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 output: Contribution to journalArticlepeer-review

8 Scopus citations

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.

Original languageEnglish
Pages (from-to)7745-7751
Number of pages7
JournalAnalytical and Bioanalytical Chemistry
Volume408
Issue number27
DOIs
StatePublished - Nov 1 2016

Bibliographical note

Publisher Copyright:
© 2016, Springer-Verlag Berlin Heidelberg.

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

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