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

Abstract

β-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 β-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 β-barrel OMPs largely via its N-domain but with β-barrel assembly machine protein BamA mainly via its satellite P2 domain, and that the nascent β-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 β-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 β-barrel OMP biogenesis.

Keywords: BamA; SurA; chaperone; complex; membrane biogenesis; membrane protein; protein folding; the Sec translocon; β-barrel outer membrane protein biogenesis.

FIGURE 1.

Identification of amino acid residues in SurA that are involved in interacting with β-barrel OMPs and BamA by Bpa-mediated in vivo photo-crosslinking. A–C, representative results of immunoblotting analyses for the in vivo photo-crosslinking products of the 58 Bpa-incorporated variants of SurA, using antibodies against OmpF (A), LamB (B), or BamA (C). The immunoblotting results of photo-crosslinking products for all the 58 Bpa variants of SurA that are displayed in supplemental Figs. S1 (probing OmpF), S2 (probing LamB), S3 (probing BamA), and S4 (probing SurA). It should be noted that the photo-crosslinked SurA-OmpF was assayed using the Ni-NTA affinity-purified SurA-Q35Bpa protein sample, while the photo-crosslinked SurA-LamB and SurA-BamA were directly assayed using the cell lysate preparations (i.e. without purification). D, summary showing the involvement of each of the 58 Bpa variants of SurA in interacting with OmpF, LamB, and BamA, with the localization of each Bpa-substituted position in the four domains (N-domain, P1 domain, P2 domain, and C-domain) of SurA indicated. A detected interaction is indicated by a red “+” sign, while a lack of interaction is indicated by a black “−” sign. E, crystal structure (PDB: 1M5Y) of SurA presented in the “surface” mode, with its four domains colored in blue (for the N-domain), green (for the P2 domain), and gray (for the P1 domain and C-domain).

FIGURE 2.

Nascent β-barrel OMPs directly interact with SurA via their N- and C-terminal regions. A and B, immunoblotting results of the in vivo photo-crosslinking products for the 17 Bpa variants of OmpF (panel A) and the 19 Bpa variants of LamB (panel B). Immunoblotting was performed using the anti-SurA antibody. Monomeric SurA, photo-crosslinked OmpF-SurA, and photo-crosslinked LamB-SurA are indicated. Cells expressing the wild-type OmpF (with no Bpa incorporation) were analyzed as a negative control (lanes 1 and 2 in panel A). The residue positions of Bpa insertion in both OmpF and LamB are numbered by referring to the mature protein (not the precursor protein).

FIGURE 3.

An OmpF-SurA-BamA ternary complex was detected by dual photo-crosslinking. A, immunoblotting results of the in vivo photo-crosslinking products for the BamA-K135Bpa variant, using the anti-SurA antibody. Cells were either untreated (lanes 1 and 2) or treated with chloramphenicol (a protein synthesis inhibitor) for 20 (lanes 3 and 4) or 40 (lanes 5 and 6) min before the in vivo photo-crosslinking. B, immunoblotting analysis for the photo-crosslinked OmpF-SurA-BamA ternary complex in cells co-expressing the BamA-K135Bpa and OmpF-Y32Bpa variant proteins. The immunoblotting was performed using antibodies against BamA (left) or SurA (right). The wild-type OmpF (with no Bpa incorporation) was also examined as a negative control (lanes 1, 2, 5, and 6). The positions of the photo-crosslinked OmpF-SurA-BamA, OmpF-SurA, and SurA-BamA complexes are indicated to the right of the gel.

FIGURE 4.

SurA is not effectively released from the periplasm by osmotic shock treatment. A, immunoblotting results probing the presence of SurA, MalE, DegP, OmpF, or GroEL in the supernatant (S) and pellet (P) fractions of the wild type cells treated by osmotic shock. The treated cells were centrifuged to separate the two fractions, resolved by SDS-PAGE and probed with antibodies against the indicated proteins. B, relative amounts of the indicated proteins released in the supernatant and remained in the pellet, being quantified (mean ± S.E.; n = 3) from the immunoblotting results shown in panel A.

FIGURE 5.

Distribution of the BamA, BamB, SurA, PpiD, SecY, SecE, or SecA protein in the inner and outer membrane fractions of the wild type, ΔsurA-, or BamA-depleted cells and the detection of a “supercomplex.” A–C, immunoblotting analysis for SurA, BamA, SecY, SecE, PpiD, SecA, or BamB in the inner and outer membrane fractions prepared from the wild-type (A), the ΔsurA (B), or the BamA-depleted (C) cells, with the membrane fractions resolved by sucrose density gradient centrifugation. The relative amounts of the indicated protein in the inner and outer membrane fractions of the three types of cells were quantified (mean ± S.E.; n = 3) from the immunoblotting results and displayed below the corresponding gels in each panel. The α-subunit of ATP synthase and OmpF were analyzed as respective internal protein markers for the inner and outer membranes. D, results of immunoblotting against BamA, OmpA, or OmpF in cells whose chromosome-encoded BamA protein was depleted via a Bpa-controlled strategy (i.e. by subculturing the cells in Bpa-free LB medium, for details see “Experimental Procedures”). E, immunoblotting analysis probing the indicated proteins (BamA, BamB, SurA, PpiD, SecY, SecE, and SecA) in the inner membrane fraction (taken from fraction No. 8, see panel A) prepared from the wild-type or BamA-depleted cells, the membrane sample was resolved by Blue Native PAGE before probed with the indicated antibodies. The molecular mass of the “supercomplex” band was estimated to be ∼400 kDa through regression analysis based on the gel mobility of the “supercomplex” and the molecular size markers.

FIGURE 6.

SurA directly interacts with PpiD as a functional partner. A, immunoblotting results of the in vivo photo-crosslinking products for two representative Bpa variants of SurA, using antibodies against PpiD. The complete immunoblotting results for all the 58 Bpa variants of SurA are displayed in supplemental Fig. S8. B, summary showing the involvement of each of the 58 Bpa substituted positions of SurA in interacting with PpiD, with the localization of each position in the four domains (N-domain, P1 domain, P2 domain, and C-domain) of SurA indicated. A detected interaction is indicated by a red “+” sign, while a lack of interaction is indicated by a black “−” sign. C, immunoblotting analysis of the photo-crosslinked product of the SurA-Q35Bpa variant in cells whose cellular protein synthesis process was suppressed by treating with the antibiotics chloramphenicol for 40 min before the in vivo photo-crosslinking was performed (lanes 3 and 4). D, immunoblotting analysis of the photo-crosslinked SurA-PpiD in the inner and outer membrane fractions. The inner and outer membrane fractions were separated by sucrose density gradient centrifugation and resolved by SDS-PAGE before probed with the indicated antibodies. The α-subunit of ATP synthase and OmpF were analyzed as respective markers of the inner and outer membranes.

FIGURE 7.

The supercomplex model for the biogenesis of β-barrel OMPs (A), in comparison with the conventional “segregated” model (B). Our model emphasizes the key role of a supercomplex that spans the inner and outer membranes and integrates the translocation, transportation, and membrane insertion events for the biogenesis of β-barrel OMPs in living cells. For comparison, the conventional model depicts SurA as a chaperone that is freely diffusible in the periplasm.