Table 1.
Genotype and sources of P. denitrificans strains.
Fig 1.
Two-dimensional BN/SDS-PAGE and Western blot analysis of COX assembly complexes.
140 μg membranes prepared from P. denitrificans cells from strains (A) WT, (B) MR31 (ΔctaDI/DII), (C) FA2 (Δsurf1c) were solubilized with digitonin at a detergent/protein ratio of 4 g/g and subjected to a first dimension 3.5–18% gradient BN-PAGE. A gel strip was excised, soaked in 1% SDS and placed on top of a 10% SDS Tricine gel for separation in the second dimension. Proteins were transferred to a nitrocellulose membrane and analyzed by Western blotting with polyclonal antibodies directed against COX (recognizing SUI and SUII), CtaG, Surf1c or CtaA. To fully ascertain the positional alignment between different Western blots, critical blots were either re-stained with a second antibody of interest, or initially developed with a mixture of different antibodies (not shown). The approximate molecular masses have been assigned based on a molecular weight marker control and a previous mass estimation for molecular complexes in P. denitrificans [49]. The positions of the COX SUI subassemblies I1-4 as well as the three supercomplexes Sa-c (nomenclature as in [49]) are indicated. In (A) the dotted line a shows COX SUI (62.4 kDa) and CtaG (21.4 kDa; known to migrate as double band on SDS PAGE [33]) on a vertical, whereas line b marks co-migration of COX SUI, SUII (28 kDa), CtaG, Surf1c (24.6 kDa) and less prominently of CtaA (41.8 kDa); in panel B these assembly complexes are not observed, however the position of lines a and b is maintained as in (A) and (C) for comparison purposes.
Fig 2.
Pull-down assays indicating interactions between COX, Surf1c and CtaG.
Interactions between COX, Surf1c, CtaG and CtaA were assayed by immunoprecipitation and subsequent Western blot analysis. Solubilized membranes from untreated (top) or crosslinked cells (bottom), including WT and deletion strains FA2 (Δsurf1c) and MR31 (ΔctaDI/DII), were used for Co-IP experiments with protein A/G magnetic beads, as described in the experimental procedures. Eluates were separated by 12% SDS PAGE, blotted and probed against Surf1c, CtaA, COX SUI, COX SUII and CtaG. As bait antibody anti-COX (recognizing SUI and II), anti-CtaG, anti-Surf1c, anti-CtaA or a control serum was employed. The hashtag indicates a non-reproducible interaction and the asterisk designates unspecific staining (different molecular weight bands compared to control).
Fig 3.
Heat map illustration of COX subunits and biogenesis factors identified by complexome profiling.
140 μg of membranes prepared from WT (top panel) or oxidase SUI double deletion strain MR31 (ΔctaDI/DII; lower panel) were solubilized with detergent (digitonin:protein 2:1), separated by BN-PAGE and subsequently analyzed by quantitative MS. The position of supercomplexes (Sa-c) and COX SUI assembly intermediates (I1-4) is indicated. For each identified protein, the data are normalized to maximum appearance over all 60 gel slices in each sample.
Fig 4.
Migration profiles of COX and metallochaperones CtaG and Surf1c obtained by complexome profiling.
One-dimensional BN-PAGE of digitonin-solubilized membranes from P. denitrificans was analyzed by MS-based complexome profiling. Each panel shows the relative abundance of identified proteins plotted against the apparent molecular mass after BN-PAGE separation. Corresponding heat maps are shown in Fig 3. All profiles are presented as indicated in the legend. (A) Migration profiles of all four COX subunits in a WT background are shown and the positions of subcomplexes I1-4 and supercomplexes Sa-c are indicated. (B) Comparison of CtaG migration pattern in membranes prepared from WT or strain MR31, deleted in both COX SUI gene loci, together with the COX SUI WT migration profile. (C) The migration profile of Surf1c is shown for WT and oxidase SUI deletion strain MR31 together with the migration pattern of WT COX SUI.
Fig 5.
Proposed model of the sequential COX assembly pathway in P. denitrificans.
COX subunits, respiratory chain complexes and assembly factors are depicted schematically. Direct interactions confirmed by our data are indicated by solid arrows, and presumed interactions by dashed arrows, respectively. Newly translated COX SUI interacts with copper chaperone CtaG to form subcomplex I1. This complex evolves into subcomplex I2, characterized by an extra mass X of approximately 40 kDa. Formation of subcomplex I2 is followed by the interaction with Surf1c. Direct interactions between Surf1c and CtaA as well as between CtaB and CtaA are indicated by dashed arrows. After association of Surf1c, COX SUII and SUIII are recruited, leading to formation of subcomplex I3. Addition of SUIV leads to formation of the holoenzyme complex, which subsequently associates with subunits of respiratory chain complexes III and I, passing through the supercomplex assembly intermediate I4 and ultimately ends up in supercomplexes Sc, Sb and Sa.