Evaluation of the roles of the cytosolic N-terminus and His-rich loop of ZNT proteins using ZNT2 and ZNT3 chimeric mutants

Abstract

The physiological roles of Zn transporter (ZNT) proteins are being increasingly recognized, and three dimensional structures of ZNT bacterial homologs have facilitated our understanding of their biochemical characteristics at the molecular level. However, the biological role of the unique structural features of vertebrate ZNTs, which are absent in their bacterial homologues, is not completely understood. These ZNT sequences include a cytosolic His-rich loop between transmembrane helices IV and V and the cytosolic N-terminus. This study investigated the contribution of these features to zinc transport by ZNT proteins. The importance of the His residues in the cytosolic His-rich loop was investigated using ZNT2 Ala substitution and deletion mutants. The presence of His residues was not essential for zinc transport, even though they possibly participate in modulation of zinc transport activity. Furthermore, we determined the role of the N-terminus by characterizing ZNT2 and ZNT3 domain-swapped and deletion mutants. Unexpectedly, the N-terminus was also not essential for zinc transport by ZNT2 and the domain-swapped ZNT2 mutant, in which the cytosolic His-rich loop was substituted with that of ZNT3. These results provide molecular insights into understanding the roles of the cytosolic parts of ZNT2, ZNT3, and probably other members of their subgroup.

Figure 1

Alignment of ZNT2 and ZNT3 amino acid sequences of the cytosolic His-rich loop and the cytosolic N-terminus. (A) Alignment of the cytosolic His-rich loop of human ZNT2 (residues 191–227) and ZNT3 (residues 193–242). Predicted TM helices IV and V (based on YiiP) are labelled and His residues of the His-rich loop are highlighted in green. (B) Alignment of human ZNT2 (residues 1–98) and ZNT3 (residues 1–100) N-terminal sequences preceding the first TM helix. The sequence (Glu2 to His62) of ZNT2 deleted is shaded in gray. In (A) and (B), sequences of E. coli and S. oneidensis YiiP were also aligned for comparison. Amino acids identical between ZNT2 and ZNT3 sequences are indicated by *.

Figure 2

His residues of the cytosolic ZNT2 His-rich loop are not essential for zinc transport. (A) Zinc transport activity of znt1^−/−mt^−/−znt4^−/− cells expressing H197R or H205D ZNT2 SNP mutants (ZNT2_(H197R), ZNT2_(H205D)) was comparable to that of WT ZNT2-expressing cells. (B) Expressing ZNT2 mutants with single His to Ala substitution in the His-rich loop (ZNT2_(H197A), ZNT2_(H201A), ZNT2_(H203A), and ZNT2_(H205A)) in znt1^−/−mt^−/−znt4^−/− cells resulted in zinc transport activity comparable to that of WT ZNT2. Similar observations were made with the double substitution mutants (ZNT2_(H201AH203A), ZNT2_(H201AH205A), and ZNT2_(H203AH205A)) (C), whereas expression of ZNT2 mutants with three or four Ala-substituted His loop residues (ZNT2_(Loop3H-3A) and ZNT2_(Loop4H-4A)), moderately decreased zinc resistance (D). (E) Expression of ZNT2 mutant with the deleted cytosolic His-rich loop (ZNT2_(Δ201-205)) in znt1^−/−mt^−/−znt4^−/− cells did not confer zinc resistance. In experiments depicted in panels (A–E), cells were grown in the presence of indicated concentrations of ZnSO₄ and the number of surviving cells was estimated using the alamarBlue assay in triplicate (representative results shown). Expression of WT or mutant ZNT2 proteins in znt1^−/−mt^−/−znt4^−/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control. (F) Comparison of the stability of ZNT2_(Loop4H-4A) and ZNT2_(Δ201-205) mutants with those of WT ZNT2 or ZNT2_(G280R) mutant. The expression levels of each protein at each time point are shown, with representative results of immunoblotting depicted in the lower panel. The results of ZNT2_(G280R) is shown as a control for the destabilized ZNT2 mutant. Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control.

Figure 3

Domain swapping analysis of ZNT2 and ZNT3 on the ZNT2 backbone. (A) Expression of ZNT3 in znt1^−/−mt^−/−znt4^−/− cells did not confer significant resistance to high zinc concentrations, compared to the zinc resistance of cells expressing ZNT2. (B–D) Expression of ZNT2_(ZNT3Nter) in znt1^−/−mt^−/−znt4^−/− cells did not confer zinc resistance, whereas expression of ZNT2_(ZNT3Loop) and ZNT2_(ZNT3Cter) conferred zinc resistance, which was similar to that of WT ZNT2-expressing cells. In (D) *indicates the position of non-specific band. (E) Expression of ZNT2_{(ZNT3Nter,ZNT3Loop)} conferred zinc resistance in znt1^−/−mt^−/−znt4^−/− cells. (F) Stabilities of ZNT2_(ZNT3Loop), ZNT2_(ZNT3Nter), and ZNT2_{(ZNT3Nter,ZNT3Loop)} were evaluated as described in Fig. 2F. Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control. (G) Cells expressing ZNT2_(ZNT2-3Nter) showed zinc resistance similar to that of ZNT2-expressing cells, whereas cells expressing ZNT2_(ZNT3-2Nter) showed moderate resistance. In (A−E) and (G), the alamarBlue assay was performed as shown in Fig. 2. Stable expression of WT and mutant ZNT2 in znt1^−/−mt^−/−znt4^−/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control.

Figure 4

Domain swapping analysis of ZNT2 and ZNT3 on the ZNT3 backbone. (A) Expression of ZNT3_(ZNT2Nter), ZNT3_(ZNT2Loop), or ZNT3_(ZNT2Cter) in znt1^−/−mt^−/−znt4^−/− cells did not result in zinc resistance. (B) Cells expressing ZNT3_{(ZNT2Nter,ZNT2Loop)} or ZNT3_{(ZNT2Nter,ZNT2Cter)}, with two grafted cytosolic segments, showed almost no resistance to high zinc concentrations. In (A,B), alamarBlue assay was performed as shown in Fig. 2. Stable expression of WT and mutant ZNT2 in znt1^−/−mt^−/−znt4^−/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control.

Figure 5

Deletion of the ZNT2 cytosolic N-terminus did not result in loss of zinc transport function. (A) Expression of ZNT2_(Nter-del) and ZNT2_{(Nter-del,ZNT3Loop)} in znt1^−/−mt^−/−znt4^−/− cells conferred zinc resistance, similar to (with minor loss) the effect of ZNT2_{(Nter-del,Loop3H-3A)} expression (B). In (A,B), the alamarBlue assay was performed as shown in Fig. 2. Stable expression of WT and mutant ZNT2 in znt1^−/−mt^−/−znt4^−/− cells was confirmed by immunoblotting (lower sub-panels). Tubulin was used as the loading control. (C) The stabilities of ZNT2_(Nter-del), ZNT2_{(Nter-del,ZNT3Loop)}, and ZNT2_{(Nter-del,Loop3H-3A)} were evaluated as described in Fig. 2F. Data show mean ± SEM of triplicate experiments (lower sub-panels). Tubulin was used as the loading control. Asterisk (*) denotes a significant difference between WT ZNT2, and ZNT2_(Nter-del) mutant protein levels (P < 0.05 by Dunnett’s test).

Figure 6

Schematic representation of the main findings of this study. His residues of the cytosolic His-rich loop (A) and the N-terminus of ZNTs (B) are not essential but are required for ZNT-mediated zinc transport. Zinc transport activities are shown above the schematic models of each construct.