Close proximity of a cytoplasmic loop of subunit a with c subunits of the ATP synthase from Escherichia coli.
Interactions between subunit a and the c subunits of the Escherichia coli ATP synthase are thought to control proton translocation through the F(o) sector. In this study cysteine substitution mutagenesis was used to define the cytoplasmic ends of the first three transmembrane spans of subunit a, as judged by accessibility to 3-N-maleimidyl-propionyl biocytin. The cytoplasmic end of the fourth transmembrane span could not be defined in this way because of the limited extent of labeling of all residues between 186 and 206. In contrast, most of the preceding residues in that region, closer to transmembrane span 3, were labeled readily. The proximity of this region to other subunits in F(o) was tested by reacting mono-cysteine mutants with a photoactivated cross-linker. Residues 165, 169, 173, 174, 177, 178, and 182-184 could all be cross-linked to subunit c, but no sites were cross-linked to b subunits. Attempts using double mutants of subunit a to generate simultaneous cross-links to two different c subunits were unsuccessful. These results indicate that the cytoplasmic loop between transmembrane spans 3 and 4 of subunit a is in close proximity to at least one c subunit. It is likely that the more highly conserved, carboxyl-terminal region of this loop has limited surface accessibility due to protein-protein interactions. A model is presented for the interaction of subunit a with subunit c, and its implications for the mechanism of proton translocation are discussed.
Zhang
Di
D
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376, USA.
Vik
Steven B
SB
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017434:Protein Structure, Tertiary
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D025261:Mitochondrial Proton-Translocating ATPases