A Novel Labeling Approach Supports the Five-transmembrane Model of Subunit a of the Escherichia coli ATP Synthase

Abstract

Cysteine mutagenesis and surface labeling has been used to define more precisely the transmembrane spans of subunit a of the Escherichia coli ATP synthase. Regions of subunit a that are exposed to the periplasmic space have been identified by a new procedure, in which cells are incubated with polymyxin B nonapeptide (PMBN), an antibiotic derivative that partially permeabilizes the outer membrane of E. coli, along with a sulfhydryl reagent, 3-(N-maleimidylpropionyl) biocytin (MPB). This procedure permits reaction of sulfhydryl groups in the periplasmic space with MPB, but residues in the cytoplasm are not labeled. Using this procedure, residues 8, 27, 37, 127, 131, 230, 231, and 232 were labeled and so are thought to be exposed in the periplasm. Using inside-out membrane vesicles, residues near the end of transmembrane spans 1, 64, 67, 68, 69, and 70 and residues near the end of transmembrane spans 5, 260, 263, and 265 were labeled. Residues 62 and 257 were not labeled. None of these residues were labeled in PMBNpermeabilized cells. These results provide a more detailed view of the transmembrane spans of subunit a and also provide a simple and reliable technique for detection of periplasmic regions of inner membrane proteins in E. coli.The ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (for reviews, see Refs. 1-3). It contains an F 1 sector, with subunits for nucleotide binding and catalysis, and an F 0 sector, which conducts protons across the membrane. Five different subunits are found in the E. coli F 1 : ␣, ␤, ␥, ␦, and ⑀, in a stoichiometry of 3:3:1:1:1. Three different subunits named a, b, and c form the E. coli F 0 with a stoichiometry of 1:2:12 (4) . The mechanism by which an electrochemical proton gradient across the membrane drives ATP synthesis is thought to involve a rotary mechanism. The crystallization of F 1 from bovine mitochondria (5) led to a high resolution structure of the ␣ 3 ␤ 3 hexamer, plus parts of ␥ in the central core . Subsequently, the hypothesis of rotation of ␥ and ⑀ and relative to ␣ 3 ␤ 3 has been supported by direct visualization of rotation of fluorescently labeled actin filaments covalently attached to ␥ (6) or ⑀ (7). It has been proposed that F 0 subunit c drives the rotation of ␥ and ⑀ as a rotor (8), whereas subunits a and b function as the stator. Recent theoretical work has indicated the feasibility of this proposal (9), but as of yet there is no direct evidence of rotation by F 0 subunits.Information about the tertiary and quaternary structure of F 0 subunits will be necessary for an understanding of how F 0 translocates protons, and how it might drive rotation of ␥ and ⑀ subunits in F 1 . Subunit b seems to be embedded in the membrane via a span of hydrophobic amino acids at its N terminus. A truncated, soluble form of b has been shown to be extended and dimeric (10) . Recent NMR studies of c have confirmed the ␣-helical hairpin structure of the two predicted transmembrane spans, and also ...

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