@article{oai:nagoya.repo.nii.ac.jp:00005483,
 author = {Mimura, Hisatoshi and Nakanishi, Yoichi and Hirono, Megumi and Maeshima, Masayoshi},
 journal = {Journal of Biological chemistry},
 month = {Aug},
 note = {The H^+-translocating pyrophosphatase (H^+-PPase) is a proton pump that is found in a wide variety of organisms. It consists of a single polypeptide chain that is thought to possess between 14 and 17 transmembrane domains. To determine the topological arrangement of its conserved motifs and transmembrane domains, we carried out a cysteine-scanning analysis by determining the membrane topology of cysteine-substitution mutants of Streptomyces coelicolor H^+-PPase expressed in Escherichia coli, using chemical reagents. First, we prepared a synthetic DNA that encoded the enzyme and constructed a functional cysteine-less mutant by substituting the four cysteine residues. We then introduced cysteine residues individually into 42 sites in its hydrophilic regions, and N- and C-terminal segments. Thirtysix of the mutant enzymes retained both pyrophosphatase and H^+-translocating activities. Analysis of 29 of these mutant forms using membrane permeable and impermeable sulfhydryl reagents revealed that S. coelicolor H^+-PPase contains 17 transmembrane domains, and that several conserved segments, such as the substrate-binding domains, are exposed to the cytoplasm. Four essential serine residues that were located on the cytoplasmic side were also identified. A marked characteristic of the S. coelicolor enzyme is a long additional sequence, which includes a transmembrane domain at the C-terminus. We propose that the basic structure of H^+-PPases has 16 transmembrane domains with several large cytoplasmic loops containing functional motifs.},
 pages = {35106--35112},
 title = {Membrane Topology of the H+-Pyrophosphatase of Streptomycescoelicolor Determined by Cysteine-Scanning Mutagenesis},
 volume = {279},
 year = {2004}
}