Assembly of the Stator in Escherichia coli ATP Synthase. Complexation of α Subunit with Other F1 Subunits Is Prerequisite for δ Subunit Binding to the N-Terminal Region of α

α subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure α was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator δ subunit and the N-terminal residue 1−22 region of α occurred normally when pure α was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure δ and isolated α subunit. Unlike in F1, the N-terminal region of isolated α was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of α subunit with other F1 subunits is prerequisite for δ subunit binding to the N-terminal region of α. We suggest that the N-terminal 1−22 residues of α are sequestered in isolated α until released by binding of β to α subunit. This prevents 1/1 δ/α complexes from forming and provides a satisfactory explanation of the stoichiometry of one δ per three α seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one δ to the α3/β3 hexagon. The cytoplasmic fragment of the b subunit (bsol ) did not bind to isolated α. It might also be that complexation of α with β subunits is prerequisite for direct binding of stator b subunit to the F1-sector.