Importance of the GP dipeptide of the antiporter motif and other membrane-embedded proline and glycine residues in tetracycline efflux protein Tet(L)

Abstract

Proline and glycine residues are well represented among functionally important residues in hydrophobic domains of membrane transport proteins, and several critical roles have been suggested for them. Here, the effects of mutational changes in membrane-embedded proline and glycine residues of Tet(L) were examined, with a focus on the conserved GP(155,156) dipeptide of motif C, a putative "antiporter motif". Mutation of Gly155 to cysteine resulted in a mutant Tet(L) that bound its tetracycline-divalent metal (Tc-Me2+) substrate but did not catalyze efflux or exchange of Tc-Me2+ or catalyze uptake or exchange of Rb+ which was used to monitor the coupling ion. These results support suggestions that this region is involved in the conformational changes required for translocation. Mutations in Pro156 resulted in reduction (P156G) or loss (P156A or P156C) of Tc-Me2+ efflux capacity. All three Pro156 mutants exhibited a K+ leak (monitored by 86Rb+ fluxes) that was not observed in wild-type Tet(L). A similar leak was observed in a mutant in a membrane-embedded proline residue elsewhere in the Tet(L) protein (P175C) as well as in a P156C mutant of related antiporter Tet(K). These findings are consistent with roles proposed for membrane-embedded prolines in tight helix packing. Patterns of Tc resistance conferred by additional Tet(L) mutants indicate important roles for another GP dipeptide in transmembrane segment (TMS) X as well as for membrane-embedded glycine residues in TMS XIII.

Figure 1

A topological model of Tet(L) highlighting the residues functionally probed in this study. The topology is based on the experimental work of others on Tet(K) (28, 29). The gray bars indicate the positions of Motifs described by Paulsen et al. (17) that are conserved either in both 12-TMS and 14-TMS Drug/H⁺ families (Motifs A,B,C) or conserved only in 14-TMS (Motifs D1, H, E, F).

Figure 2

Transport assays of Tet(L) Gly¹⁵⁵ mutants in comparison with wild type Tet(L). Left panel: Uptake of [³H]Tc-Co²⁺ into everted vesicles from the indicated transformants of E. coli DH5α were carried out as described under Materials and Methods. The values shown, with standard deviations, are the averages of duplicate determinations on at least two independent vesicle preparations. The values shown were corrected for the binding control of de-energized (CCCP-treated) vesicles from each of the same strains. Right panels: Uptake of ⁸⁶Rb⁺ was assayed in RSO vesicles of transformants expressing the same plasmids used in assays of [³H]Tc-Co² uptake but the bacterial strain was E. coli TK2420. The results for each transformant are shown in separate panels because assays were conducted under several different conditions that distinguish an electrogenic leak from antiport (the conditions are listed in the box above the wild type panel).

Figure. 3

Assays of [³H]Tc-Co²⁺:Tc-Co²⁺ and K⁺-⁸⁶Rb⁺: K⁺-Rb⁺ exchange in RSO vesicles expressing Tet(L) G155C. Left: A diagram of the reaction cycle for Tet(L): E_i and E_o respectively represent the inward and outward facing conformations of the transporter; A is the efflux substrate (e.g. Tc-Co2⁺) and B is a coupling ion which could be either H⁺ or Rb⁺-K⁺. The B:A ratio is greater than unity (42); the precise stoichiometry is not known and is shown here as 2B:A for diagrammatic purposes. The reaction cycle illustrates an alternating access or ping-pong model in which efflux substrate A binds to E_i in step 1 followed by hypothetical tight binding (step 2) that may be detected in the G155C mutant because of a defect in subsequent translocation (step 3) that usually occurs immediately after tight binding. This is followed by hypothesized release from tight binding (step 4) and final substrate release (step 5). The Tc:Co²⁺ exchange reaction assesses this set of reactions (steps 1-5), starting with unlabeled Tc:Co²⁺ inside and labeled Tc:Co²⁺ outside and proceeding forward and reverse. A comparable half reaction, going in both directions, with the top half cycle (reactions 6-10, dotted box) is assessed by the K⁺-⁸⁶Rb⁺: K⁺-Rb⁺ exchange. Middle and Right: the experimental data for the two exchange reactions. The assays were carried out as described under Materials and Methods in comparison with wild type Tet(L) and control membranes. The data are the averages from two independent preparations and are shown with standard deviations.

Figure 4

Transport assays of Tet(L) Pro¹⁵⁶ mutants in comparison with wild type Tet(L). The assays were precisely as described in the legend to Figure 2.

Figure 5

Assays of ⁸⁶Rb⁺ uptake by RSO vesicles of E. coli TK2420 expressing wild type Tet(K) or Tet(K) mutants G155C or P156C. Assays were conducted as described for the right-hand panels of Figure 2.

Figure 6

Assays of ⁸⁶Rb⁺ uptake by RSO vesicles of E. coli TK2420 expressing wild type Tet(L) or Tet(L) P175C. Assays were conducted as described for the right-hand panels of Figure 2.

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