Subunit δ Is the Key Player for Assembly of the H+-translocating Unit of Escherichia coli FOF1 ATP Synthase

The ATP synthase (FOF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane to the synthesis or hydrolysis of ATP. This nanomotor is composed of the rotor c(10)gamma epsilon and the stator ab(2)alpha(3)beta(3)delta. To study the assembly of this multimeric enzyme complex consisting of membraneintegral as well as peripheral hydrophilic subunits, we combined nearest neighbor analyses by intermolecular disulfide bond formation or purification of partially assembled FOF1 complexes by affinity chromatography with the use of mutants synthesizing different sets of FOF1 subunits. Together with a time-delayed in vivo assembly system, the results demonstrate that FOF1 is assembled in a modular way via subcomplexes, thereby preventing the formation of a functionalH(+)-translocating unit as intermediate product. Surprisingly, during the biogenesis of FOF1, F-1 subunit delta is the key player in generating stable F-O. Subunit delta serves as clamp between ab(2) and c(10)alpha(3)beta(3)gamma epsilon and guarantees that the open H+ channel is concomitantly assembled within coupled FOF1 to maintain the low membrane proton permeability essential for viability, a general prerequisite for the assembly of multimeric H+-translocating enzymes.