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.

Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter

“…there are no direct interactions between the zinc ion (or its first hydration shell water molecules) and the histidine side chains of the GHGHSH motif. A very recent publication strengthen our findings, showing that deletion or substitution of these His residues to Ala in ZnT2 did not affect zinc transport activity [ 71 ]. However, an allosteric regulatory role of this motif cannot be excluded.…”

Zinc transporter 1 (ZNT1) expression on the cell surface is elaborately controlled by cellular zinc levels

“…We first confirmed whether our anti-ZNT1 mAb, which was generated against the C-terminal cytosolic portion of the protein, specifically detects ZNT1 using an N-terminally FLAGtagged ZNT1 (FLAG-ZNT1) protein, because it detected ZNT1 at a greater molecular size than that calculated based on its cDNA in our previous study (26 -28). The anti-ZNT1 mAb detected FLAG-ZNT1 stably expressed in DT40 cells deficient in znt1, mt, and znt4 (znt1 (27,29) as two bands (mainly ϳ75 and ϳ63 kDa) in immunoblotting (Fig. 1A, left), which was also detected in the same manner by an anti-FLAG antibody (Fig.…”

Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a ‘locked‐in’ configuration

“…The fine tuning in the regulation might be evolution‐related, since more highly evolved organisms emerge more sophisticated and complex regulation mechanisms. For example, recent studies of eukaryotic CDF proteins (plant MTP proteins and human ZnT proteins) suggest that other elements such as a His‐rich loop – a cytoplasmatic loop between two transmembrane helices that is not found in the structurally characterized CDF proteins – might play a role in protein regulation . Further biochemical and structural studies of CDF proteins that are not Zn 2+ or Fe 2+ transporters proteins, and of eukaryotic CDF proteins, would be required to decipher the evolutionary pathway of the regulation mechanism in CDF proteins.…”