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

Abstract

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, competent in nucleotide binding, and had normal N-terminal sequence. In F 1 -subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F 1 -depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the Nterminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F 1 subunits. On the other hand, three different types of experiment showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F 1 , the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F 1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming, and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F 1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon.The cytoplasmic fragment of the b subunit (b sol ) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F 1 -sector.ATP synthase is the terminal enzyme of oxidative phosphorylation and photophosphorylation, which synthesizes ATP from ADP and phosphate (Pi). The energy for ATP synthesis comes from transmembrane movement of protons down an electrochemical gradient, generated by substrate oxidation or by light capture. Initially, as the protons move through the interface between a and c subunits in the membrane-bound F o -sector of the enzyme, the realized energy is transduced into mechanical rotation of a group of subunits (γεc ring ) which comprise the "rotor". A helical coiled coil domain of γ projects into the central space of the α 3 β 3 hexagon, in the membrane-extrinsic F 1 -sector. α 3 β 3 hexagon contains three catalytic sites at α/β interfaces. In a manner that is not yet understood, rotation of γ vis-à-vis the three α/β subunit pairs brings about ATP synthesis at the three catalytic sites using a sequential reaction scheme (1). Detailed reviews of ATP synthase mechanism may be found in (2-5).Contact information: Alan E. Senior, Department of Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, Rochester NY 14642. Phone: 585-275-6645, Fax: 585-271-2683, Email: alan_senior@urmc.rochester "Stator" subunits b 2 and δ are present to prevent co-rotation of α 3 β 3 with the rotor (6). The interaction between δ and α subunits is known to be of major importance for this function. In...

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

“…Until now, only little experimental data regarding the order of the assembly of the E. coli F 1 part have been available. However, in vitro reconstitution experiments (13,62,63) support an assembly in the cytoplasm prior to its binding to the c ring comparable with that described for yeast mitochondrial F 1 (19,64,65). Therefore, an assembly of the F O F 1 core complex from two individual subcomplexes c 10 and ␣ 3 ␤ 3 ␥⑀ is proposed, with ␣ 3 ␤ 3 ␥ forming the minimal unit exhibiting ATPase activities (12,62,66) and subunit ⑀ being necessary for a stable binding to the membrane via c 10 (63,67).…”

“…Furthermore, a stable insertion of subunit a into the membrane is strictly dependent upon the co-insertion of the other F O subunits, whereas b and c are inserted independently (11). Overexpression of subunits ␣, ␤, and ␥ allowed complex formation with high ATPase activity in the cytoplasm (12), and for interaction between subunits ␦ and ␣, the assembly of ␣ with other F 1 subunits is a prerequisite (13).…”

Assembly of F1F0-ATP synthases

“…1B). Whether δ binds first to ab 2 to form an ab 2 δ pre-complex or associates with the N-terminal region of the α-subunit in F 1 F 0 core complexes [191,192] needs to be clarified in future experiments.…”

Two ATPases

“…67. We found that ␦ would not bind to isolated ␣-subunit; rather, it would bind only to ␣ that was pre-incorporated into F 1 .…”