The membrane topology of the a subunit of the F 1 F 0 ATP synthase from Escherichia coli has been probed by surface labeling using 3-(N-maleimidylpropionyl) biocytin. Subunit a has no naturally occurring cysteine residues, allowing unique cysteines to be introduced at the following positions: 8, 24, 27, 69, 89, 128, 131, 172, 176, 196, 238, 241, and 277 (following the COOH-terminal 271 and a hexahistidine tag). None of the single mutations affected the function of the enzyme, as judged by growth on succinate minimal medium. Membrane vesicles with an exposed cytoplasmic surface were prepared using a French pressure cell. Before labeling, the membranes were incubated with or without a highly charged sulfhydryl reagent, 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid. After labeling with the less polar biotin maleimide, the samples were solubilized with octyl glucoside/cholate and the subunit a was purified via the oligohistidine at its COOH terminus using immobilized nickel chromatography. The purified samples were electrophoresed and transferred to nitrocellulose for detection by avidin conjugated to alkaline phosphatase. Results indicated cytoplasmic accessibility for residues 69, 172, 176, and 277 and periplasmic accessibility for residues 8, 24, 27, and 131. On the basis of these and earlier results, a transmembrane topology for the subunit a is proposed.The F 1 F 0 ATP synthase from Escherichia coli is typical of the ATP synthases found in mitochondria, chloroplasts, and many other bacteria (for recent reviews, see Refs.
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 ...
Most of what is known about the structure and function of subunit a, of the ATP synthase, has come from the construction and isolation of mutations, and their analysis in the context of the ATP synthase complex. Three classes of mutants will be considered in this review. (1) Cys substitutions have been used for structural analysis of subunit a, and its interactions with subunit c. (2) Functional residues have been identified by extensive mutagenesis. These studies have included the identification of second-site suppressors within subunit a. (3) Disruptive mutations include deletions at both termini, internal deletions, and single amino acid insertions. The results of these studies, in conjunction with information about subunits b and c, can be incorporated into a model for the mechanism of proton translocation in the Escherichia coli ATP synthase.
The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(Nmaleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Crosslinking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunit b, and the crosslinking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40 -64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits.
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