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Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase

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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.

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Acknowledgments

This work was supported by grant R01GM40508 from the National Institutes of Health (USA). The authors thank SMU students Uju Rochas, Mai Bedair and Damilola Salako for constructing some of the plasmids necessary for the final experiments.

This work was supported by grants from the NIH (GM-40508) and the Welch Foundation (N-1378) to S.B.V.

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Author notes

  1. Robert R. Ishmukhametov

    Present address: Clarendon Laboratory, Department of Physics, University of Oxford, Oxford, OX1 3PU, UK

  2. Shaotong Zhu

    Present address: Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX, 75390, USA

Authors and Affiliations

  1. Department of Biological Sciences, Southern Methodist University, PO Box 750376, Dallas, TX, 75275-0376, USA

    Jessica DeLeon-Rangel & Steven B. Vik

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  1. Robert R. Ishmukhametov
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Correspondence to Steven B. Vik.

Additional information

While this manuscript was under review a new structure of the E. coli ATP synthase was reported: Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states. Sobti M, Smits C, Wong AS, Ishmukhametov R, Stock D, Sandin S, Stewart AG. Elife. 2016 Dec 21;5. pii: e21598. doi:10.7554/eLife.21598

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Ishmukhametov, R.R., DeLeon-Rangel, J., Zhu, S. et al. Analysis of an N-terminal deletion in subunit a of the Escherichia coli ATP synthase. J Bioenerg Biomembr 49, 171–181 (2017). https://doi.org/10.1007/s10863-017-9694-z

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