The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin‐arginine translocase

Abstract

Mitochondrial respiratory complex III ⁄ cytochrome bc 1 is among the best-characterized membrane proteins, with structures elucidated from several species [1][2][3][4]. These structures revealed the organization of three catalytic subunits (SU) in the homodimeric complex; these are cytochrome b, cytochrome c 1 , and the Rieske ironsulfur protein (ISP). Cytochrome b forms eight transmembrane (TM) helices that bind two hemes and is largely contained within the membrane bilayer. Both cytochrome c 1 and the ISP are single-spanning TM proteins with globular hydrophilic domains located in the periplasmic space; these ecto-domains carry the heme and [2Fe)2S] cofactors, respectively. As the first cytochrome bc 1 structures were characterized, two features of the ISP came as a major surprise: (a) the ISP intertwines between the monomeric halves of the enzyme, such that the N-terminal TM helix of a given ISP is anchored within one monomer, whereas the The Rieske [2Fe)2S] protein (ISP) is an essential subunit of cytochrome bc 1 complexes in mitochondrial and bacterial respiratory chains. Based on the presence of two consecutive arginines, it was argued that the ISP of Paracoccus denitrificans, a Gram-negative soil bacterium, is inserted into the cytoplasmic membrane via the twin-arginine translocation (Tat) pathway. Here, we provide experimental evidence that membrane integration of the bacterial ISP indeed relies on the Tat translocon. We show that targeting of the ISP depends on the twin-arginine motif. A strict requirement is established particularly for the second arginine residue (R16); conservative replacement of the first arginine (R15K) still permits substantial ISP transport. Comparative sequence analysis reveals characteristics common to Tat signal peptides in several bacterial ISPs; however, there are distinctive features relating to the fact that the presumed ISP Tat signal simultaneously serves as a membrane anchor. These differences include an elevated hydrophobicity of the h-region compared with generic Tat signals and the absence of an otherwise well-conserved '+5'-consensus motif lysine residue. Substitution of the +5 lysine (Y20K) compromises ISP export and ⁄ or cytochrome bc 1 stability to some extent and points to a specific role for this deviation from the canonical Tat motif. EPR spectroscopy confirms cytosolic insertion of the [2Fe)2S] cofactor. Mutation of an essential cofactor binding residue (C152S) decreases the ISP membrane levels, possibly indicating that cofactor insertion is a prerequisite for efficient translocation along the Tat pathway.Abbreviations EPR, electron paramagnetic resonance spectroscopy; ISF, Rieske iron-sulfur protein soluble fragment; ISP, Rieske iron-sulfur protein; SU, subunit; Tat, twin-arginine translocation; TM, transmembrane.

Plant mitochondria contain the protein translocase subunits TatB and TatC

“…This is fully consistent with our observations that mutation of the AtRip Tat targeting signal and also that AtRip requires a ΔpH for assembly. Rip proteins of chloroplasts and bacteria have also been demonstrated previously to require a Tat pathway for proper insertion (Molik et al, 2001;Aldridge et al, 2008;Bachmann et al, 2006;De Buck et al, 2007). These observations are strengthened by the correlation of mitochondrial Tat proteins with the absence of a complete Bcs1 protein in a variety of organisms.…”

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

“…It is not clear whether the iron-sulfur cluster is inserted into the Rieske protein at this stage, since we were unable to obtain enough material from the ⌬tatC strain to look for its presence by electron paramagnetic resonance (EPR). However, it should be noted that Bachmann et al (18) showed that the cytosolic fraction of a Paracoccus denitrificans strain in which the twin arginine motif of the Rieske protein had been inactivated by substituting twin lysines gave an EPR signature of the Rieske iron-sulfur cluster, consistent with cofactor insertion in the cytoplasm prior to interaction with the Tat machinery. It is difficult to envisage how the complex we isolated from the membranes of the S. coelicolor ⌬tatC strain would be stable to detergent solubilization and purification if the first two TMD of the Rieske protein were not interacting with one or both of the cytochromes.…”

“…In contrast, in bacteria and plants, the Rieske iron-sulfur protein is inserted into the membrane by the Tat machinery (17,18,19,20,21). The Tat pathway transports folded substrate proteins (see references 22 and 23 for recent reviews), and the iron-sulfur cluster is inserted into the Rieske protein in the cytoplasm prior to transport.…”

Organophosphate Hydrolase Is a Lipoprotein and Interacts with Pi-specific Transport System to Facilitate Growth of Brevundimonas diminuta Using OP Insecticide as Source of Phosphate

“…In bacteria these usually have either a single transmembrane domain at the C terminus (35) or an N-terminal non-cleaved twin-arginine signal anchor sequence (37). However, bioinformatic analysis supported by structural studies show that OPH has no C-terminal hydrophobic helical domain (38) and the N-terminal signal peptide of OPH is cleaved off during biosynthesis and therefore cannot serve as a signal anchor (15).…”