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).
Prokaryotic and eukaryotic proteins cluster separately but probably function with the same polarity by similar mechanisms. These proteins are secondary carriers which utilize the pmf and function by H+ antiport (for metal efflux). One member, CzcD of Bacillus subtilis, has been shown to exchange the divalent cation (Zn2+ or Cd2+ ) for two monovalent cations (K+ and H+ ) in an electroneutral process energized by the transmembrane pH gradient (Guffanti et al., 2002). Another, ZitB of E. coli (TC #2.A.4.1.4), has been reconstituted in proteoliposomes and studied kinetically (Chao and Fu, 2004a). It appears to function by simple Me2+:H antiport with a 1:1 stoichiometry.
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).
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.
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.
The generalized transport reaction for CDF family members is:
Me2+ (in) H+ (out) ± K+ (out) → Me2+ (out) H+ (in) ± K+ (in)
ZitB of E. coli (P75757)
The major Zn2+ resistance determinant, ZitA (Grover and Sharma, 2006)
ZitA of Mycobacterium smegmatis (A0QQH3)
Dimeric Zn2+ efflux carrier of 299 aas and 5 TMSs, CzcD (Martin and Giedroc 2016).
CzcD of Streptococcus pneumoniae
Cotl of Saccharomyces cerevisiae
Zrclp (ZnrP) of Saccharomyces cerevisiae
Plasma membrane Zn2+ efflux permease. Substituing H43 with N (asn) changed the specificity from Zn2+ to Mn2+, and this difference was found in the Mn2+-specific metal ion exporter, ZnT10 (TC# 2.A.4.2.5) (Nishito et al. 2016). Expression on the cell surface is controlled by cellular zinc levels (Nishito and Kambe 2019).
ZnT1 of Rattus norvegicus
CDF-1 of Caenorhabditis elegans (Q95QW4)
Solute carrier family 30, member 10, ZnT10. Manganese efflux transporteer of 485 aas and 6 TMSs in a 4 + 2 TMS arrangement with two long hydrophilic regions between residues 135 and 240, and residues 300 and 485 (Nishito et al. 2016). Homozygous mutations lead to the development of familial manganese Mn2+-induced parkinsonism; it is a cell surface-localized Mn2+ efflux transporter, and parkinsonism-causing mutations block its trafficking and efflux activity. Residues in the transmembrane and C-terminal domains together confer optimal Mn2+ transport capability (Zogzas et al. 2016). Residues involved in Mn2+ binding in the transmembrane domain have been identified, and they differ in position and nature from the Zn2+ binding site in Zn2+ transporting members of the CDF family (Zogzas and Mukhopadhyay 2018). Loss of ZnT10 expression caused by autosomal mutations in the ZnT10 gene leads to hypermanganesemia in multiple organs (Levy et al. 2019). The cellular transport of Mn2+ is coupled to a reciprocal movement of Ca2+. Replacing a single asparagine residue in ZnT10 (Asn-43) with threonine (ZnT10 N43T) converted the Mn2+/Ca2+ exchanger into an uncoupled channel permeable to both Ca2+ and Mn2+ (Levy et al. 2019). It is expressed in the CNS (Sreedharan et al. 2011).
SLC30A10 or ZnT10 of Homo sapiens
Zinc transporter 1 (ZnT-1) (Solute carrier family 30 member 1). The loss of the sialic acid transporter SLC35A1/CST (TC# 2.A.7.12.11) and the zinc transporter, ZnT1, affected cell survival upon infection with cytolytic vesicular stomatitis virus (VSV). Both of these transporters seem to play a role in the apoptotic response induced by VSV (Moskovskich et al. 2019).
SLC30A1 of Homo sapiens
ZnT63C Zinc exporter, ZnT1, of 545 aas and 5 putative TMSs (Lye et al. 2013; Dechen et al. 2015).
ZnT63C of Drosophila melanogaster
Vacuolar CDF1 transporter. Transports Zn2+, Co2+ and Cd2+ but not Mn2+, of 487 aas and 6 TMSs (Sácký et al. 2016).
CDF1 of Russula atropurpurea
The plasma membrane zinc efflux system, ZnT-1, of 485 aas and 6 predicted TMSs with a large histidine-rich intracellular loop between TMSs 4 and 5 and intracellular C- and N-termini (Balesaria and Hogstrand 2006).
ZnT-1 of Takifugu rubripes (Japanese pufferfish) (Fugu rubripes)
Vesicular Zn2+ uptake (into endosomal/lysosomal vesicles) permease, ZnT2. There are two isoforms due to alternative splicing, 35 kDa (plasma membrane localized) and 42 kDa (endosome/secretory compartment localized) (Lopez and Kelleher, 2009).
ZnT2 of Rattus norvegicus
CDF transporter, MTP1 of 640 aas and 6 TMSs. It transports zinc (Zn2+) and cobalt (Co2+) (Ibuot et al. 2020).
MTP1 of Chlamydomonas reinhardtii
Zn2+ Transporter, LbrM31, of 443 aas and 6 or 7 TMSs.
LbrM31 of Leishmania braziliensis (A4HJM3)
Vesicular Zn2+ uptake (into synaptic vesicles) permease, ZnT3 (SLC30A subfamily). ZnT-3 regulates presynaptic Erk1/2 signaling and hippocampus-dependent memory (Sindreu et al., 2011).
Plant root and leaf vacuolar Zn2+ transporter, ZAT-1 or MTP1 (metal tolerance protein 1) (Desbrosses-Fonrouge et al., 2005). Loss of the cytoplasmic histidine-rich loop, where 4 Zn2+ (or Ni2+ or Co2+) bind, stimulates transport activity (Kawachi et al., 2008), presumably providing a feedback sensor of cytoplasmic Zn2+ (Tanaka et al. 2013). The barley orthologue has broader metal specificity (Podar et al., 2012). Critical residues for function, ion selectivity and structure have been identified (Kawachi et al., 2012). In Citrus sinensis, expression is induced by Cd2+ (Fu et al. 2017).
MTP1 of Arabidopsis thaliana
Homodimeric solute carrier family 30 (zinc transporter), member 8, ZnT8 or ZnT-8. An inherited R325W mutant gives aberant zinc transport in pancreatic beta cells (Weijers 2010). It is chiefly expressed in pancreatic islet cells, where it mediates zinc (Zn2+) uptake into secretory granules. It plays a role in glucose tolerance (Mitchell et al. 2016). ZnT8 is one of four human vesicular zinc transporters. It supplies millimolar zinc concentrations to insulin granules in pancreatic beta-cells, affecting insulin processing, crystallisation, and secretion. ZnT8 has a transmembrane and a C-terminal cytosolic domain; the latter has important functions and purportedly mediates protein-protein interactions, senses cytosolic zinc, and channels zinc to the transport site in the transmembrane domain (Parsons et al. 2018). The variant, W325R, in the C-terminal domain (CTD) increases the risk to develop type 2 diabetes and affects autoantibody specificity in type 1 diabetes. The CTDs of the WT and mutant proteins form tetramers which are stabilized by zinc binding and exhibit negligible differences in their secondary structural content and zinc-binding affinities in solution (Ullah et al. 2020).
SLC30A8 of Homo sapiens
Metal tolerance protein 4, MTP4, of 386 aas. Exports Zn2+ and Cd2+ (Migocka et al. 2015). MTP1 transports the same two ions but is less restrictive with respect to the tissues in which the protein is synthesized.
MTP4 of Cucumis sativus (Cucumber)
Co2+ resistance protein, DmeF, of 382 aas and 6 TMSs. Co2+ export appears to be its dominant physiological function, but it may also export other heavy metal ions such as Zn2+ and Cd2+ (Munkelt et al. 2004).
DmeF of Cupriavidus metallidurans (Ralstonia metallidurans)
Heteromeric nuclear/ER Zn2+ uptake permease, Msc2/Zrg17 (Ellis et al., 2005). Zrg17 is also called Meiotic sister-chromatid recombination-related protein or metal cation transporter Msc2p of 724 aas and 15 TMSs in a 7 + 2 + 6 TMS arrangement. Only the last 6 TMSs are homologous to CDF transporters.
Msc2/Zrg17 heteromeric Zn2+ transporter of Saccharomyces cerevisiae
Golgi/secretory granule Zn2+ uptake (into Golgi or granules) permease, ZnT5 or ZTL1 (ZnT5 forms heterooligomers with ZnT6) (Ellis et al., 2005; Ishihara et al., 2006; Fukunaka et al. 2009) (Variant B catalyzes bidirectional transport (Valentine et al., 2007) )
SLC30A5 or ZnT5 of Homo sapiens
Golgi/secretory granule/endoplasmic reticulum Zn2+ uptake (into Golgi, the ER, or granules) permease, ZnT6. ZnT6 forms heterooligomers with ZnT5 (Ellis et al., 2005; Ishihara et al., 2006; Fukunaka et al. 2009).
SLC30A6 of Homo sapiens
Metal tolerance protein C2 (AtMTPc2) (AtMTP5). Forms a large, active, heteromeric complex with MTP12 in the golgi and pumps Zn2+ into this organelle (Fujiwara et al. 2015). MTP12 has 798 aas and 14 TMSs. Its UniProt acc # is A0A0A8IL98. In Citrus sinensis, expression is induced by Zn2+ in roots (Fu et al. 2017).
MTPC2 or MTP5 of Arabidopsis thaliana
Lead (Pb2+) efflux transporter of 211 aas, PbtF (Hložková et al. 2013).
PbtF of Achromobacter xylosoxidans
Uncharacterized protein of 701 aas and 16 TMSs, where the last 6 TMSs are homologous to CDF transporters.
UP of Eremothecium cymbalariae (Yeast)
Metal tolerance protein 12, MTP12 of 300 aas and 6 TMSs. In Citrus sinensis, expression is induced by Cd2+ in roots (Fu et al. 2017).
MTP12 of Arabidopsis thaliana
Golgi/endomembrane Mn2+-specific CDF transporter (394 aas) (Peiter et al., 2007). A rice homologue, MTP8.1, has been characterized (Chen et al. 2016).
MTP11.1 of Populus trichocarpa (A4ZUV2)
CDF2 transporter for Zn2+ and Co2+ but not Cd2+ or Mn2+ of 417 aas and 5 TMSs (Sácký et al. 2016).
CDF2 of Russula atropurpurea
MTP8.1 is a tonoplast-localized manganese transporter of 400 aas and 6 TMSs. Critical residues for function in the rice orthologue have been identified (Chen et al. 2016).
MTP8.1 of Hordeum vulgare (Barley)
Metal tolerance protein 8, MTP8, of 411 aas and 6 TMSs. In Citrus sinensis, expression is induced by Mn2+ in roots (Fu et al. 2017). In A. thaliana, MPT8 determines the localization of manganese and iron in seeds (Chu et al. 2017). The endodermal vacuole is the iron and manganese storage compartment in the A. thaliana embryo (Roschzttardtz et al. 2009).
MTP8 in Arabidopsis thaliana
Metal tolerance protein 11, MTP11, of 298 aas and 5 TMSs. In Citrus sinensis, expression is induced by Zn2+ in leaves (Fu et al. 2017).
MTP11 of Arabidopsis thaliana
CDF transporter, MTP3, of 458 aas and 7 TMSs. It transports Mn2+, Zn2+ and Co2+ (Ibuot et al. 2020).
MTP3 of Chlamydomonas reinhardtii (Chlamydomonas smithii)
Mn2+ transporting CDF porter, MTP4, of 538 aas and 6 TMSs.
MTP4 of Chlamydomonas reinhardtii (Chlamydomonas smithii)
CDF transporter, ZnT9 (568 aas) (Montanini et al., 2007). Mutations cause autosomal recessive cerebro-renal syndrome and affect intracellular Zinc homeostasis (Perez et al. 2017).
SLC30A9 of Homo sapiens
Metal tolerance protein C4, MTPC4 of 457 aas and 6 TMSs. It is involved in sequestration of excess metal ions, including Zn2+ in the cytoplasm into vacuoles to maintain metal homeostasis.
MTPC4 of Arabidopsis thaliana (Mouse-ear cress)
Zn2+/Cd2+/Hg2+/Fe2+:H+ antiporter, YiiP or FieF (Chao and Fu, 2004b; Grass et al., 2005; Wei et al., 2004; Wei and Fu, 2006). The structure (3.8 Å resolution) reveals a homodimer interconnected at the cytoplasmic domain through four Zn2+ ions. A 6 TMS bundle features of a tetrahedral Zn2+ binding site (Lu and Fu, 2007). The gated water access to the transport site enables a stationary proton gradient to facilitate the conversion of zinc-binding energy to the kinetic power stroke of a vectorial zinc transport (Gupta et al. 2014). Of the two cavities of the dimer, only one was accessible from the cytosol during transport. Zinc(II) binding to D49 of the transport site triggered a rearrangement of the TMS that closed the accessible cavity. The free-energy profiles of metal transit in the channel suggested the existence of a high barrier preventing release from the transport site (Sala et al. 2019).
YiiP of E. coli (P69380)
Cobalt/zinc resistance protein B, CzrB, of 291 aas and 6 TMSs. It has a cytosolic extramembranal C-terminus. This 92-residue fragment may function independently of the full-length integral membrane protein. X-ray analyses of this fragment to 2.2 A resolution with and 1.7 A without zinc ions have been solved. The former has at least two zinc ions bound per monomer (Höfer et al. 2007). Full-length variants of CzrB in the apo and zinc-loaded states were generated by homology modeling with the Zn2+/H+ antiporter YiiP. The model suggests a way in which zinc binding to the cytoplasmic fragment creates a docking site to which a metallochaperone can bind for delivery and transport of zinc. A proposal was advanced that it functions as a metallochaperone and regulates the zinc-transporting activity of the full-length protein. The latter requires that zinc binding becomes uncoupled from the creation of a metallochaperone-docking site on CzrB (Cherezov et al. 2008).
CzrB of Thermus thermophilus
Magnetosome protein, MamV, of 322 aas and 5 TMSs. Putative Co/Zn/Cd cation transporter (Smalley et al. 2015).
MamV of Magnetospirillum magnetotacticum MS-1
Putative magnetosome membrane iron transporter, MamB. Forms a heterodimeric stable complex with MamM which stabilizes MamB. MamB also interacts with other proteins including the PDZ1 domain of MamE (Q6NE61). Both MamB and MamM are essential for magnetite biomineralization and are involved in several steps in magnetosome formation, but only MamB is essential for formation of magnetosome membrane vesicles (Uebe et al. 2011). Implicated in iron uptake due to homology with other CDF transporters.
MamB of Magnetospirillum gryphiswaldense
Putative magnetosome membrane iron transporter, MamM. Forms a heterodimeric stable complex with MamB which it stabilizes. MamB; see 2.A.4.7.3) also interacts with other proteins including the PDZ1 domain of MamE (Q6NE61). Both MamB and MamM are essential for magnetite biomineralization and are involved in several steps in magnetosome formation, but only MamB is essential for formation of magnetosome membrane vesicles (Uebe et al. 2011). Implicated in iron uptake due to homology with other CDF transporters. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). A MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein, ZnT-10 causes severe structural changes in its CTD, resulting in abnormally reduced function.Thus, the CTD fold is critical for the CDF proteins' proper function and suggest that the CDF cytoplasmic domain is a CDF regulatory element (Barber-Zucker et al. 2016).
MamM of Magnetospirillum gryphiswaldense
Cd2+/Zn2+ efflux pump, YiiP or FieF. A low resolution structure in the open configuration has been determined by cryoelectron microscopy (Coudray et al. 2013).
YiiP of Shewanella oneidensis (Q8E919)
Dimeric Mn2+ efflux transporter, MntE of 394 aas (Rosch et al. 2009; Lisher et al. 2013; Jakubovics and Valentine 2009; Martin and Giedroc 2016).
MntE of Streptococcus pneumoniae
Mn2+ efflux pump, YiiP, probably a Mn2+:H+ antiporter. Necessary for efficient nodulation of alfalfa plants (Raimunda and Elso-Berberián 2014)
YiiP of Sinorhizobium meliloti
CDF protein, exporting Zn2+ and Cd2+ (323 aas). It lacks the C-terminal hydrophilic domain (CTD) common to many CDF homologues (Kolaj-Robin et al. 2015).
CDF homologue of Maricaulis maris
Ferrous iron detoxifying protein, FieF, of 337 aas and 6 TMSs. Also probably exports Zn2+, Co2+, Cd2+ and Ni2+ (Munkelt et al. 2004).
FieF of Cupriavidus metallidurans (Ralstonia metallidurans)
CDF Family homologue with MMT1 domain
CDF porter of Streptomyces coelicolor
CDF Family member
CDF cation efflux carrier of Mycobacterium tuberculosis
Zn2+ transporter, TMEM163 or Znt11, of 289 aas and 6 TMSs. Interacts with TrpML1 (TC# 1.A.5.3.1), but by itself, it can pump out Zn2+ (Sanchez et al. 2019). It may be involved in the human lipid storage disorder, mucolipidosis type IV (MLIV), caused by Zn2+ overload (Cuajungco et al. 2014). TMEM163 is found in synaptic vesicles where it is called SV31 (Burré et al. 2007). It plays a role in Zn2+ uptake into lysosomes (Cuajungco and Kiselyov 2017).
TMEM163 of Homo sapiens