2.A.4 The Cation Diffusion Facilitator (CDF) Family

The CDF (ZnT) family is a ubiquitous family, members of which are found in bacteria, archaea and eukaryotes (Paulsen and Saier, 1997). They transport heavy metals including cobalt, cadmium, iron, zinc and possibly nickel, copper and mercuric ions. There are 9 mammalian paralogues, ZnT1 - 8 and 10 (Cousins et al., 2006; Kambe 2012). Most members of the CDF family possess six putative transmembrane spanners with N- and C-termini on the cytoplasmic side of the membrane, but MSC2 of S. cerevisiae (TC #2.A.4.4.1) and Znt5 and hZTL1 (2.A.4.4.3) of H. sapiens exhibit 15 and 12 putative TMSs, respectively (Cragg et al., 2002). The homologs of this family exhibit an unusual degree of sequence divergence and size variation (300-750 residues). Eukaryotic proteins exhibit differences in cell localization. Some catalyze heavy metal uptake from the cytoplasm into various intracellular eukaryotic organelles (ZnT2-7) while others (e.g., ZnT1) catalyze efflux from the cytoplasm across the plasma membrane into the extracellular medium. Thus, some are found in plasma membranes while others are in organellar membranes such as vacuoles of plants and yeast and the golgi of animals (Chao and Fu, 2004b; Haney et al., 2005; MacDiarmid et al., 2003). They catalyze cation:proton antiport, have a single essential zinc-binding site within the transmembrane domains of each monomer within the dimer, and have a binuclear zinc-sensing and binding site in the cytoplamsic C-terminal region (Kambe 2012). Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD) (Barber-Zucker et al. 2016). Mutation of the CTD fold is critical for CDF proteins' proper function, supporting a role of the CDF cytoplasmic domain as a CDF regulatory element (Barber-Zucker et al. 2016).  CDF or ZnT zinc transporters include ten family members in mammals such as Homo sapiens. They show a unique architecture characterized by a Y-shaped conformation and a large cytoplasmic domain (Bin et al. 2018). There are 10 ZnT (CDF) and 15 Zip (TC#2.A.5) 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, perhaps, 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).

Montanini et al (2007) have conducted phylogenetic analysis of CDF family members. Their analysis revealed three major and two minor phylogenetic groups. They suggest that the three major groups segregated according to metal ion specificity: (1) Mn²⁺ , (2) Fe²⁺  and Zn²⁺  as well as other metal ions, and (3) Zn²⁺  plus other metals, but not Iron. CDF proteins have 6 TMSs with three 2 TMSs repeats. They are related to CRAC Ca²⁺  channels (TC#1.A.52) which has 4 TMSs (Matias et al., 2010). Members of this family have the YiiP fold (Ferrada and Superti-Furga 2022). In eucalyptus, there are many CDF metal-tolerance proteins that antiport Me²⁺ in vacuoles, H⁺ and K⁺, and these antiporters protect against Mn²⁺, Zn²⁺, Cd²⁺ and Cu²⁺ (Shirazi et al. 2023).

At least two metal binding sites have been identified in the E. coli paralogue, YiiP (TC #2.A.4.1.5), and one plays a role in H⁺ binding as well (Chao and Fu, 2004b). The two Zn²⁺/Cd²⁺  binding sites consist of two interacting conserved aspartyl residues (Asp-157 and Asp-49), both in 2 fold symmetry-related TMS 5 and TMS 2, respectively, at the dimer interface of the homodimer (Wei and Fu, 2006). The (Asp-49 and Asp-157) may form a bimetal binding center. Two bound Cd²⁺  were transported cooperatively with sigmoidal dependency on the Cd²⁺  concentration. A translocation pathway for metal ions at the dimer interface has been proposed (Wei and Fu, 2006). CDF family members may generally be homodimeric (Haney et al., 2005; Wei et al., 2004). ZNT sequences include a cytosolic His-rich loop between TMSs IV and V and histidyl residues in the cytosolic N-terminus, but neither is required for transport activity (Fukue et al. 2018).

Lu and Fu (2007) have reported the x-ray structure of YiiP of E. coli (2.A.4.7.1) in complex with zinc at 3.8 angstrom resolution. YiiP is a homodimer held together in a parallel orientation through four Zn²⁺  ions at the interface of the cytoplasmic domains.  The two transmembrane domains swing out to yield a Y-shaped structure. In each protomer, the cytoplasmic domain adopts a metallochaperone-like protein fold. The transmembrane domain features a bundle of six transmembrane helices and a tetrahedral Zn²⁺  binding site located in a cavity that is open to both the membrane outer leaflet and the periplasm. 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).

Coudray et al. (2013) used cryoelectron microscopy to determine a 13-Å resolution structure of a YiiP homolog from Shewanella oneidensis within a lipid bilayer in the absence of Zn²⁺. Starting from the X-ray structure in the presence of Zn²⁺, they used molecular dynamic flexible fitting to build a model. Comparison of the structures suggested a conformational change that involves pivoting of a transmembrane, four-helix bundle (M1, M2, M4, and M5) relative to the M3-M6 helix pair. Although accessibility of transport sites in the X-ray model indicates that it represents an outward-facing state, their model was consistent with an inward-facing state, suggesting that the conformational change is relevant to the alternating access mechanism for transport. They speculated that the dimer may coordinate rearrangement of the transmembrane helices,

Involved in metal tolerance/resistance by efflux, most CDF proteins share a two-modular architecture consisting of a transmembrane domain (TMD) and a C-terminal domain (CTD) that protrudes into the cytoplasm. A Zn²⁺ and Cd²⁺ CDF transporter from the marine bacterium, Maricaulis maris, that does not possess the CTD is a member of a new, CTD-lacking subfamily of CDFs. TMEM163 proteins are members of the CDF family (see 2.A.4.8.3 (Styrpejko and Cuajungco 2021)). Wheat (Triticum urartu) has nine CDF porters, three Zn-CDFs, two Fe/Zn-CDFs, and four Mn-CDFs (Wang et al. 2021).

The generalized transport reaction for CDF family members is:

Me²⁺  (in) H⁺ (out) ± K⁺ (out) → Me²⁺  (out) H⁺ (in) ± K⁺ (in)