2.A.5 The Zinc (Zn²⁺)-Iron (Fe²⁺) Permease (ZIP) Family

Most members of the ZIP family consist of 220-650 amino acyl residues with 8 TMSs. However, LIV1 of man has been reported to have only 6 TMSs, although it exhibits 8 hydrophobic peaks, and the IAA-alanine resistance protein 1 (Iar1 of A. thaliana) also exhibits 8 TMSs (Lasswell et al., 2000). They are derived from animals, plants, yeast, bacteria and archaea. They comprise a diverse family, with several paralogues in any one organism (e.g., 14 in mammals such as humans, at least 5 in Caenorhabditis elegans and Arabidopsis thaliana, 9 in maize and two in Saccharomyces cervisiae). Zinc homeostasis in plants has been reviewed (Ricachenevsky et al. 2015).  ZIP proteins form homo- or heterodimers with 8 transmembrane domains and extra-/intracellular domains of various lengths. Several ZIP members show specific extracellular domains composed of two subdomains, a helix-rich domain and proline-alanine-leucine (PAL) motif-containing domain (Bin et al. 2018).  ZIP genes in peanuts play crucial roles in the uptake and transport of Fe, Zn and Mn (Zhang et al. 2022). The structural bases for zinc transport through ZIP and ZnT porters, including the molecular mechanisms of zinc binding and transport, have been reviewed (Yin et al. 2022). Genome-wide functional studies of the ZIP family proteins in wild emmer wheat have been summarized (Gong et al. 2022). These proteins have the ZIP fold (Ferrada and Superti-Furga 2022).

The various mammalian paralogues fall into four subfamilies and are found in a variety of cell types, cell locations and tissues, and some are responsive to hormones and cytokines (Dempski 2012). Some mammalian Zip genes apparently do not  play critical roles in zinc homeostasis when zinc is replete, but they play important, noncompensatory roles when this metal is deficient (Kambe et al. 2008).  Zip6 (LIV1) is estrogen responsive in breast cancer cells and is related to metastasis in lymph nodes. Zip8 (Big M103) is TNFα and endotoxin induced in monocytes. The two S. cerevisiae proteins, Zrt1 and Zrt2, both probably transport Zn²⁺ with high specificity, but Zrt1 transports Zn²⁺ with ten-fold higher affinity than Zrt2. In cacao (Theobroma cacao L.) there are 11 ZIP homologs, and their physicochemical properties, evolution, duplication, gene structure, promoter region and TcZIP family three-dimensional protein structures are described in the plasma membrane and chloroplast (Pacheco et al. 2023).

Some members of the ZIP family have been shown to transport Zn²⁺ while others transport Fe²⁺, and a few have been shown to transport a range of metal ions. One human protein member of the ZIP family is designated ''growth arrest inducible gene product,'' but its presumed transport activity has not been identified. A second human protein, Zip4, is a Zn²⁺ uptake permease and a disease protein (Cousins et al., 2006). Histidine-rich repeats are found in extracellular N- and C-termini as well as a long intracellular loop, and Zip14 has an extra extracellular his-rich loop. One family of mammalian Zip proteins (the LZT family) has a metaprotease motif (HEXPHEXGD) that may allow them to function as matrix metaloproteases. Zip10 has C₂H₂ zinc finger and cytochrome c motifs in its first TMS (Cousins et al., 2006).

The energy source for transport has not been characterized, but these systems probably function as secondary carriers. They do not require ATP (Cousins et al., 2006). In one study, uptake of Zn²⁺ via the hZip2 permease was energy independent, independent of Na⁺ and K⁺ gradients, but stimulated by HCO₃^- (Gaither and Eide, 2000). The authors propose a Zn²⁺:HCO₃^- symport mechanism. hZip1 is the major Zn²⁺ uptake system in many human tissues (Gaither and Eide, 2001). The N-terminal regions are novel substrate selectors in the ZIP family of transporters (Nishida et al., 2011). An inward-open metal-free BbZIP structure differs substantially in the relative positions of the two separate domains of ZIPs. With accompanying coevolutional analyses, mutagenesis, and uptake assays, the data point to an elevator-type transport mechanism, likely shared within the ZIP family, unifying earlier functional data. Moreover, the structure reveals a previously unknown ninth transmembrane segment that is important for activity in vivo (Wiuf et al. 2022).

Mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth, and connective tissues, reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS), of some features of osteogenesis imperfecta and Zn deficient disorders. The Zip13 knockout (Zip13-KO) mice show defects in the function of osteoblasts, chondrocytes, odontoblasts and fibroblasts. Zip13 protein is localized to the Golgi in the corresponding cells. Impairment in BMP and TGF-beta signaling were observed in Zip13-KO cells (Fukada et al., 2008).  ZIP5, ZIP6, ZIP7, and ZIP10 in rat liver are regulated by iron. They may play a role in hepatic iron/metal homeostasis during iron deficiency and overload (Nam and Knutson, 2012).  In maize, IRT1 is induced by zinc and iron deficiency, ZIP4 is induced during early embryogeneis, ZIP5 is induced during middle embryogeneis, and IRT1 and ZIP6 are induced during late embryogenesis (Li et al. 2013). 

12 members of the Zn/Fe-regulated transporters (ZRT/IRT) (ZIP Family) have been identified and isolated from Poncirus trifoliata, and they were named PtZIPs according to the sequence and functional similarity to Arabidopsis thaliana ZIPs (Fu et al. 2017). The 12 PtZIPs are of 334-419 aas, harboring 6-9 putative TMSs. All contain the conserved ZIP signature sequences in TMS4, and nine of them showed a variable region rich in histidine residues between TMS3 and TMS4.  PtZIPs fall into four phylogenetic groups as for ZIPs of A. thaliana. Expression analyses showed that PtZIP genes are differently induced in roots and leaves under conditions of Zn²⁺, Fe²⁺ and Mn²⁺ deficiency. PtIRT1, PtZIP1, PtZIP2, PtZIP3, and PtZIP12  complement a zrt1 zrt2 mutant, which was deficient in Zn²⁺ uptake; PtIRT1 and PtZIP7 complement a fet3 fet4 mutant, deficient in Fe²⁺ uptake, and PtIRT1 complements a smf1 mutant, deficient in Mn²⁺ uptake, suggesting their respective functions in Zn²⁺, Fe²⁺, and Mn²⁺ transport (Fu et al. 2017).

There are 10 ZnT (CDF) (TC#2.A.4) and 15 Zip transporters in humans. They appear to play opposite roles in cellular zinc homeostasis. CDF transporters reduce intracellular zinc availability by promoting zinc efflux from cells or into intracellular vesicles, while Zip transporters increase intracellular zinc availability by promoting extracellular zinc uptake and vesicular zinc release into the cytoplasm. Both the ZnT and Zip transporter families exhibit unique tissue-specific expression, differential responsiveness to dietary zinc deficiency and excess, and differential responsiveness to physiologic stimuli via hormones and cytokines (Liuzzi and Cousins 2004).

The apo state structure in an inward-facing conformation from Bordetella bronchiseptica revealed a disassembled transport site, altered inter-helical interactions, and a rigid body movement of a 4 TMS bundle relative to the other TMSs (Zhang et al. 2023). The computationally generated and biochemically validated outward-facing conformation model revealed a slide of the 4-TMS bundle, which carries the transport site(s), by approximately 8 Å toward the extracellular side against the static TMSs which mediate dimerization. Thus, BbZIP is an elevator-type transporter. Pang et al. 2023 reported a cryo-EM structure of a ZIP-family transporter from Bordetella bronchiseptica at 3.05 Å resolution in an inward-facing, inhibited conformation. The transporter forms a homodimer, each protomer containing nine transmembrane helices and three metal ions. Two metal ions form a binuclear pore structure, and the third ion is located at an egress site facing the cytoplasm. The egress site is covered by a loop, and two histidine residues on the loop interact with the egress-site ion and regulate its release. Cell-based Zn²⁺ uptake and cell growth viability assays revealed negative regulation of Zn²⁺ uptake through sensing intracellular Zn²⁺ status using a built-in sensor. These analyses provided mechanistic insight into the autoregulation of zinc uptake across membranes (Pang et al. 2023).

The generalized transport reaction for members of the ZIP family is:

Me²⁺ (out) (pmf) → Me²⁺ (in)