A conserved function of YidC in the biogenesis of respiratory chain complexes - PubMed (original) (raw)

A conserved function of YidC in the biogenesis of respiratory chain complexes

M van der Laan et al. Proc Natl Acad Sci U S A. 2003.

Abstract

The Escherichia coli inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase. YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins. Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC. The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force. This reduction is due to defects in the functional assembly of cytochrome o oxidase and the F(1)F(o) ATPase complex, which is reminiscent of the effects of mutations in the yidC homologue OXA1 in the yeast mitochondrial inner membrane. The integration of CyoA (subunit II of the cytochrome o oxidase) and F(o)c (membrane subunit of the F(1)F(o) ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.

PubMed Disclaimer

Figures

Figure 1

YidC depletion induces PspA expression. IMVs were isolated from JS7131 cells depleted of YidC (YidC↓) for 3.5 h or grown under control conditions (WT) as described in Materials and Methods. IMVs (10 μg) were analyzed by SDS/PAGE and stained with Coomassie brilliant blue (A) or immunostained with anti- (α)YidC (B Upper) or αPspA (B Lower). (A, ♦) The ≈25-kDa protein PspA. (C) Effects of YidC depletion on the expression of stress-related proteins. Cells were depleted for 1.5, 2.5, 3.5, and 5 h or grown under control conditions; 0.05 OD660 unit of cells was analyzed by immunoblotting using antibodies against YidC, PspA, DnaK, SecA, and DegP. (D) Effects of YidC depletion on the expression of translocase components. IMVs were isolated from cells depleted of YidC for 1.5, 2.5, and 3.5 h; 10 μg of protein was analyzed by SDS/PAGE and immunoblotting with antibodies against YidC, SecY, SecE, SecD, and SecF.

Figure 2

YidC depletion affects the PMF. (A) Cells were grown as described in Materials and Methods. After 1.5, 2.5, or 3.5 h of depletion, cells were harvested and pretreated, and TPP+ uptake was measured as described in Materials and Methods. The WT signals are shown in black and the YidC↓ signals are shown in gray. (B) Cells were grown overnight in the presence of 0.2% arabinose, washed, and diluted in LB medium containing no sugars, 0.1% glucose, or arabinose. After 3.5 h, the cells were harvested and treated as described above. (C) Cell samples of B were taken and analyzed by SDS/PAGE and Western blotting with antibodies against YidC and PspA.

Figure 3

YidC-depleted IMVs are defective in PMF generation. JS7131 IMVs were prepared from cultures grown for 1.5 (A and D), 2.5 (B and E), or 3.5 (C and F) h on 0.1% glucose (YidC↓, dotted line) or arabinose (WT, continuous line). The generation of ΔpH was determined by monitoring the fluorescence quenching of ACMA. Where indicated, 1 mM ATP (A–C) or 1.25 mM NADH (D–F) was added to the IMVs to generate a PMF. Valinomycin was used at 0.75 μM to convert the generated Δψ into a ΔpH. Subsequently, the ΔpH was dissipated by the addition of 1.5 μM nigericin.

Figure 4

Depletion of YidC leads to a decreased amount of functional F1Fo ATPase complex. JS7131 IMVs were prepared from cultures grown for 1.5, 2.5, or 3.5 h on 0.1% glucose (black bars, YidC↓) or arabinose (white bars, WT). (A) Total ATPase activity of IMVs. The data points are the average of three independent measurements. (B) SDS/PAGE and immunoblot analysis of the Fob and Foc subunit levels in IMVs.

Figure 5

Depletion of YidC leads to a decreased activity of the respiratory chain. JS7131 IMVs were prepared from cultures grown for the indicated times on 0.1% glucose (YidC↓, black bars) or arabinose (WT, white bars). (A) NADH consumption was monitored photometrically as decrease of absorption at 340 nm. (B) NADH dehydrogenase activities were determined by using Triton X-100-solubilized IMVs and the artificial electron acceptor DCIP. NADH-dependent reduction of DCIP was monitored photometrically as decrease of absorption at 600 nm. (C) Cytochrome o oxidase activities were determined by using the artificial electron donor system phenazine methosulfate/ascorbate. Phenazine methosulfate/ascorbate-dependent O2 consumption was measured by using an oxygen electrode. The data points are the average of three independent measurements.

Figure 6

Depletion of YidC leads to a decreased amount of functional cytochrome o oxidase complex. JS7131 IMVs were prepared from cultures grown for 1.5, 2.5, or 3.5 h on 0.1% glucose (YidC↓) or arabinose (WT). Difference spectra (dithionite-reduced minus air-oxidized) of YidC-depleted (3.5 h on 0.1% glucose, dotted line) or WT (3.5 h on 0.1% arabinose, continuous line) IMVs were recorded. (Inset) IMVs were analyzed by SDS/PAGE and immunostaining with antibodies against cytochrome o oxidase.

References

1. Herskovits A A, Bochkareva E S, Bibi E. Mol Microbiol. 2000;38:927–939. - PubMed
1. Manting E H, Driessen A J M. Mol Microbiol. 2000;37:226–238. - PubMed
1. Scotti P A, Urbanus M L, Brunner J, de Gier J W, von Heijne G, van der Does C, Driessen A J M, Oudega B, Luirink J. EMBO J. 2000;19:542–549. - PMC - PubMed
1. Nouwen N, Driessen A J M. Mol Microbiol. 2002;44:1397–1405. - PubMed
1. Kuhn A. FEMS Microbiol Rev. 1995;17:185–190. - PubMed

A conserved function of YidC in the biogenesis of respiratory chain complexes - PubMed (original) (raw)