Transmembrane 163 (TMEM163) protein effluxes zinc

Abstract

Recent investigations of rodent Tmem163 suggest that it binds to and transports zinc as a dimer, and that alanine mutagenesis of its two species-conserved aspartate (D123A/D127A) residues proposed to bind zinc, perturbs protein function. Direct corroboration, however, is lacking whether it is an influx or efflux transporter in cells. We hypothesized that human TMEM163 is a zinc effluxer based on its predicted protein characteristics. We used cultured human cell lines that either stably or transiently expressed TMEM163, and pre-loaded the cells with zinc to determine transport activity. We found that TMEM163-expressing cells exhibited significant reduction of intracellular zinc levels as evidenced by two zinc-specific fluorescent dyes and radionuclide zinc-65. The specificity of the fluorescence signal was confirmed upon treatment with TPEN, a high-affinity zinc chelator. Multiple sequence alignment and phylogenetic analyses showed that TMEM163 is related to distinct members of the cation diffusion facilitator (CDF) protein family. To further characterize the efflux function of TMEM163, we substituted alanine in two homologous aspartate residues (D124A/D128A) and performed site-directed mutagenesis of several conserved amino acid residues identified as non-synonymous single nucleotide polymorphism (S61R, S95C, S193P, and E286K). We found a significant reduction of zinc efflux upon cellular expression of D124A/D128A or E286K protein variant when compared with wild-type, suggesting that these particular amino acids are important for normal protein function. Taken together, our findings demonstrate that TMEM163 effluxes zinc, and it should now be designated ZNT11 as a new member of the mammalian CDF family of zinc efflux transporters.

Keywords: Cation diffusion facilitator; SV31; Single nucleotide polymorphism; Zinc transporter; ZnT protein.

Fig. 1.. Functional expression of TMEM163 stably expressed in HeLa cells.

A) Cell membrane impermeant Fluozin-3 (MI-FZ3) fluorescence analysis revealed a significant increase in extracellular RFU levels in the milieu of cells expressing TMEM163-mCherry upon exposure to zinc. Data are represented as mean ± SEM (****p < 0.0001, Student’s t-test, unpaired, two-tailed; n = 5 independent trials). B) Cell membrane permeant Newport Green (MP-NG) fluorescence analysis revealed a significant decrease in intracellular RFU levels of cells expressing TMEM163-mCherry upon exposure to zinc when compared with cells expressing the pmCherry vector control. Data are represented as mean ± SEM of each kinetic time point analyzed (*p = 0.01; Student’s t-test, unpaired, two-tailed; n = 6 independent trials). C) Concentration–d-dependent and saturable uptake of ⁶⁵Zn by TMEM163-and mCherry-expressing cells. The presence of TMEM163 resulted in significant reduction of intracellular ⁶⁵Zn levels (p = 0.01; Student’s t-test, paired, two-tailed; n = 3 independent trials).

Fig. 2.. Multiple sequence alignment of TMEM163 and select CDF family members.

A) Clustal alignment of TMEM163 and CDF amino acid sequences showing predicted transmembrane domains 1 through 6 (TM1-TM6; bold text). The alignment was done using the MAFFT website (https://mafft.cbrc.jp/alignment/server/) for the following full-length protein sequences obtained from NCBI: CzcD (YP_002515680.1), TMEM163 (NP_112185.1), YiiP (NP_462942), ZitB (WP_000951292), ZNT1 (NP_067017.2), ZNT2 (NP_001004434.1), ZNT3 (NP_003450.2), ZNT4 (NP_037441.2), ZNT5 (NP_075053.2), ZNT6 (NP_001180442.1), ZNT7 (NP_598003.2), ZNT8 (NP_776250.2), ZNT9 (NP_006336.3), and ZNT10 (NP_061183.2). For simplicity, only TM domains (bold text) and consensus or conserved amino acid residues predicted to bind zinc ions are shown. Histidine (green), Aspartic acid (red), and Glutamic acid (blue). B) Phylogram of the evolutionary relationship among specific CDF family members and TMEM163 protein. The phylogenetic tree diagram was generated using the Java-based Archaeopteryx visualization and analysis of phylogenetic trees via the MAFFT web interface.

Fig. 2.. Multiple sequence alignment of TMEM163 and select CDF family members.

A) Clustal alignment of TMEM163 and CDF amino acid sequences showing predicted transmembrane domains 1 through 6 (TM1-TM6; bold text). The alignment was done using the MAFFT website (https://mafft.cbrc.jp/alignment/server/) for the following full-length protein sequences obtained from NCBI: CzcD (YP_002515680.1), TMEM163 (NP_112185.1), YiiP (NP_462942), ZitB (WP_000951292), ZNT1 (NP_067017.2), ZNT2 (NP_001004434.1), ZNT3 (NP_003450.2), ZNT4 (NP_037441.2), ZNT5 (NP_075053.2), ZNT6 (NP_001180442.1), ZNT7 (NP_598003.2), ZNT8 (NP_776250.2), ZNT9 (NP_006336.3), and ZNT10 (NP_061183.2). For simplicity, only TM domains (bold text) and consensus or conserved amino acid residues predicted to bind zinc ions are shown. Histidine (green), Aspartic acid (red), and Glutamic acid (blue). B) Phylogram of the evolutionary relationship among specific CDF family members and TMEM163 protein. The phylogenetic tree diagram was generated using the Java-based Archaeopteryx visualization and analysis of phylogenetic trees via the MAFFT web interface.

Fig. 3.. Spectrofluorometric assay and fluorescence microscopy of HEK-293 cells transiently expressing wild-type TMEM163, TMEM163-D124A/128A, ZIP1, and pmCherry empty vector control.

A) Cell membrane impermeant Fluozin-3 fluorescence analysis reveals a significant increase in extracellular fluorescence upon expression of TMEM163 protein when compared with controls. Data are represented as mean ± SEM of each kinetic time point analyzed (**p=0.002, ****p < 0.0001; Tukey’s multiple comparisons post-hoc test; n ≥ 5 independent trials). B) Representative fluorescent micrographs showing subcellular distribution of pmCherry (mCh) empty vector, TMEM163-mCherry, ZIP1-mCherry, and TMEM163-D124A/D128A-mCherry. TMEM163 and TMEM163-D124A/D128A localize within the plasma membrane and membrane compartments (arrowhead). ZIP1 is also detected within the plasma membrane and intracellular membrane compartment (arrowhead). The pmCherry vector exclusively localizes in the cytoplasm (arrow). Scale bar: 50 μm.

Fig. 4.. Spectrofluorometric assay and fluorescence microscopy of HEK-293 cells transiently expressing wild-type TMEM163 and non-synonymous SNPs: S61R, S95C, S193P, and E286K.

A) Cell membrane impermeant Fluozin-3 fluorescence analysis reveals a significant increase in extracellular fluorescence upon expression of TMEM163 protein. Non-synonymous SNPs negatively affect the efflux function of TMEM163, particularly the E286K clone. Data are represented as mean ± SEM of each kinetic time point analyzed (*p=0.04, **p=0.004, ***p = 0.0004; Tukey’s multiple comparisons post-hoc test; n ≥ 4 independent trials). B) Representative fluorescent micrographs of expression constructs with mCherry (mCh) fluorescent protein tag (top panel) and Myc-DDK peptide tag probed with Alexa Fluor-488 (A488) (bottom panel). TMEM163-S61R, TMEM163-S95C, and TMEM163-E286K exhibit localization within the plasma membrane and intracellular membrane compartment (arrowhead), while TMEM163-S193P shows mostly cytoplasmic and compartmental localization (arrow). Scale bar: 50 μm.

Fig. 5.. Schematic diagram summarizing the main observations on the zinc efflux function of TMEM163.

Heterologous expression of the wild-type TMEM163 results in cytoplasmic zinc extrusion in the presence of high zinc levels inside the cells. Expression of the non-synonymous SNPs S61R, S95C, and S193P produces marked reduction of efflux activity when compared with wild-type TMEM163. S61R showed altered protein band migration pattern on WB, suggesting that S61 is a PTM target. S193P is mostly expressed in the cytoplasm and MC as evidenced by CSB and WB, while the other variants are found mostly in the PM and MC. The E286K protein variant has the lowest efflux activity in comparison with other SNPs and wild-type TMEM163. Similar to E283K, the D124A/D128A protein variant has comparably the lowest efflux activity relative to the wild-type TMEM163 and other SNPs. PM, plasma membrane; MC, membrane compartment.