Oligomerization of H(+)-pyrophosphatase and its structural and functional consequences.
The H(+)-pyrophosphatase (H(+)-PPase) consists of a single polypeptide, containing 16 or 17 transmembrane domains. To determine the higher order oligomeric state of Streptomyces coelicolor H(+)-PPase, we constructed a series of cysteine substitution mutants and expressed them in Escherichia coli. Firstly, we analyzed the formation of disulfide bonds, promoted by copper, in mutants with single cysteine substitutions. 28 of 39 mutants formed disulfide bonds, including S545C, a substitution at the periplasmic side. The formation of intermolecular disulfide bonds suppressed the enzyme activity of several, where the substituted residues were located in the cytosol. Creating disulfide links in the cytosol may interfere with the enzyme's catalytic function. Secondly, we prepared double mutants by introducing second cysteine substitutions into the S545C mutant. These double-cysteine mutants produced cross-linked complexes, estimated to be at least tetramers and possibly hexamers. Thirdly, we co-expressed epitope-tagged, wild type, and inactive mutant H(+)-PPases in E. coli and confirmed the formation of oligomers by co-purifying one subunit using the epitope tag used to label the other. The enzyme activity of these oligomers was markedly suppressed. We propose that H(+)-PPase is present as an oligomer made up of at least two or three sets of dimers.
Mimura
Hisatoshi
H
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Nakanishi
Yoichi
Y
Maeshima
Masayoshi
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics
D004220:Disulfides; D000939:Epitopes; D021122:Protein Subunits; D011994:Recombinant Proteins; D003300:Copper; D043564:Inorganic Pyrophosphatase; D003545:Cysteine