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. 2006 Nov 15;25(22):5241-9.
doi: 10.1038/sj.emboj.7601402. Epub 2006 Nov 2.

Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin

Ingrid Guilvout  1 , Mohamed Chami, Andreas Engel, Anthony P Pugsley, Nicolas Bayan
Affiliations

Bacterial outer membrane secretin PulD assembles and inserts into the inner membrane in the absence of its pilotin

Ingrid Guilvout et al. EMBO J. .

Abstract

Dodecamerization and insertion of the outer membrane secretin PulD is entirely determined by the C-terminal half of the polypeptide (PulD-CS). In the absence of its cognate chaperone PulS, PulD-CS and PulD mislocalize to the inner membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD-CS purified from the inner membrane revealed apparently normal dodecameric complexes. Electron microscopy of PulD-CS and PulD in inner membrane vesicles revealed inserted secretin complexes. Mislocalization of PulD or PulD-CS to this membrane induces the phage shock response, probably as a result of a decreased membrane electrochemical potential. Production of PulD in the absence of the phage shock response protein PspA and PulS caused a substantial drop in membrane potential and was lethal. Thus, PulD-CS and PulD assemble in the inner membrane if they do not associate with PulS. We propose that PulS prevents premature multimerization of PulD and accompanies it through the periplasm to the outer membrane. PulD is the first bacterial outer membrane protein with demonstrated ability to insert efficiently into the inner membrane.

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Figures

Figure 1
Figure 1
SDS–PAGE analysis of PulDhis and PulD-CShis purified by cobalt affinity chromatography from envelopes of E. coli cells with and without PulS or PulShis. The strains used were PAP105(pCHAP585) (with PulS) or PAP5198 (without PulS). PulDhis and PulD-CShis multimers ((PulDhis)12 and (PulD-CShis)12, respectively) were dissociated into monomers (PulDhis and PulD-CShis) with phenol. Proteins were separated on a 4–15% acrylamide gradient gel and stained with Coomassie blue. The positions of molecular size markers (kDa) are shown on the left. Arrows indicate the multimeric forms of PulDhis and PulD-CShis. Note that PulDhis and PulD-CShis migrate more slowly than expected from their mass (68.7 and 45.6 kDa) in this electrophoresis system.
Figure 2
Figure 2
Electron microscopy of purified PulD-CShis and PulDhis particles negatively stained with uranyl acetate. (A) PulD-CShis purified from membranes of E. coli PAP105 with PulS; inset, averaged images of two major classes: top views (n=171) and side views (n=233). (B) PulD-CShis purified from membranes of E. coli PAP5198 without PulS; inset, averaged images of two major classes: top views (n=177) and side views (n=300). (C) PulDhis purified from E. coli PAP5198 without PulS; inset, averaged images of two major classes: top views (n=272) and side views (n=253). The scale bar corresponds to 50 nm and the inset baseline corresponds to 25 nm.
Figure 3
Figure 3
Inner membrane localization of PulD (upper panel) and PulD-CS (lower panel) multimers produced in the absence of PulS and DegP detected by separating membranes in sucrose floatation gradients. Fractions from the gradients were loaded onto 7.5 or 10% acrylamide–SDS gels and stained with Coomassie blue (note that PulD-CS multimers were detected using the 7.5% acrylamide gel whereas PulD multimers were detected in the stacking gel). SecG was detected by immunoblotting with specific antibodies. The outer membrane fractions contain proteins OmpF, OmpC and OmpA, whereas inner membrane fractions contain PspA and SecG. The band migrating close to the position of PspA but in the outer membrane fraction is not PspA.
Figure 4
Figure 4
Electron microscopy of inner membrane vesicles purified from protease-deficient E. coli producing PulD (A) or PulD-CShis (B) without PulS. Arrows indicate ring-like particles corresponding to secretin particles. The scale bar corresponds to 100 nm.
Figure 5
Figure 5
Extraction of PulD-CShis, PspA and SecG with urea. Samples were treated with phenol and then subjected to SDS–PAGE and immunoblotting with specific antibodies. Samples were derived from the same amount of starting material. Size markers (kDa) are indicated on the left.
Figure 6
Figure 6
Induction of PspA caused by production of PulD or PulD-CS in the absence of PulS. Proteins from pooled inner membrane fractions (Supplementary Figure S1) were separated by SDS–PAGE and immunoblotted with PspA-specific antibodies.
Figure 7
Figure 7
PspA induction by PulD derivatives with linker insertions in the C or S domains. Total cell extracts were analysed by SDS–PAGE with or without phenol treatment. Immunoblots were developed with antibodies against PulD and PspA. All extracts are derived from the same amount of starting material.
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