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

Abstract

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.