Role of the Twin Arginine Protein Transport Pathway in the Assembly of the Streptomyces coelicolor Cytochrome bc ₁ Complex

Abstract

The cytochrome bc 1 -cytochrome aa 3 complexes together comprise one of the major branches of the bacterial aerobic respiratory chain. In actinobacteria, the cytochrome bc 1 complex shows a number of unusual features in comparison to other cytochrome bc 1 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 bc 1 complex is dependent upon an active Tat pathway. However, the polytopic Rieske protein is still integrated into the membrane in a ⌬tatC 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 bc 1 complex can be isolated from S. coelicolor M145 membranes by affinity chromatography. Surprisingly, a stable cytochrome bc 1 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.

Cytochrome <b><i>bcc-aa3</i></b> Oxidase Supercomplexes in the Aerobic Respiratory Chain of <b><i>Streptomyces coelicolor</i></b> A3(2)

“…All three QcrB paralogs are predicted to have nine transmembrane helices [Sawers et al, 2016], in contrast to the usual eight found in other organisms [Iwata et al, 1998], and they share roughly 50% amino acid sequence identity. These QcrB paralogs have an additional 100-amino acid C-terminal extension, which is also commonly found within the actinobacteria Hopkins et al, 2014]. Preliminary RNA analyses suggest that transcripts of qcrB3 are present in spores [Falke and Sawers, unpubl.…”

“…All three subunits of the bcc complex (QcrCAB) of the actinobacteria carry modifications compared with the bc 1 complex of most other bacteria or mitochondria [Sakamoto et al, 2001]. The QcrB cytochrome b (QcrB) has nine transmembrane helices as opposed to the typical eight in the same subunit in other bacteria [Xia et al, 1997;Bott and Niebisch, 2003] and it has an approximate 100 amino acid-extension at its C-terminus [Hopkins et al, 2014]. Examination of the genome of S. coelicolor indicates that it has two additional copies of the qcrB gene [Bentley et al, 2002] and, although these gene products have currently no demonstrated function, they possibly represent cytochromes b that form alternative bcc complexes.…”

“…Examination of the genome of S. coelicolor indicates that it has two additional copies of the qcrB gene [Bentley et al, 2002] and, although these gene products have currently no demonstrated function, they possibly represent cytochromes b that form alternative bcc complexes. The Rieske iron-sulfur protein (QcrA) has three transmembrane helices, as opposed to the usual single helix [Hopkins et al, 2014] and the cytochrome c component (QcrC) is a diheme, membrane-anchored cytochrome [Niebisch and Bott, 2001;Sone et al, 2001]. The CtaC subunit of the aa 3 -type oxidase has an additional 30 amino acids, many of which are charged residues, and these have been suggested to mediate an interaction between QcrC of the bcc complex and the aa 3 oxidase [Sakamoto et al, 2001;Niebisch and Bott, 2003].…”

Bacterial Electron Transfer Chains Primed by Proteomics

“…The Sec system is assisted by the YidC protein, which correctly inserts TMHs (Dalbey, Kuhn, Zhu, & Kiefer, 2014). In contrast, in the 'twin-arginine translocation' (TAT) pathway proteins are taken across the lipid bilayer in a preassembled and prefolded manner (Cline, 2015;Fr€ obel, Rose, & Müller, 2012;Goosens, Monteferrante, & van Dijl, 2014a, 2014bGoosens et al, 2013;Hopkins, Buchanan, & Palmer, 2014;Robinson et al, 2011). The pathway is named after two adjacent arginines that typically are located about 20 positions after the translation start.…”

Co-factor Insertion and Disulfide Bond Requirements for Twin-arginine Translocase-dependent Export of the Bacillus subtilis Rieske Protein QcrA

“…1). Apart from QcrA of B. subtilis, other Rieske proteins also share a strong association with the Tat pathway as was demonstrated in various bacteria and chloroplasts of green plants (43)(44)(45)(46)(47). Notably, the Gram-negative bacterium Escherichia coli does not contain a cytochrome bc 1 complex, which precludes studies on Rieske protein assembly in this important model organism for research on Tat-dependent protein translocation (32,33).…”