Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function

DipZ is a bacterial cytoplasmic membrane protein that transfers reducing po wer from the cytoplasm to the periplasm so as to facilitate the formation o f correct disulphide bonds and c-type cytochromes in the latter compartment . Topological analysis using gene fusions between the Escherichia coli dipZ and either E. coli phoA or lacZ shows that DipZ has a highly hydrophobic c entral domain comprising eight transmembrane alpha-helices plus periplasmic globular N-terminal and C-terminal domains. The previously assigned transl ational start codon for the E. coli DipZ was shown to be incorrect and the protein to be larger than previously thought. The experimentally determined translational start position indicates that an additional alpha-helix at t he N-terminus acts as a cleavable signal peptide so that the N-terminus of the mature protein is located in the periplasm. The newly assigned 5' end o f the dipZ gene was shown to be preceded by a functional ribosome-binding s ite. The hydrophobic central domain and both of the periplasmic globular do mains each have a pair of highly conserved cysteine residues, and it was sh own by site directed mutagenesis that all six conserved cysteine residues c ontribute to DipZ function.