Abstract
In this work, we report the phenotypic and biochemical effects of deleting the C-terminal cytoplasmic portion of the NhaP2 cation/proton antiporter from Vibrio cholerae. While the deletion changed neither the expression nor targeting of the Vc-NhaP2 in an antiporter-less Escherichia coli strain, it resulted in a changed sensitivity of the host to sodium ions at neutral pH, indicating an altered Na+ transport through the truncated variant. When assayed in inside-out sub-bacterial vesicles, the truncation was found to result in greatly reduced K+/H+ and Na+/H+ antiport activity at all pH values tested and a greater than fivefold decrease in the affinity for K+ (measured as the apparent K m) at pH 7.5. Being expressed in trans in a strain of V. cholerae bearing a chromosomal nhaP2 deletion, the truncated nhaP2 gene was able to complement its inability to grow in potassium-rich medium at pH 6.0. Thus the residual K+/H+ antiport activity associated with the truncated Vc-NhaP2 was still sufficient to protect cells from an over-accumulation of K+ ions in the cytoplasm. The presented data suggest that while the cytoplasmic portion of Vc-NhaP2 is not involved in ion translocation directly, it is necessary for optimal activity and substrate binding of the Vc-NhaP2 antiporter.
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Acknowledgments
This research was supported by Grants from the Natural Sciences and Engineering Research Council of Canada (to JS, PD, JLW, and CTR). J. Stetefeld is a Canada Research Council Chair in Structural biology.
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EJW, CTR and GLO performed most of the experiments, contributed to data organization and manuscript review; JLW performed part of the gene construction, as well as growth experiments, reviewed the manuscript; JS designed some experiments, critically reviewed data, contributed to the writing of manuscript; PD designed most of the experiments, critically edited data, wrote and reviewed manuscript.
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Wiens, E.J., Winogrodzki, J.L., Resch, C.T. et al. The C-terminal cytoplasmic portion of the NhaP2 cation–proton antiporter from Vibrio cholerae affects its activity and substrate affinity. Mol Cell Biochem 389, 51–58 (2014). https://doi.org/10.1007/s11010-013-1926-x
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DOI: https://doi.org/10.1007/s11010-013-1926-x