Abstract
The 43.1-kDa tetracycline-cation/proton antiporter TetA from Tn10 comprises two equal-sized domains, alpha and beta (amino-terminal and carboxyl-terminal halves, respectively). An inactivating mutation in the alpha domain can complement a mutation on a second polypeptide in the beta domain to restore partial tetracycline resistance in bacterial cells, suggesting that intermolecular interactions permit this transport protein to act as a multimer. In the present studies, multimer formation was examined in mixtures of dodecylmaltoside extracts of membranes from Escherichia coli cells containing different TetA derivatives. TetA, TetA alpha, and TetA beta were each fused genetically to a six-histidine carboxyl-terminal tail. The ability of these fusions, immobilized on a nickel affinity column, to bind wild type TetA or other Tet fusions was determined. An interaction between alpha domains on different polypeptides which resulted in multimerization was seen. The binding was specific for Tet protein and did not occur with other membrane proteins or another polyhistidine fusion protein. No alpha-beta interactions were detected by this method, although they are postulated to occur in the intact cell based on the alpha-beta genetic complementations. A dimeric model for TetA having intermolecular alpha-alpha and alpha-beta interactions is presented.
Highlights
TetA(B), a cytoplasmic membrane protein encoded by Tn10, is a member of a family of related tetracycline efflux proteins in Gram-negative bacterial cells (1, 2)
The B/C and C/B chimeras together in the same cell, showed about 20% complementation of tetracycline resistance, indicating multimer formation (13). ␣- interaction was suggested by the ability of the cloned ␣ half to stabilize the cloned  half when both were present on separate polypeptides in the same cell (14)
The results showed that the Tet-Tet interaction might be between the ␣ domains of the two different polypeptides, or between an ␣ and a  domain, but possibly not between the  domains, since Tet279-LacZ had the entire ␣ domain but only the first two helices and associated loops of the  region. - Interactions Were Not Required for Multimer Formation—In Tet␣-6H the  domain is completely absent
Summary
Construction and Description of Plasmids—See Table I for summary of plasmids and Fig. 1 for diagrams of protein constructs. PACT7 (encoding T7 RNA polymerase regulated by the lacUV5 promoter) (Kan,R p15A origin) This plasmid (16) was used in trans with pET21b-Tet, pLY17, and pLY22. The same tetA PCR product used to make pET21b-Tet was restricted with EcoRI (in the central loop of TetA) and XhoI (at the end of TetA) and cloned into identically restricted pET21b. This put the TetA domain in-frame with both the upstream “T7 tag” and the downstream polyhistidine tail encoded by pET21b. A tetA PCR product having BamHI sites on each end was restricted with BamHI and cloned into BamHI-restricted pMAL-C2 (New England BioLabs) This created an in-frame fusion between maltose-binding protein MalE
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