title: Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method.
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.
authors.affiliation: Department of Chemistry, University of Kentucky, 305 Chemistry-Physics Building, Lexington, KY, 40506, USA.
authors.forename: Qian
authors.initials: Q
authors.lastname: Chai
authors.affiliation: Department of Molecular & Cellular Biochemistry, University of Kentucky, Lexington, KY, 40536, USA.
authors.forename: Stacy R
authors.initials: SR
authors.lastname: Webb
authors.affiliation: Department of Chemistry, University of Kentucky, 305 Chemistry-Physics Building, Lexington, KY, 40506, USA.
authors.forename: Zhaoshuai
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Molecular & Cellular Biochemistry, University of Kentucky, Lexington, KY, 40536, USA.
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Dutch
authors.affiliation: Department of Chemistry, University of Kentucky, 305 Chemistry-Physics Building, Lexington, KY, 40506, USA. yinan.wei@uky.edu.
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D057930:Click Chemistry / Q000379:methods; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D008074:Lipoproteins / Q000172:deficiency / Q000235:genetics*; D026901:Membrane Transport Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000172:deficiency / Q000235:genetics*; D011485:Protein Binding; D055550:Protein Stability; D059748:Proteolysis; D013194:Staining and Labeling / Q000379:methods*; D013462:Sulfur Radioisotopes
chemical_list: D000409:Alanine; D000900:Anti-Bacterial Agents; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008074:Lipoproteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D013462:Sulfur Radioisotopes; C083497:AcrA protein, E coli; C083496:AcrB protein, E coli; C506098:azidohomoalanine; C073126:tolC protein, E coli
keywords: Azidohomoalanine; Click chemistry; Integral membrane protein; Multidrug efflux pump; Protein lifetime; Pulse chase