Co-factor insertion and disulfide bond requirements for twin-arginine translocase-dependent export of the Bacillus subtilis Rieske protein QcrA

Abstract

The twin-arginine translocation (Tat) pathway can transport folded and co-factor-containing cargo proteins over bacterial cytoplasmic membranes. Functional Tat machinery components, a folded state of the cargo protein and correct co-factor insertion in the cargo protein are generally considered as prerequisites for successful translocation. The present studies were aimed at a dissection of these requirements with regard to the Rieske iron-sulfur protein QcrA of Bacillus subtilis. Notably, QcrA is a component of the cytochrome bc1 complex, which is conserved from bacteria to man. Single amino acid substitutions were introduced into the Rieske domain of QcrA to prevent either co-factor binding or disulfide bond formation. Both types of mutations precluded QcrA translocation. Importantly, a proofreading hierarchy was uncovered, where a QcrA mutant defective in disulfide bonding was quickly degraded, whereas mutant QcrA proteins defective in co-factor binding accumulated in the cytoplasm and membrane. Altogether, these are the first studies on Tat-dependent protein translocation where both oxidative folding and co-factor attachment have been addressed in a single native molecule.

Keywords: Bacillus; Cytochromes; Disulfide; Iron-Sulfur Protein; Protein Translocation; QcrA; Rieske Protein; TatAy; Twin-arginine Translocation.

FIGURE 1.

The Rieske domain of B. subtilis QcrA. a, annotated amino acid residues involved in co-factor attachment and disulfide bond formation. b, schematic representation of 2Fe-2S co-factor-binding by Cys-100, His-102, Cys-123, and His-124.

FIGURE 2.

Translocation and quality control of QcrA mutant proteins defective in co-factor binding or disulfide bond formation. a, translocation of QcrA in strains with a deleted chromosomal qcrA gene that ectopically express wild-type qcrA or site specifically mutated qcrA genes from pHB-201. The mutant qcrA genes specify the H102L or H124L mutant QcrA proteins impaired in co-factor binding, or the C123S mutant QcrA protein defective in disulfide bond formation. All strains were grown in LB with 1% NaCl. Cells were separated from the growth medium, and crude whole cell extracts and growth medium fractions were analyzed by Western blotting using specific antibodies against QcrA, BdbD, or LipA. The positions of the 18-kDa full-size QcrA protein in the whole cell fraction and the 14-kDa processed QcrA protein (QcrA*) in the growth medium fraction, both separated on the same gel, are marked with arrows. The membrane protein BdbD and the secreted protein LipA were used as positive controls. b, subcellular fractionation was performed to separate the cytoplasmic and membrane proteins of tat mutant cells and qcrA mutant cells producing site specifically mutated QcrA proteins as indicated. The positions of QcrA, the cytoplasmic marker protein TrxA, and the membrane protein BdbD are marked with arrows. c, pulse-chase labeling of wild-type QcrA and QcrA-H102L or QcrA-C123S mutant proteins in tat-proficient cells (upper panel) or tatAy-tatCy-deficient cells (lower panel). Cells were labeled for 2 min with a [³⁵S]methionine/cysteine mixture prior to chase with an excess of non-radioactive methionine/cysteine. At the time of chase (t = 0) and 5 and 30 min after the chase, samples were collected in which the presence of labeled QcrA was assessed by immunoprecipitation, NuPAGE, and autoradiography. The position of QcrA is marked with an arrow. d, the possibility to severely overexpress QcrA-C123S was assessed with the subtilin-inducible “SURE” expression plasmid pNZ8910. In this case, wild-type cells and cells over-expressing QcrA-H102L were used as controls. The positions of QcrA and the control protein BdbD are marked with arrows. UI, uninduced; I, subtilin-induced expression.

FIGURE 3.

Bdb-independent biogenesis of QcrA and cross-linking with AMS. a, translocation of QcrA by bdbA-bdbB, bdbB-bdbC, or bdbC-bdbD mutant cells was assessed by Western blotting as described in the legend to Fig. 2a. All protein samples were loaded on the same gel. The positions of QcrA, QcrA*, TrxA, and LipA are marked with arrows. b, AMS cross-linking of QcrA in protoplasts of B. subtilis 168. AMS cross-linking was detectable by an up-shift of the QcrA band upon NuPAGE and Western blotting only if Triton X-100 was present during the incubation of protoplasts with AMS. TrxA was used as a cytoplasmic control protein. Note that under the tested conditions only a minor fraction of TrxA was cross-linkable with AMS upon protoplast lysis with Triton X-100. The positions of QcrA, QcrA-AMS, TrxA, and TrxA-AMS are indicated.