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The role of the amino‐terminal periplasmic domain of subunit a of the E. coli ATP synthase in function and assembly
Author(s) -
Vik Steven B,
Ishmukhametov Robert R
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a518-d
Subject(s) - periplasmic space , atp synthase gamma subunit , atp synthase , biochemistry , atp hydrolysis , protein subunit , cysteine , transmembrane domain , biology , amino acid , chemistry , atpase , gene , escherichia coli , enzyme
Subunit a is the largest integral membrane protein in the ATP synthase, and it provides an interface to rotary subunits. Approximately 40 amino‐terminal residues of subunit a reside in the periplasmic space of E. coli , including a conserved sequence motif in residues 7–19. Several studies have indicated that this region may be important for function or assembly. To examine the role of this segment, we have replaced residues 6–20 by a single alanine, using a vector pFV2, that expresses all 8 genes of the ATP synthase. This plasmid was used to transform DK8, a strain deleted for all 8 genes. In membrane vesicles prepared from this strain, rates of ATP hydrolysis were about one‐half the wild type, and stimulation of ATP hydrolysis by LDAO (lauryl dimethylamine oxide) was found to be about two times higher. The mutant was much less sensitive to DCCD (dicyclohexyl carbodiimide) and the rate of ATP‐driven proton translocation was extremely low as compared to the wild type membranes. For further analysis, the ATP synthase was purified using a His‐tag attached at the amino‐terminus of the beta subunits. Rates of ATP hydrolysis and stimulation by LDAO were very similar to the wild type, but sensitivity to DCCD was still reduced. The possibility that the first transmembrane span fails to insert into the membrane was addressed by double mutants that contain unique cysteine substitutions in addition to the deletion. Cysteine residues that are normally found in the periplasm will be examined for localization by biotin‐maleimide labeling studies. This work was supported by grants from the NIH and the Welch Foundation.