ABSTRACT
Recent investigations of rodent Tmem163 protein suggest that it binds to and transports zinc as a dimer, and that alanine mutagenesis of its two conserved aspartate (D123A/D127A) residues perturbs its function. Direct evidence, however, is lacking whether it is an influx or efflux transporter. We hypothesized that human TMEM163, like its rodent counterpart, is a zinc effluxer. Using cultured human cell lines that either stably or transiently expressed TMEM163 protein, we determined the direction of zinc flux using two distinct fluorescent dyes. Spectrofluorometric assays showed that TMEM163-expressing cell lines loaded with zinc markedly reduced intracellular zinc levels when compared with controls. The specificity of the fluorescence signal was confirmed upon treatment with TPEN, a high-affinity zinc chelator. To further dissect the function of TMEM163, we performed alanine mutagenesis of two similarly conserved aspartate residues (D124A/D128A) to inactivate zinc efflux. We also performed site-directed mutagenesis of other conserved residues in TMEM163 (i.e. S61R, S95C, S193P, and E286K) to test their effects on the function of TMEM163. We found a significant reduction of zinc efflux activity upon expression of D124A/D128A or E286K mutation. Taken together, our findings demonstrate that TMEM163 is a zinc effluxer that contributes to zinc homeostasis in cells.
Background Previous studies using rodent Tmem163 showed that it binds and transports zinc. However, it is not known whether the protein serves as an influx or efflux transporter. Using human TMEM163, we set out to show its function in cells.
Results Zinc flux assays using Newport Green and Fluozin-3 fluorescence dyes revealed that cells stably or transiently expressing TMEM163 exhibited marked decrease of cytoplasmic zinc levels. Site-directed mutagenesis of key amino acid residues known as zinc-binding sites or are predicted as post-translational modification sites disrupted zinc efflux.
Conclusion TMEM163 is a zinc efflux transporter, but is predicted to be structurally distinct to known ZnT efflux transporters.
Significance TMEM163 may represent a new subclass member of the cation diffusion facilitator family of zinc transporter that is vital for zinc homeostasis in specific cell types.