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Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method

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Abstract

Proteins are constantly synthesized and degraded in living cells during their growth and division, often in response to metabolic and environmental conditions. The synthesis and breakdown of proteins under different conditions reveal information about their mechanism of function. The metabolic incorporation of non-natural amino acid azidohomoalanine (AHA) and subsequent labeling via click chemistry emerged as a non-radioactive strategy useful in the determination of protein kinetics and turnover. We used the method to monitor the degradation of two proteins involved in the multidrug efflux in Escherichia coli, the inner membrane transporter AcrB and its functional partner membrane fusion protein AcrA. Together they form a functional complex with an outer membrane channel TolC to actively transport various small molecule compounds out of E. coli cells. We found that both AcrA and AcrB lasted for approximately 6 days in live E. coli cells, and the stability of AcrB depended on the presence of AcrA but not on active efflux. These results lead to new insight into the multidrug resistance in Gram-negative bacteria conferred by efflux.

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Acknowledgments

This work was supported by the National Science Foundation (MCB 1158036, YW) and National Institute of Allergy and Infectious Diseases (1R21AI103717, YW; R01 AI051517, RED; and F31 fellowship AI120653-01, SRW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Authors and Affiliations

  1. Department of Chemistry, University of Kentucky, 305 Chemistry-Physics Building, Lexington, KY, 40506, USA

    Qian Chai, Zhaoshuai Wang & Yinan Wei

  2. Department of Molecular & Cellular Biochemistry, University of Kentucky, Lexington, KY, 40536, USA

    Stacy R. Webb & Rebecca E. Dutch

Authors
  1. Qian Chai
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  2. Stacy R. Webb
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  3. Zhaoshuai Wang
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  4. Rebecca E. Dutch
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  5. Yinan Wei
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Correspondence to Yinan Wei.

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Chai, Q., Webb, S.R., Wang, Z. et al. Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method. Anal Bioanal Chem 408, 7745–7751 (2016). https://doi.org/10.1007/s00216-016-9871-7

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  • DOI: https://doi.org/10.1007/s00216-016-9871-7

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