The Rieske protein from Paracoccus denitrificans is inserted into the cytoplasmic membrane by the twin-arginine translocase.
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.
Bachmann
Julie
J
Institut für Biochemie, Johann Wolfgang Goethe-Universität, D-60438 Frankfurt, Germany.
Bauer
Brigitte
B
Zwicker
Klaus
K
Ludwig
Bernd
B
Anderka
Oliver
O
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D014450:Electron Transport Complex III / Q000235:genetics / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007425:Intracellular Membranes / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000502:physiology*; D008969:Molecular Sequence Data; D010231:Paracoccus denitrificans / Q000378:metabolism*; D021382:Protein Sorting Signals; D021381:Protein Transport; D013104:Spheroplasts / Q000378:metabolism
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; C037989:Rieske iron-sulfur protein; D001120:Arginine; D014450:Electron Transport Complex III