Structural evidence for a constant c₁₁ ring stoichiometry in the sodium F‐ATP synthase

Abstract

The Na+‐dependent F‐ATP synthases of Ilyobacter tartaricus and Propionigenium modestum contain membrane‐embedded ring‐shaped c subunit assemblies with a stoichiometry of 11. Subunit c from either organism was overexpressed in Escherichia coli using a plasmid containing the corresponding gene, extracted from the membrane using detergent and then purified. Subsequent analyses by SDS/PAGE revealed that only a minor portion of the c subunits had assembled into stable rings, while the majority migrated as monomers. The population of rings consisted mainly of c11, but more slowly migrating assemblies were also found, which might reflect other c ring stoichiometries. We show that they consisted of higher aggregates of homogeneous c11 rings and/or assemblies of c11 rings and single c monomers. Atomic force microscopy topographs of c rings reconstituted into lipid bilayers showed that the c ring assemblies had identical diameters and that stoichiometries throughout all rings resolved at high resolution. This finding did not depend on whether the rings were assembled into crystalline or densely packed assemblies. Most of these rings represented completely assembled undecameric complexes. Occasionally, rings lacking a few subunits or hosting additional subunits in their cavity were observed. The latter rings may represent the aggregates between c11 and c1, as observed by SDS/PAGE. Our results are congruent with a stable c11 ring stoichiometry that seems to not be influenced by the expression level of subunit c in the bacteria.

Engineering rotor ring stoichiometries in the ATP synthase

“…A few c-rings in these images were seen to lack individual c-subunits; however, instead of closing this gap so as to form smaller c-rings, these incomplete oligomers have the same shape and diameter as the complete ones (10,25). Furthermore, c-subunits from I. tartaricus and Bacillus TA2.A1 expressed in E. coli were found to assemble correctly into c 11 and c 13 rings, respectively (20,23,24,48), despite the preferred c 10 stoichiometry of the native E. coli c-ring (46). The c-ring sizes also are independent of external factors such as medium pH, host protein expression (48) or host membrane composition (23,24), the source of carbon used by the cell (48,49), and, importantly, the rate of ATP synthesis itself (50,51).…”

“…Vector pt7cIT harbors the atpE gene of the I. tartaricus ATP synthase and allows the expression of its c 11 ring in E. coli host cells in the native stoichiometry (23,24). To study the impact of single amino acid residues within the glycine motif of the I. tartaricus c-subunit (G 25 IGPGVGQG 33 ), a set of alanine and serine substitutions was introduced by site-directed mutagenesis: G25A, G25S, G27A, P28A, G29A, G31A, Q32A, and G33A.…”

“…Furthermore, c-subunits from I. tartaricus and Bacillus TA2.A1 expressed in E. coli were found to assemble correctly into c 11 and c 13 rings, respectively (20,23,24,48), despite the preferred c 10 stoichiometry of the native E. coli c-ring (46). The c-ring sizes also are independent of external factors such as medium pH, host protein expression (48) or host membrane composition (23,24), the source of carbon used by the cell (48,49), and, importantly, the rate of ATP synthesis itself (50,51). Instead, the results presented in this and previous work consistently demonstrate that the c-ring stoichiometries are determined and constrained by the primary structure of the c-subunits (25,52).…”

“…After subsequent incubation at 20°C for 20 min, the contaminating proteins were removed by a second precipitation using 65% (wt/vol) (NH 4 ) 2 SO 4 , followed by centrifugation (39,000 × g for 20 min at 25°C). The filtrated supernatant containing the c-oligomers was dialyzed, and the c-rings were concentrated and stored (23).…”

“…The preparations of the mutant c-rings heterologously expressed in E. coli cells were performed as previously described (23,24), with some modifications. Briefly, after cells were disrupted (French pressure at 12,000 psi) and ultracentrifugation (200,000 × g for 45 min at 4°C), the membrane pellet was solubilized for 45 min at room temperature in buffer A [10 mM Tris·HCl (pH 8), 5 mM EDTA (pH 8)] containing 1% DDM.…”