Abstract
Second site suppressor mutations at position 31 of F1 subunit epsilon recouple ATP-driven H+ translocation in the uncoupled Q42E mutant of subunit c of the Escherichia coli F1F0 ATP synthase (Zhang, Y., Oldenburg, M., and Fillingame, R. H. (1994) J. Biol. Chem. 269, 10221-10224). This finding suggests a functional interaction between subunit c and subunit epsilon during the coupling of H+ transport through F0 to ATP synthesis of F1. However, the physical proximity of the two subunits remained to be defined. In this study, Cys residues were introduced into residues in the polar loop region of subunit c surrounding Gln42 and at position 31 of subunit epsilon to see whether the subunits could be cross-linked. Disulfide bridge formation between subunit c and subunit epsilon was observed in membranes of three double mutants, i.e. cA40C/epsilon E31C, cQ42C/epsilon E31C, and cP43C/epsilon E31C, but not in wild type membranes or in membranes of the cA39C/epsilon E31C double mutant. These results indicate that the polar loop of subunit c and the region around residue 31 of subunit epsilon are physically close to each other in the F1F0 complex and support the hypothesis that these two subunits interact directly in the coupling of H+ transport to ATP synthesis. Disulfide cross-linking of the Q42C subunit c and E31C subunit epsilon leads to inhibition of ATPase coupled H+ transport, as might be expected in a model where the catalytic sites of the F1 ATPase alternate during H+ transport-coupled ATP hydrolysis/synthesis. However, a quantitative relationship between the extent of inhibition of transport and the extent of cross-linking could not be established by the methods used here, and the possibility remains that the epsilon-c cross-linked F1F0 complex retains residual H+ transporting activity.
Highlights
The Hϩ-transporting, F1F0 ATP synthase of Escherichia coli utilizes an Hϩ electrochemical gradient to drive ATP synthesis during oxidative phosphorylation (Senior, 1988)
These results indicate that the polar loop of subunit c and the region around residue 31 of subunit ⑀ are physically close to each other in the F1F0 complex and support the hypothesis that these two subunits interact directly in the coupling of H؉ transport to ATP synthesis
Disulfide cross-linking of the Q42C subunit c and E31C subunit ⑀ leads to inhibition of ATPase coupled H؉ transport, as might be expected in a model where the catalytic sites of the F1 ATPase alternate during H؉ transport-coupled ATP hydrolysis/synthesis
Summary
Oligonucleotide-directed Mutagenesis and Plasmid Construction— All the plasmids used in this study are derivatives of plasmid pBR322. Plasmid pYZ201 (Fig. 1), which carries the eight structural genes of unc operon (bases 870-10172), is an equivalent of plasmid pMO142 (Zhang et al, 1994) except that it lacks an NcoI site at base 1305 due to a silent mutation introduced in the subunit a His codon. The subunit c polar loop Cys substitutions and subunit ⑀E31C mutation were introduced using the polymerase chain reaction, as described by Herlitze and Koenen (1990), with several 21–27-base oligonucleotides (Table I). The mutated polymerase chain reaction fragments for the uncE gene were digested with PstI (1561) and HpaI (2162) and ligated into the equivalent sites of pDF163 (Fig. 1). The mutated polymerase chain reaction fragment for the uncC gene, containing the ⑀E31C mutation, was di-.
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