrane by the barrel assembly machinery (Bam) complex by an unknown mechanism. OMP assembly has been reconstituted in vitro, but assembly reactions have involved the use of urea-denatured protein purified from inclusion bodies. Here we show that the E. coli Bam complex catalyzes the efficient assembly of OMPs synthesized de novo in a coupled in vitro transcription/translation system. Interest ...[more]

PMID: 32165713

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Project description:Membrane proteins that are integrated into the outer membrane of Gram-negative bacteria typically contain a unique "β barrel" structure that serves as a membrane spanning segment. A conserved "β signal" motif is located at the C terminus of the β barrel of many outer membrane proteins (OMPs), but the function of this sequence is unclear. We found that mutations in the β signal slightly delayed the assembly of three model Escherichia coli OMPs by reducing their affinity for the barrel assembly machinery (Bam) complex, a heterooligomer that catalyzes β barrel insertion, and led to the degradation of a fraction of the protein in the periplasm. Interestingly, the absence of the periplasmic chaperone SurA amplified the effect of the mutations and caused the complete degradation of the mutant proteins. In contrast, the absence of another periplasmic chaperone (Skp) suppressed the effect of the mutations and considerably enhanced the efficiency of assembly. Our results reveal the existence of two parallel OMP targeting mechanisms that rely on a cis-acting peptide (the β signal) and a trans-acting factor (SurA), respectively. Our results also challenge the long-standing view that periplasmic chaperones are redundant and provide evidence that they have specialized functions.IMPORTANCE Proteins that are embedded in the outer membrane of Gram-negative bacteria (OMPs) play an important role in protecting the cell from harmful chemicals. OMPs share a common architecture and often contain a conserved sequence motif (β motif) of unknown function. Although OMPs are escorted to the outer membrane by proteins called chaperones, the exact function of the chaperones is also unclear. Here, we show that the β motif and the chaperone SurA both target OMPs to the β barrel insertion machinery in the outer membrane. In contrast, the chaperone Skp delivers unintegrated OMPs to protein degradation complexes. Our results challenge the long-standing view that chaperones are functionally redundant and strongly suggest that they have specialized roles in OMP targeting and quality control.

Dataset Information

Bam complex-mediated assembly of bacterial outer membrane proteins synthesized in an in vitro translation system.

Publications

Bam complex-mediated assembly of bacterial outer membrane proteins synthesized in an in vitro translation system.

Similar Datasets

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