title: Membrane topology of the H+-pyrophosphatase of Streptomyces coelicolor determined by cysteine-scanning mutagenesis.
abstract: 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. Thirty-six 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 that 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.
authors.affiliation: Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
authors.forename: Hisatoshi
authors.initials: H
authors.lastname: Mimura
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Nakanishi
authors.forename: Megumi
authors.initials: M
authors.lastname: Hirono
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Maeshima
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011522:Protons; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D010455:Peptides; D011522:Protons; D043564:Inorganic Pyrophosphatase; D003545:Cysteine