Analytical and Bioanalytical Chemistry

, Volume 408, Issue 27, pp 7745–7751 | Cite as

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

  • Qian Chai
  • Stacy R. Webb
  • Zhaoshuai Wang
  • Rebecca E. Dutch
  • Yinan Wei
Research Paper

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.

Keywords

Pulse chase Protein lifetime Integral membrane protein Azidohomoalanine Click chemistry Multidrug efflux pump 

Supplementary material

216_2016_9871_MOESM1_ESM.pdf (197 kb)
ESM 1(PDF 197 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Qian Chai
    • 1
  • Stacy R. Webb
    • 2
  • Zhaoshuai Wang
    • 1
  • Rebecca E. Dutch
    • 2
  • Yinan Wei
    • 1
  1. 1.Department of ChemistryUniversity of KentuckyLexingtonUSA
  2. 2.Department of Molecular & Cellular BiochemistryUniversity of KentuckyLexingtonUSA

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