Single amino acid insertions probe the alpha subunit of the Escherichia coli F1F0-ATP synthase.

Single amino acid insertions of alanine or aspartate have been introduced into the alpha subunit of the F1F0-ATP synthase at seven different sites, after residues 187, 193, 198, 202, 212, 217, and 222. These sites span a highly conserved region of the alpha subunit, parts of which are thought to be located in transmembrane spanning regions. Alanine insertions have little or no effect on function after positions 187, 193, 198, and 202, indicating that the region spanned by these residues is not essential for function. Alanine insertions after residues 212 and 217 disrupt ATP synthase function without grossly affecting the assembly of the enzyme, while the alanine insertion after residue 222 disrupts both ATP synthase function and assembly. All of the aspartate insertions are deleterious to ATP synthase function, except after residue 198. At the other six sites, aspartate insertions prevent growth on succinate minimal medium, indicating an inability to synthesize ATP. Aspartate insertions after residues 187 and 193 result in alpha subunits that do not fractionate with membranes, as indicated by immunoblotting. These results support a model of the alpha subunit in which residues 187-193 and residues 212-222 are part of distinct transmembrane spans, separated by a short extramembrane loop. The results are consistent with an important interaction between residues 212-222 of the alpha subunit and b or c subunits. General aspects of "insertion scanning mutagenesis" are also discussed.