Characterization of the first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase by surface labeling, cross-linking, and mutagenesis.
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.
Long
Julie C
JC
Department of Biological Sciences, Southern Methodist University, Dallas, TX 75275, USA.
DeLeon-Rangel
Jessica
J
Vik
Steven B
SB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry; D015151:Immunoblotting; D007930:Leucine / Q000737:chemistry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D003432:Cross-Linking Reagents; D005973:Glutamine; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; D007930:Leucine; D003545:Cysteine; D000409:Alanine