The preferred stoichiometry of c subunits in the rotary motor sector of Escherichia coli ATP synthase is 10

Abstract

The stoichiometry of c subunits in the H(+)-transporting F(o) rotary motor of ATP synthase is uncertain, the most recent suggestions varying from 10 to 14. The stoichiometry will determine the number of H(+) transported per ATP synthesized and will directly relate to the P/O ratio of oxidative phosphorylation. The experiments described here show that the number of c subunits in functional complexes of F(o)F(1) ATP synthase from Escherichia coli can be manipulated, but that the preferred number is 10. Mixtures of genetically fused cysteine-substituted trimers (c(3)) and tetramers (c(4)) of subunit c were coexpressed and the c subunits crosslinked in the plasma membrane. Prominent products corresponding to oligomers of c(7) and c(10) were observed in the membrane and purified F(o)F(1) complex, indicating that the c(10) oligomer formed naturally. Oligomers larger than c(10) were also observed in the membrane fraction of cells expressing c(3) or c(4) individually, or in cells coexpressing c(3) and c(4) together, but these larger oligomers did not copurify with the functional F(o)F(1) complex and were concluded to be aberrant products of assembly in the membrane.

Figure 1

Rotary model for ATP synthase based on the subunit composition of the E. coli enzyme (8). The F₁ portion of the complex is bound at the cytoplasmic face of the membrane. The proton-motive force drives rotation of a ring composed of c subunits, the number (n) of which is uncertain. Protons enter the assembly through a periplasmic inlet channel and bind to the Asp⁶¹ carboxylate (open circle) of a subunit c at the a₁b₂ stator interface. The protonated binding site then moves from the stator interface into the lipid phase of the membrane, and after n steps the proton is released at an outlet channel on the F₁-binding side of the membrane. The γ and ɛ subunits remain fixed to the top of a set of c subunits so that rotation of the c oligomer also drives rotation of subunit γ within the α₃β₃ hexamer of F₁ to alternatively promote ADP + P_i binding and ATP product release in the β catalytic subunits. The b₂ and δ subunits of the stator hold the α₃β₃ subunits in a fixed position as the γ subunit turns inside them to drive ATP synthesis. The coupled reaction catalyzed by the complex is reversible so that ATP hydrolysis will drive proton transport in the reverse of the direction shown. This figure is modified from ref. .

Figure 2

Strategy for testing the preferred number of subunit c in E. coli F_oF₁ complex. (A) System used for coexpression of c₃(I30C) and c₄(I30C) substituted proteins in one cell. The eight genes of the atp (ATP synthase) operon are deleted from the chromosome of the cell shown. In the scheme shown, the c₄ subunit is expressed from a pBR322-derived plasmid carrying genes for the whole atp operon (pWOc4), with transformant cells selected on the basis of plasmid-encoded ampicillin resistance (Amp^r). The c₃ subunit is expressed by itself from a pACYC184-derived plasmid with transformant cells being selected for via chloramphenicol resistance (Cm^r). (B) Coexpression of c₃(I30C) and c₄(I30C) in the same cell should lead to formation of a c₁₀ decamer if that is the preferred structure in F_o. The figure shows the positions of the I30C-substituted cysteine in the first and last subunit of the c₃ trimer and c₄ tetramer. The approximate shape of a cross section of subunit c at the position of the I30C substitution is indicated (21). Cys–Cys crosslinking is expected to yield c₆, c₇, and c₁₀ products.

Figure 3

ATP-driven proton translocation with membrane vesicles from cells expressing c₃ or c₄ by themselves, or cells coexpressing c₃ and c₄. Coexpression in the pWOc4/pCOc3 and pWOc3/pCOc4 configurations are indicated as (c₄ + c₃)I30C and (c₃ + c₄)I30C, respectively. The quenching of ACMA fluorescence was used as an indicator the pH gradient established by ATPase-coupled proton pumping. At the times indicated, ATP was added to 0.92 mM and, to terminate the quenching response, the protonophore SF6847 was added to 0.25 μM to make the membranes proton permeable.

Figure 4

Crosslinked products generated in membranes from cells expressing c₃(I30C) and c₄(I30C) subunits. All samples were prepared after crosslinking of membranes with I₂. The oligomeric size of the crosslinked product is indicated on the vertical axis. (A) Membranes prepared from cells expressing pWOc3(I30C) or pWOc4(I30C) individually or cells expressing both and pWOc4(I30C) and pCOc3(I30C). The deoxycholate/cholate extract and glycerol gradient purified F_oF₁ fractions from the pWOc4(I30C)/pCOc3(I30C) membranes is also shown. (B) Glycerol gradient fraction shown in A after prolonged development. (C) Glycerol gradient F_oF₁ fractions prepared from the c₃ or c₄ membranes shown in A. (D) Scan of lanes of (c₄ + c₃) membrane and glycerol gradient shown in A.

Figure 5

Crosslinked products generated in membranes from cells expressing both the pWOc3(I30C) and pCOc4(I30C) plasmids. Membranes were treated with I₂ and the F_oF₁ complex extracted and purified by glycerol gradient centrifugation as described in the text. (A) Lanes from immunoblot. (B) Scan of crosslinked products ≥c₆ in the three lanes shown.

Figure 6

Cu(II)-phenanthroline catalyzed crosslinking of monomeric subunit c with double Cys substitutions. (A) Membrane fraction of c₁(Cys²¹/Cys⁶⁵) with (+) and without (−) crosslinking. (B) The crosslinked membrane, deoxycholate/cholate extract, and purified F_oF₁ from the c₁(Cys¹¹/Cys⁷⁵) mutant are shown. Crosslinking was carried out as described (15).