Transmembrane topology of Escherichia coli H(+)-ATPase (ATP synthase) subunit a.
Escherichia coli H(+)-ATPase subunit a is a hydrophobic F0 subunit. To investigate the topology of the subunit in the membrane, we prepared site-specific polyclonal antibodies against amino-terminal (Ser-3 to Leu-16), middle loop (Lys-167 to Gln-181), and carboxyl-terminal (Thr-259 to His-271) peptide segments. Enzyme-linked immunosorbent assay revealed that these antibodies specifically reacted with subunit a of inside-out membrane vesicles, but not with that of right-side-out spheroplasts. Full reactivity appeared when spheroplasts were disrupted with Triton X-100 (0.5%) or by sonication. These results suggest that at least parts of the three peptide segments of subunit a face the cytoplasm. Based on these observations, we propose a novel transmembrane topology of subunit a.
Yamada
H
H
Department of Biological Science, Institute of Scientific and Industrial Research, Osaka University, Japan.
Moriyama
Y
Y
Maeda
M
M
Futai
M
M
D000595:Amino Acid Sequence; D000906:Antibodies; D000918:Antibody Specificity; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D046911:Macromolecular Substances; D008961:Models, Structural; D008969:Molecular Sequence Data; D009876:Operon; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013104:Spheroplasts / Q000201:enzymology
D000906:Antibodies; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases