title: Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase.
abstract: Subunit a is a membrane-bound stator subunit of the ATP synthase and is essential for proton translocation. The N-terminus of subunit a in E. coli is localized to the periplasm, and contains a sequence motif that is conserved among some bacteria. Previous work has identified mutations in this region that impair enzyme activity. Here, an internal deletion was constructed in subunit a in which residues 6-20 were replaced by a single lysine residue, and this mutant was unable to grow on succinate minimal medium. Membrane vesicles prepared from this mutant lacked ATP synthesis and ATP-driven proton translocation, even though immunoblots showed a significant level of subunit a. Similar results were obtained after purification and reconstitution of the mutant ATP synthase into liposomes. The location of subunit a with respect to its neighboring subunits b and c was probed by introducing cysteine substitutions that were known to promote cross-linking: a_L207C + c_I55C, a_L121C + b_N4C, and a_T107C + b_V18C. The last pair was unable to form cross-links in the background of the deletion mutant. The results indicate that loss of the N-terminal region of subunit a does not generally disrupt its structure, but does alter interactions with subunit b.
authors.affiliation: Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, OX1 3PU, UK.
authors.forename: Robert R
authors.initials: RR
authors.lastname: Ishmukhametov
authors.affiliation: Department of Biological Sciences, Southern Methodist University, PO Box 750376, Dallas, TX, 75275-0376, USA.
authors.forename: Jessica
authors.initials: J
authors.lastname: DeLeon-Rangel
authors.affiliation: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA.
authors.forename: Shaotong
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Biological Sciences, Southern Methodist University, PO Box 750376, Dallas, TX, 75275-0376, USA. svik@smu.edu.
authors.forename: Steven B
authors.initials: SB
authors.lastname: Vik
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D000595:Amino Acid Sequence; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017136:Ion Transport; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D029968:Escherichia coli Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; C074428:atpB protein, E coli; D003545:Cysteine
keywords: ATP synthase; ATPase; F1Fo; Proton translocation; Rotary motor; Subunit a