Transmembrane topology of Escherichia coli H+-ATPase (ATP synthase) subunit a

Escherichia coli H+-ATPase subunit alpha is a hydrophobic F-0 subunit. To investigate the topology of the subunit in the membrane, we prepared site-specific polyclonal antibodies against amino-terminal (Ser3 to Leu-16), middle loop (Lys-167 to Gln-181), and carboxyl-terminal (Thr-259 to His-271) peptide segments. Enzyme-linked immunosorberat assay revealed that these antibodies specifically reacted with subunit a of inside-out membrane vesicles, but not with that of Fight-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.