title: Function of the MexB efflux-transporter divided into two halves.
abstract: The MexA-MexB-OprM efflux pump exports structurally and functionally diverse xenobiotics and confers multi-drug resistance on Pseudomonas aeruginosa cells. The MexB transporter traverses the inner membrane twelve times, bears two large periplasmic domains and has two homologous tandem repeats. To test whether two homologous halves of MexB function independently or interdependently, the protein was divided medially into two halves, each consisting of six amino- and carboxyl-proximal transmembrane segments. When two halves of MexB were coexpressed from independent open reading frames, the cells lacking chromosomal mexB exhibited restored antibiotic resistance at a level close to that in the cells producing a full-length MexB. In contrast, MexB protein containing either an amino- or carboxyl-half fragment failed to transport antibiotics. To test whether the amino- and carboxyl-proximal halves were present in a complex, we purified the histidine-tagged carboxyl-proximal half molecule using nickel-chelate chromatography from the cells that coexpressed two halves. The results showed that the nonhistidine-tagged amino-proximal half was co-purified with the carboxyl-proximal half, thereby indicating that the amino-proximal half fragment was tightly associated with the carboxyl-proximal half molecule. These findings suggest that the presence of both amino- and carboxyl-halves of MexB in a complex is essential for transport activity.
authors.affiliation: Department of Molecular Life Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, 259-1193, Japan.
authors.forename: Shima
authors.initials: S
authors.lastname: Eda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Yoneyama
authors.forename: Taiji
authors.initials: T
authors.lastname: Nakae
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004352:Drug Resistance, Microbial; D015151:Immunoblotting; D026901:Membrane Transport Proteins; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000187:drug effects / Q000235:genetics; D015262:Xenobiotics / Q000378:metabolism / Q000493:pharmacokinetics
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C118597:MexB protein, Pseudomonas aeruginosa; D015262:Xenobiotics