A Supercomplex Spanning the Inner and Outer Membranes Mediates the Biogenesis of β-Barrel Outer Membrane Proteins in Bacteria

Abstract

␤-barrel outer membrane proteins (OMPs) are ubiquitously present in Gram-negative bacteria, mitochondria and chloroplasts, and function in a variety of biological processes. The mechanism by which the hydrophobic nascent ␤-barrel OMPs are transported through the hydrophilic periplasmic space in bacterial cells remains elusive. Here, mainly via unnatural amino acid-mediated in vivo photo-crosslinking studies, we revealed that the primary periplasmic chaperone SurA interacts with nascent ␤-barrel OMPs largely via its N-domain but with ␤-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent ␤-barrel OMPs interact with SurA via their N-and C-terminal regions. Additionally, via dual in vivo photo-crosslinking, we demonstrated the formation of a ternary complex involving ␤-barrel OMP, SurA, and BamA in cells. More importantly, we found that a supercomplex spanning the inner and outer membranes and involving the BamA, BamB, SurA, PpiD, SecY, SecE, and SecA proteins appears to exist in living cells, as revealed by a combined analyses of sucrose-gradient ultra-centrifugation, Blue native PAGE and mass spectrometry. We propose that this supercomplex integrates the translocation, transportation, and membrane insertion events for ␤-barrel OMP biogenesis.

Mitochondrial-bacterial hybrids of BamA/Tob55 suggest variable requirements for the membrane integration of β-barrel proteins

“…In the absence of such a domain, SurA can bind a substrate protein but does not release it for further assembly into the membrane and hence altogether has a rather inhibiting effect. This assumption is supported by a recent report describing a ternary complex formed by BamA (mainly via POTRA domain 2), SurA, and β-barrel nascent protein35. Our observations are also in line with previous works suggesting that the individual or combinatorial deletion of periplasmic chaperones or non-essential Bam components differentially affect the assembly of various groups of β-barrel proteins, proposing that there are classes of substrates with variable dependencies for efficient assembly363738.…”

“…In contrast, SurA was suggested to interact with POTRA domains of BamA45, and it was proposed that secretion of the passenger domain of autotransporter proteins might involve a stepwise transfer of polypeptide segments from SurA to the POTRA domains46. Supporting this notion is a recent report describing the detection of a ternary complex formed by BamA (via POTRA domain 2), SurA, and β-barrel substrate protein35. Hence, we hypothesize that in the mitochondrial system SurA might still bind its substrate proteins.…”

Profiling the E. coli Membrane Interactome Captured in Peptidisc Libraries

“…Interactions between SecYEG and the BamA, BamC and BamD subunits in particular are present in our interaction list at high precision. This Sec-Bam interaction has been suggested previously based on indirect evidence from membrane fractionation experiments and Western blotting (Wang et al 2016). To further confirm these interactions, the SecYEG complex was trapped in peptidisc and analyzed following the SILAC AP/MS workflow (Fig.…”

Global landscape of cell envelope protein complexes in Escherichia coli

“…Similarly, interactions between the periplasmic hydrogenase 2 small subunit (HybO) and cytoplasmic fumarate reductase (FrdB) suggest coordination in substrate reduction (such as quinones) during anaerobic respiration 29 . The β-barrel assembly OMP (BamA) and OM lipoproteins (BamCDE) also co-purified with the IM Sec translocon (SecDFGY), consistent with a primary role in β-barrel translocation, transport and membrane insertion OMP biogenesis 30 .…”