Reconstitution of the metal‐tetracycline/H+ antiporter of Escherichia coli in proteoliposomes including F0F1‐ATPase

Someya, Yuichi; Moriyama, Yoshinori; Futai, Masamitsu; Sawai, Tetsuo; Yamaguchi, Akihito

doi:10.1016/0014-5793(95)01079-t

Cited by 10 publications

(6 citation statements)

References 29 publications

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“…Thus, NorA itself can contribute to drug resistance and in some conditions may be sufficient alone to cause drug resistance. Other multidrug transporters functionally reconstituted in proteoliposomes include Smr of S. aureus (8), LmrP and LmrA of Lactococcus lactis (14,23), EmrE of E. coli (31), and mammalian P-glycoprotein (25), in addition to the unidrug transporter TetA of E. coli (26).…”

Section: Discussionmentioning

confidence: 99%

NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

Grinius

Hooper

2002

J Bacteriol

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Section: Discussionmentioning

confidence: 99%

NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

Grinius

Hooper

2002

J Bacteriol

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“…It is also the first successful reconstitution among the family of 14-transmembrane segment, drug-resistance antiporters. Two other purified preparations of Tet proteins, both of the Gram-negative TetA(B) version, have been described (27,28), but the only reported reconstitution of Tc transport activity necessitated the coreconstitution of an F 1 F 0 -ATPase complex to effect successful energization (28). That co-reconstituted TetA(B) preparation had modest activity compared with the activity assayed in membrane vesicles.…”

Section: Discussionmentioning

confidence: 99%

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H ⁺ and Na ⁺ (K ⁺ )/H ⁺ exchange

Cheng

Hicks

Krulwich

1996

Proc. Natl. Acad. Sci. U.S.A.

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Recent work has suggested that the chromosomally encoded TetA(L) transporter of Bacillus subtilis, for which no physiological function had been shown earlier, not only confers resistance to low concentrations of tetracycline but is also a multifunctional antiporter protein that has dominant roles in both Na ؉ -and K ؉ -dependent pH homeostasis and in Na ؉ resistance during growth at alkaline pH. To rigorously test this hypothesis, TetA(L) has been purified with a hexahistidine tag at its C terminus and reconstituted into proteoliposomes. The TetA(L)-hexahistidine proteoliposomes exhibit high activities of tetracycline-cobalt͞H ؉ , Na ؉ ͞ H ؉ , and K ؉ ͞H ؉ antiport in an assay in which an outwardly directed proton gradient is artificially imposed and solute uptake is monitored. Tetracycline uptake depends on the presence of cobalt and vice versa, with the cosubstrates being transported in a 1:1 ratio. Evidence for the electrogenicity of both tetracycline-cobalt͞H ؉ and Na ؉ ͞H ؉ antiports is presented. K ؉ and Li ؉ inhibit Na ؉ uptake, but there is little cross-inhibition between Na ؉ and tetracycline-cobalt uptake activities. The results strongly support the conclusion that TetA(L) is a multifunctional antiporter. They expand the roster of such porters to encompass one with a complex organic substrate and monovalent cation substrates that may have distinct binding domains, and provide the first functional reconstitution of a member of the 14-transmembrane segment transporter family.

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“…In order to elucidate the structure, function, and mechanism of multidrug transporters at the molecular level, methods have been developed for overexpression, purification, and functional reconstitution into liposomes (72,73,147,203,227,275,280). Although the overexpression of membrane proteins is often deleterious for the cells (48,119,203,243), drug transporters could be overexpressed up to 5 to 30% of total membrane protein using homologous expression systems and inducible promoters, such as the T7 polymerase promoter (275), the lac promoter (236), and the nisA promoter (147,203). The first step in the purification of membrane proteins involves extraction of the protein from the membrane.…”

Section: Reconstitutionmentioning

confidence: 99%

Molecular Properties of Bacterial Multidrug Transporters

Putman

Veen

Konings

2000

Microbiol Mol Biol Rev

695

586

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SUMMARY One of the mechanisms that bacteria utilize to evade the toxic effects of antibiotics is the active extrusion of structurally unrelated drugs from the cell. Both intrinsic and acquired multidrug transporters play an important role in antibiotic resistance of several pathogens, including Neisseria gonorrhoeae, Mycobacterium tuberculosis, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Vibrio cholerae. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors which block the multidrug transporter and allow traditional antibiotics to be effective. This review gives an extensive overview of the currently known multidrug transporters in bacteria. Based on energetics and structural characteristics, the bacterial multidrug transporters can be classified into five distinct families. Functional reconstitution in liposomes of purified multidrug transport proteins from four families revealed that these proteins are capable of mediating the export of structurally unrelated drugs independent of accessory proteins or cytoplasmic components. On the basis of (i) mutations that affect the activity or the substrate specificity of multidrug transporters and (ii) the three-dimensional structure of the drug-binding domain of the regulatory protein BmrR, the substrate-binding site for cationic drugs is predicted to consist of a hydrophobic pocket with a buried negatively charged residue that interacts electrostatically with the positively charged substrate. The aromatic and hydrophobic amino acid residues which form the drug-binding pocket impose restrictions on the shape and size of the substrates. Kinetic analysis of drug transport by multidrug transporters provided evidence that these proteins may contain multiple substrate-binding sites.

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Reconstitution of the metal‐tetracycline/H⁺ antiporter of Escherichia coli in proteoliposomes including F₀F₁‐ATPase

Cited by 10 publications

References 29 publications

NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H ⁺ and Na ⁺ (K ⁺ )/H ⁺ exchange

Molecular Properties of Bacterial Multidrug Transporters

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Reconstitution of the metal‐tetracycline/H+ antiporter of Escherichia coli in proteoliposomes including F0F1‐ATPase

Cited by 10 publications

References 29 publications

NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H + and Na + (K + )/H + exchange

Molecular Properties of Bacterial Multidrug Transporters

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Reconstitution of the metal‐tetracycline/H⁺ antiporter of Escherichia coli in proteoliposomes including F₀F₁‐ATPase

The purified Bacillus subtilis tetracycline efflux protein TetA(L) reconstitutes both tetracycline–cobalt/H ⁺ and Na ⁺ (K ⁺ )/H ⁺ exchange