Assembly of the Streptomyces coelicolor Rieske protein
Final Report Abstract
Protein export and assembly is essential for the bacterial cell and is facilitated in general by two distinct operating translocases, called the Sec and the Tat systems. Protein translocation by the Sec pathway proceeds by a threading mechanism in which the substrate is maintained in an unfolded conformation prior to transport. In contrast proteins are transported through the Tat (twin arginine translocation) pathway in a folded state and targeted to the Tat apparatus by N-terminal signal peptides within an S-R-R-x-F-L-K consensus motif. One of the most important Tat-dependent membrane proteins in bacteria is the Rieske protein, a fundamental component of the essential energy transducing cytochrome bc1 complex in the respiratory chains of many bacteria. Usually the bacterial Rieske protein is composed of a single transmembrane helix at its N-terminus which is preceded by the Tat (twin arginine) motif and followed by an iron sulphur domain. Interestingly, in Actinobacteria like Streptomyces coelicolor (S.co.) and pathogenic relatives like Mycobacteria tuberculosis the Rieske protein has three transmembrane domains (TM) prior to the iron-sulphur cluster. As a model for the actinobacterial Rieske protein we investigated the assembly process of the three transmembrane domains of the Streptomyces coelicolor Rieske protein. We demonstrate using different reporter systems that the actinobacterial Rieske protein requires the Tat pathway for its assembly into the cytoplasmic membrane. We showed that the Tat system recognizes an internal twin arginine motif, which is localized adjacent to transmembrane helix three. Protease treatment on sphaeroplasts demonstrated that in the absence of the Tat machinery the globular domain remains at the cytoplasmic side of the membrane. Surprisingly fractionation and membrane protein extraction revealed that the protein resides stably in the cytoplasmic membrane in the absence of the Tat machinery due to the Tat independent insertion of its first two transmembrane domains. Excitingly in vitro insertion experiments demonstrated that the Sec pathway inserts the first two transmembrane domains of the protein into the membrane, most likely in a co-translational manner. As pulse chase experiments suggested that depletion of YidC had an effect on the stability of that membrane protein in vivo, in vitro insertion experiments finally confirmed a role for YidC in the assembly of the first two transmembrane helices of the S. co. Rieske protein. Here we demonstrated for the first time that the Sec/YidC and the Tat pathways co-operate together to assemble a single membrane protein into the cytoplasmic membrane.
Publications
- Assembly of the Streptomyces coelicolor Rieske protein - An unusual Tat target: Gordon Research Conference: Bacterial Cell Surface (USA): June 2010
Keller R. & Palmer T.
- Biosynthesis of an unusual Tat-dependent membrane protein: Tat meeting Oxford (UK): Sept. 2010
Keller R. & Palmer T.
- Assembly of Actinobacterial Rieske protein: VAAM (GER) April 2011
Keller R. et al.
- Assembly of an unusual membrane protein by the Tat machinery: Tat meeting at the University of Dundee (UK): Okt. 2011
Keller R et al.
