Zinc transporters and their functional integration in mammalian cells

Abstract

Zinc is a ubiquitous biological metal in all living organisms. The spatiotemporal zinc dynamics in cells provide crucial cellular signaling opportunities, but also challenges for intracellular zinc homeostasis with broad disease implications. Zinc transporters play a central role in regulating cellular zinc balance and subcellular zinc distributions. The discoveries of two complementary families of mammalian zinc transporters (ZnTs and ZIPs) in the mid-1990s spurred much speculation on their metal selectivity and cellular functions. After two decades of research, we have arrived at a biochemical description of zinc transport. However, in vitro functions are fundamentally different from those in living cells, where mammalian zinc transporters are directed to specific subcellular locations, engaged in dedicated macromolecular machineries, and connected with diverse cellular processes. Hence, the molecular functions of individual zinc transporters are reshaped and deeply integrated in cells to promote the utilization of zinc chemistry to perform enzymatic reactions, tune cellular responsiveness to pathophysiologic signals, and safeguard cellular homeostasis. At present, the underlying mechanisms driving the functional integration of mammalian zinc transporters are largely unknown. This knowledge gap has motivated a shift of the research focus from in vitro studies of purified zinc transporters to in cell studies of mammalian zinc transporters in the context of their subcellular locations and protein interactions. In this review, we will outline how knowledge of zinc transporters has been accumulated from in-test-tube to in-cell studies, highlighting new insights and paradigm shifts in our understanding of the molecular and cellular basis of mammalian zinc transporter functions.

Keywords: endoplasmic reticulum stress (ER stress); enzyme processing; protein–protein interaction; proteomics; signal transduction; transport metal; transporter; zinc.

Figure 1

Two complementary families of mammalian zinc transporters. A, subcellular localization of ZIP (light pink) and ZnT (light blue) that are generally thought to be zinc-specific transporters. ZIP8 and ZIP14 (gold) and ZnT10 (pale green) transport manganese in addition to zinc. B, phylogenetic trees of ZnT (upper) and ZIP (lower). C, sequence alignments of key coordinating residues in the transport sites in bacterial YiiP (upper) and ZIPB (lower). Light coral shading highlights conserved His residues. Light blue shading highlights conserved Asp and Glu residues. His and Asp residues are reversed in TM4 of ZIP13. Pale turquoise shading highlights the conserved Cys residue in cell surface located LIV-1 family members of ZIPs that have been postulated to bind to the N-terminal CPALLY motif. Light green shading highlights loss of the conserved His, which in the case of ZnT10, ZIP8, and ZIP14 results in loss of zinc specificity.

Figure 2

Structural basis for zinc selectivity and mobility. A, crystal structure of YiiP and close-up views of zinc binding sites in TMD (upper) and at the CTD interface (lower). Red arrows indicate directions of zinc transport. TMs and coordinating residues are labeled. B, crystal structure of ZIPB and close-up views of the binuclear metal center with a Zn and Cd ion (upper) and two Cd ions (lower). C, schematic model depicting zinc-for-proton exchange in YiiP. ΔΦ denotes the membrane potential. The tetrahedral transport site in YiiP is formed by D45, D57, H153, and D157. The conserved L152 is a part of a hydrophobic gate between two solvent-filled cavities. D, water-mediated diffusion of metal ions in ZIPB.

Figure 3

ZIP-centric interactome. A, topological structures of ZIPs with increased His-abundance in ZIP6 and ZIP10. His residues in the N-terminus, extracellular loop TM2-3 and intracellular loop TM3-4 are shown in light green with those greater than 20 are highlighted in light pink. TMs are numbered 1–8. The CPALLY motif and the PEST cleavage sites are indicated. Note, conserved His residues in TM2 and TM5 of ZIP14 and ZIP8 are replaced with Q and E, respectively. B, zinc-mediated disassembly of focal adhesion complexes. Cell attachment is provided by NCAM1 binding to integrin, which is attached to the extracellular matrix and tubulin bundle when the integrin is phosphorylated by the active form of GSK3. During EMT, a ZIP6–ZIP10 heterodimer in the focal adhesion complex promotes zinc influx, which is captured by the cytosolic His-rich loop between TM3 and TM4 of ZIP6 and ZIP10. An increase of the local zinc concentration inhibits GSK3 (lighter blue), resulting in reduced NCAM1 phosphorylation. A reduced phospho-occupancy of NCAM1 sites promotes integrin disassociation from the NCAM1-bound focal adhesion complex, triggering cell detachment. PrP denotes prion protein domain. 14-3-3 proteins are proposed to engage the NCAM1–integrin interaction.

Figure 4

Zinc regulation of receptor signaling. Zinc is an activator of diverse protein kinases (PKs) and also a reversible inhibitor of tyrosine phosphatases (TPs). The dual controls of PKs and TPs by the cytosolic zinc concentration improve the fidelity of signal transduction in a phosphorylation cascade leading to transcription factor activation and gene expression. CK2 activates ZIP7 by phosphorylation at Ser residues in the cytosolic His-rich loop, triggering zinc release from the ER store. The increased local zinc concentration upregulates the CK2 activity in a positive feedback loop of ZIP7 activation. Likewise, zinc-dependent PKs, CK2, and GSK3 may activate ZIPs in the plasma membrane to increase zinc influx. Increased local zinc concentrations activate CK2, but inhibit GSK3 to regulate zinc influx in a positive and negative feedback loop, respectively. An increase of the cytosolic zinc concentration could inhibit TPs to shift the balance of ZIP activity to a more activated (phosphorylated) form to augment zinc influx. The depleted ER zinc store may be replenished by joint actions of ZIPs on the cell surface and ZnTs in the ER. A putative zinc metallochaperone is proposed to shuttle cytosolic zinc from ZIP to ZnT to maintain a low cytosolic zinc concentration.