Interaction with monomeric subunit c drives insertion of ATP synthase subunit a into the membrane and primes a-c complex formation.

Subunit a is the main part of the membrane stator of the ATP synthase molecular turbine. Subunit c is the building block of the membrane rotor. We have generated two molecular fusions of a and c subunits with different orientations of the helical hairpin of subunit c. The a/c fusion protein with correct orientation of transmembrane helices was inserted into the membrane, and co-incorporated into the F(0) complex of ATP synthase with wild type subunit c. The fused c subunit was incorporated into the c-ring tethering the ATP synthase rotor to the stator. The a/c fusion with incorrect orientation of the c-helices required wild type subunit c for insertion into the membrane. In this case, the fused c subunit remained on the periphery of the c-ring and did not interfere with rotor movement. Wild type subunit a inserted into the membrane equally well with wild type subunit c and c-ring assembly mutants that remained monomeric in the membrane. These results show that interaction with monomeric subunit c triggers insertion of subunit a into the membrane, and initiates formation of the a-c complex, the ion-translocating module of the ATP synthase. Correct assembly of the ATP synthase incorporating topologically correct fusion of subunits a and c validates using this model protein for high resolution structural studies of the ATP synthase proton channel.