Role of the twin arginine protein transport pathway in the assembly of the Streptomyces coelicolor cytochrome bc1 complex

Abstract

The cytochrome bc1-cytochrome aa3 complexes together comprise one of the major branches of the bacterial aerobic respiratory chain. In actinobacteria, the cytochrome bc1 complex shows a number of unusual features in comparison to other cytochrome bc1 complexes. In particular, the Rieske iron-sulfur protein component of this complex, QcrA, is a polytopic rather than a monotopic membrane protein. Bacterial Rieske proteins are usually integrated into the membrane in a folded conformation by the twin arginine protein transport (Tat) pathway. In this study, we show that the activity of the Streptomyces coelicolor M145 cytochrome bc1 complex is dependent upon an active Tat pathway. However, the polytopic Rieske protein is still integrated into the membrane in a ΔtatC mutant strain, indicating that a second protein translocation machinery also participates in its assembly. Difference spectroscopy indicated that the cytochrome c component of the complex was correctly assembled in the absence of the Tat machinery. We show that the intact cytochrome bc1 complex can be isolated from S. coelicolor M145 membranes by affinity chromatography. Surprisingly, a stable cytochrome bc1 complex containing the Rieske protein can be isolated from membranes even when the Tat system is inactive. These findings strongly suggest that the additional transmembrane segments of the S. coelicolor Rieske protein mediate hydrophobic interactions with one or both of the cytochrome subunits.

FIG 1

The major aerobic respiratory components in S. coelicolor M145 as deduced from the genome sequence. (A) Two homologues of a Nuo-type NADH dehydrogenase are encoded by S. coelicolor. The genome also reveals two potential routes to the oxygen-dependent reoxidation of quinol, via the cytochrome bc₁-cytochrome aa₃ oxidase or through a higher-affinity cytochrome bd oxidase. (B) Schematic representation of the three subunits of the S. coelicolor cytochrome bc₁ complex. Domains that show homology to the related components from mitochondria and Gram-negative bacteria are shown in gray. Domains that appear unique to the actinobacteria are shown in white. FeS, iron sulfur cluster; b, b-type heme; c, c-type heme; in, cytoplasm; out, cell exterior.

FIG 2

Comparison of the S. coelicolor M145 and ΔqcrCAB and ΔtatC mutant strains. (A) Growth of S. coelicolor M145 (WT), APH2 (ΔqcrCAB), and TP4 (ΔtatC) on SFM or YEME agar plates. Spores of each strain were streaked and incubated at 30°C for 14 days. (B) Growth in liquid culture of the same strains used in the experiments whose results are shown in panel A. An amount of 10⁸ spores of each strain, in triplicate, was inoculated into 100 ml TSB and grown at 30°C for 40 h with shaking at 200 rpm. Every 3 h, 1-ml samples were pelleted, cytosolic proteins released by boiling in 1 M NaOH for 10 min, and the protein content of each sample measured. (C) Absorption difference spectra of DDM-solubilized membranes (each at a protein concentration of 1.25 mg/ml) of strains M145 (WT), APH2 (ΔqcrCAB), and APH8 (ΔtatC ϕC31 [P_hrdB-qcrCAB_His10]). Spectra were initially collected under air oxidation, and then samples were reduced by the addition of dithionite. Note that the relevant genotype for each strain is given in the figure. (D) Expanded view of the 590- to 610-nm region of the same absorption difference spectra as shown in panel C.

FIG 3

The Rieske protein, QcrA, is integrated into S. coelicolor M145 membranes in the absence of the Tat machinery. Membrane fractions were prepared from hyphae of strains M145 and TP4 (Tat + and −, respectively) (A) and strain APH5-KK (ΔqcrCAB::Hyg^r ϕC31 P_hrdB-qcrCA_{R161K R162K}B_His10, producing a QcrA protein with the conserved twin arginines mutated to twin lysines [QcrA_KK]) (B) as described in Materials and Methods. The membranes were incubated with either 0.2 M Na₂CO₃ or 4 M urea, followed by recovery of the membrane pellet by ultracentrifugation. The presence of QcrA in the wash supernatant (S) and pelleted membrane (P) was analyzed by immunoblotting using anti-QcrA antiserum.

FIG 4

Copurification of the Rieske protein, QcrA, with decahistidine-tagged cytochrome b. Washed membrane fractions (150 mg protein) of the wild-type strain M145 or strain APH5 (ΔqcrCAB::hyg ϕC31 [P_hrdB-qcrCAB_His10]) were solubilized in the presence of 1% DDM. The solubilized membranes were incubated with Ni²⁺-charged NTA resin, and the unbound fraction was collected. Bound proteins were washed successively in buffer containing 50, 250, or 500 mM imidazole. Samples (3 μl of each indicated fraction) were separated by SDS-PAGE (14% acrylamide) and electroblotted, and the presence of QcrA or His-tagged QcrB was detected with anti-QcrA or anti-His tag antiserum, respectively.

FIG 5

The cytochrome bc₁ complex is stably assembled in both a Tat⁺ and ΔtatC mutant strain. Solubilized membranes from 2 g of cells of strain APH5 (ΔqcrCAB P_hrdB-qcrCAB_His10) (A) or strain APH8 (ΔtatC ΔqcrCAB::hyg ϕC31 [P_hrdB-qcrCAB_His10]) (E) were loaded onto a 5-ml Ni²⁺-charged NTA column and washed, and bound protein was eluted with a gradient of 30 to 500 mM imidazole (indicated by the sloping line). Fractions indicated by the arrows were analyzed by Western blotting. mAU, milli-absorbance unit. (B and C) Ten-microliter samples (from a total of 1 ml) of fractions 1 to 3 from the experiment whose results are shown in panel A. (F and G) Ten-microliter samples (from a total of 1 ml) of fractions 1 to 3 from the experiment whose results are shown in panel E. The samples were separated by SDS-PAGE (14% acrylamide), electroblotted, and incubated with anti-QcrA (B and F) or anti-His tag antibodies (C and G). The asterisk in panels B and F indicates a cross-reacting band that is recognized by the anti-QcrA antiserum, which may be His-tagged QcrB since the polyclonal QcrA antiserum was raised against a His-tagged QcrA (12). (D and H) The fractions numbered 2 in panels A and E, respectively, were concentrated from 8 ml to 150 μl, and 20 μl of each sample was analyzed by SDS-PAGE and silver staining. The identity of the indicated protein bands was confirmed by tryptic mass fingerprinting.

FIG 6

Models showing the predicted organization of the cytochrome bc₁ complex components in a tat⁺ and a Δtat strain. White oblong, heme group; black diamond, 2Fe 2S cluster; RR, twin arginines of the Tat recognition motif; in, cytoplasm; out, cell exterior.