His(15) of subunit a of the Escherichia coli ATP synthase is important for the structure or assembly of the membrane sector F(o).
Approximately 37 amino acids at the amino-terminus of subunit a of the Escherichia coli ATP synthase are found localized to the periplasm. Results indicate that a single amino acid substitution, H15D, disrupts assembly of subunit a and causes a loss of ATP synthase function. In this study, a conserved region of nine amino acids, 11-19, was initially mutagenized randomly, generating no mutants that could grow on succinate-minimal medium. Subsequent mutagenesis, confined to residues His(14), His(15), and Asn(17), indicated that constructs containing H15D were the most deleterious. Four single mutants were constructed and analyzed: H15A, H14D, H15A, and H15D. Only H15D was significantly impaired, with respect to ATP-driven proton translocation, passive proton permeability through F(o), and sensitivity of membrane-bound ATPase to DCCD. Immunoblot analysis indicated very low levels of subunit a from H15D. Cysteine mutations were constructed at positions 14, 15, 17, and 18. Residues 14, 15, and 17 were shown to be accessible in the periplasmic space, while residue 18 was not, indicating that this region was stably folded. While both His(14) and His(15) are conserved among a group of bacteria, results presented here indicate that they are not equivalent, and that a specific role for His(15) in the assembly or structure of the ATP synthase is supported.
Department of Biological Sciences, Southern Methodist University, Dallas, Texas, 75275, USA.
A R
AR
Patterson
T
T
Wada
S B
SB
Vik
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D017354:Point Mutation; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D006639:Histidine; D006180:Proton-Translocating ATPases