Membrane insertion of NuoK is affected by YidC and SecYEG levels in the membrane. A, membrane topology of NuoK indicating the glutamate residues in TMSs 2 and 3. B and C, in vitro synthesis of NuoK were carried out at 37 °C for 30 min in the presence of 10 μg of IMVs. After the synthesis reaction, the membranes were collected through a sucrose cushion and resuspended in 50 mm HEPES-KOH, pH 8. Proteinase K was added to 1.6 mg/ml in the absence (lanes 2 and 5) or presence of SDS (lanes 3 and 6). The standards of 10% of the synthesis reactions are shown (lanes 1 and 4). The synthesis reactions were performed in the presence of YidC⁺ or YidC⁻ IMVs (B) or wild type (WT) or SecYEG⁺ IMVs (C). D, NuoK insertion was quantified from the amount of protease-protected material in the presence of IMVs. The background signal of proteinase K-resistant NuoK observed when no liposomes were present was deducted from the data. All of the data points shown are the averages of three independent experiments. The bars indicate the standard errors of the mean. MW, molecular mass.

Insertion of NuoK is stimulated in the absence of a PMF. Upper panel, insertion assays with YEG+ IMVs were performed as described in the legend to Fig. 1 in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 3 μm nigericin/valinomycin (nig/val) to dissipate the PMF. As a control, insertion assays with in vitro synthesized FtsQ were performed (lower panel) in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 3 μm nigericin/valinomycin. The 10% standards of the synthesis reactions are shown (lanes 1 and 2). MW, molecular mass.

NuoK associates with YidC. E. coli FTL10 was transformed with plasmids pEH1YidC and pTrcYidC, allowing the overexpression of His-tagged YidC (lane 2) and nontagged YidC (lane 1), respectively. Overexpression was performed under anaerobic growth conditions in media containing fumarate and glycerol. IMVs were isolated and solubilized in 2% N-dodecyl-β-d-maltoside whereupon YidC was purified by Ni²⁺-NTA chromatography. Elution fractions were run on SDS-PAGE, blotted onto polyvinylidene difluoride membrane, and probed with antibodies against NuoK. MW, molecular mass.

Both YidC and SecYEG are essential for NuoK insertion into proteoliposomes. A, NuoK insertion reactions were performed in the presence (lanes 1–4 and 6–9) or absence (lanes 5 and 10) of (proteo)liposomes. For the proteoliposomes, E. coli lipids were reconstituted with YidC and SecYEG at protein/lipid ratios of 0.125 and 0.055 (w/w), respectively. Insertion reactions (lanes 6–10) were performed by proteinase K digestion essentially as described in the legend to Fig. 1. The 5% standards of the synthesis reactions are shown (lanes 1–6). B, NuoK insertion was quantified from the amount of protease-protected material in the presence and absence of (proteo)liposomes. All of the data points shown are the averages of three independent experiments. The bars indicate the standard errors of the mean. MW, molecular mass.

Mutation of glutamates at positions 36 and 72 to lysines renders NuoK independent of YidC for insertion. A, insertion reactions were performed as described in the legend of Fig. 1. Synthesis reactions were performed in the presence of YidC⁺ or YidC⁻ IMVs, and proteinase K was added in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of SDS. Synthesis reactions were performed with plasmids containing single mutants NuoK E36K (top panel) and NuoK E72K (middle panel) and the double mutant NuoK E36K,E72K (bottom panel). The 10% standards of the synthesis reactions are shown (lanes 1 and 4). B, NuoK insertion was quantified from the amount of protease-protected material in the presence of YidC⁺ (indicated in white) and YidC⁻ (indicated in dark gray) IMVs. All of the data points shown are the averages of three independent experiments. The bars indicate the standard errors of the mean. C, NuoK insertion reactions were performed in the presence (lanes 1–4 and 6–9) or absence (lanes 5 and 10) of (proteo)liposomes reconstituted with or without purified SecYEG and YidC as indicated. The insertion reactions (lanes 6–10) were performed essentially as described in the legend to Fig. 1 using plasmids containing single substitutions NuoK E36K (top panel) and NuoK E72K (middle panel) and double substitutions NuoK E36K,E72K (bottom panel). 5% standards of the synthesis reactions are shown (lanes 1–5). D, NuoK insertion was quantified from the amount of protease-protected material in the presence and absence of (proteo)liposomes. The background signal of proteinase K-resistant NuoK observed when no liposomes were present was deducted from the data. All of the data points shown are averages of three independent experiments. The bars indicate the standard errors of the mean.