Abstract
The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunit b, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40-64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits.
Highlights
The mechanism by which an electrochemical proton gradient across the membrane drives ATP synthesis involves the rotation of the subunit c oligomer of F0, which drives the rotation of ␥ and ⑀ as a rotor with subunits a and b functioning as the stator along with ␦, ␣, and 
The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(Nmaleimidylpropionyl)-biocytin
The results indicate that the first transmembrane span of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop is probably near the cytoplasmic surface, perhaps in contact with b subunits
Summary
Materials—Restriction endonucleases and T4 DNA ligase were obtained from New England Biolabs. Mutagenesis, Growth, and Expression—Plasmids pLN6HisHA [21] and pLN7-HisHA (see below) were used for the construction of mutants. These plasmids produce versions of subunit a that include both a His tag and a HA epitope at the C terminus of the protein. RH305 produces a form of subunit a, which is truncated near Pro-240 and cannot be detected in cells [36] It can be complemented by plasmids containing a wild type uncB gene. Detection of Bound MPB and Immunoblotting—The purification of subunit a following procedures for labeling or cross-linking was performed according to methods described previously [19, 21]
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