No abstract

In the cation diffusion facilitator (CDF) family, the transported substrates are confined to divalent metal ions, such as Zn 2+ , Fe 2+ , and Mn 2+ . However, this study identifies a novel CDF member designated MceT from the moderate halophile Planococcus dechangensis . MceT functions as a Na + (Li + , K + )/H + antiporter, together with its capability of facilitated Zn 2+ diffusion into cells, which have not been reported in any identified CDF transporters as yet. MceT is proposed to represent a novel CDF group, Na-CDF, which shares significantly distant phylogenetic relationship with three known CDF groups including Mn-CDF, Fe/Zn-CDF, and Zn-CDF. Variation of key function-related residues to “Y44-S48-Q150” in two structural motifs explains a significant discrimination in cation selectivity between Na-CDF group and three major known CDF groups. Functional analysis via site-directed mutagenesis confirms that MceT employs Q150, S158, and D184 for the function of MceT as a Na + (Li + , K + )/H + antiporter, and retains D41, D154, and D184 for its facilitated Zn 2+ diffusion into cells. These presented findings imply that MceT has evolved from its native CDF family function to a Na + /H + antiporter in an evolutionary strategy of the substitution of key conserved residues to “Q150-S158-D184” motif. More importantly, the discovery of MceT contributes to a typical transporter model of CDF family with the unique structural motifs, which will be utilized to explore the cation-selective mechanisms of secondary transporters.

Multidrug resistance (MDR) transporters of the major facilitator superfamily (MFS) were previously believed to drive the extrusion of multiple antimicrobial drugs through the coupling to proton translocation. Here, we present the identification of the first Na +coupled MFS-MDR transporter, MdrP, which also can achieve H +-coupled drug efflux independently of Na +. Importantly, we propose that MdrP can extrude norfloxacin in a mode of drug/Na + antiport, which has not yet been reported in any MFS member. On this basis, we further provide the insights into a novel Na + and H + coupling mechanism of MFS-MDR transporters, even for all secondary transporters. The most important finding lies in that D223 should mainly act as a key determinant in the Na + translocation coupled to norfloxacin efflux. Furthermore, our results partially modify the knowledge of the conformational stability-related residues in the motif A of MFS transporters and imply the importance of a new positively charged residue, R361, for the stabilization of outward-facing conformation of MFS transporters. These novel findings positively contribute to the knowledge of MFS-MDR transporters, especially about Na + and H + coupling mechanism. This study is based mainly on measurements in intact cells or everted membranes, and a biochemical assay with a reconstituted MdrP protein should be necessary to come to conclusion to be assured.