Copper, iron, zinc, cobalt, nickel and manganese are essential metal cations in cellular processes, since they act as important cofactors for many enzymes, are components of transcription factors and other proteins, and are essential for both mitochondrial and chloroplast functions. However, when present at high concentration, along with non-essential metals such as cadmium, mercury, silver and lead, essential metals can become extremely toxic, since they can cause oxidative damages or compete with other essential ions. A network of uptake, extrusion, chelation, trafficking and storage mechanisms ensures the maintenance of metal homeostasis at the cellular level. Specific transporters, encoded by multigenic families, are responsible for the uptake and secretion of metal ions, and for their sequestration into organelles [1–4].
Cation diffusion facilitator (CDF, TC 2.A.4) transporters, first identified by Nies and Silver , are ubiquitous, spanning all three kingdoms of life: Archaea, Eubacteria and Eukaryotes. Studies on CDF transporters may inform strategies for bioremediation and human nutrition and health [1, 2, 4]. All known CDF substrates are divalent metal cations with ionic radii of 72 (Zn2+) to 97 (Cd2+) pm . Most CDFs are Me2+/H+ (K+) antiporters [6–9] that catalyse the efflux of transition metal cations, including Zn2+, Co2+, Fe2+, Cd2+, Ni2+, or Mn2+, from the cytoplasm to the outside of the cell or into subcellular compartments [1, 2, 4, 7, 10–13]. The majority of CDF proteins possess six putative transmembrane domains (TMDs), with cytoplasmic N and C termini, as experimentally demonstrated for bacterial members [10, 14]. A signature sequence between TMDs I and II was proposed in 1997 , and a characteristic C-terminal cation efflux domain is present in all members  (Pfam 01545, ). The most conserved regions are the amphipathic TMDs I, II, V and VI, which are likely involved in metal transfer . However, some CDF members exhibit different types of secondary structure: for example the Msc2 protein of Saccharomyces cerevisiae is predicted to have 12 TMDs , and the human protein ZnT5 and its splice variant ZTL1 display 15 and 12 TMD topology, respectively [20, 21]. Plant CDF members are usually called Metal Tolerance Proteins (MTPs), while vertebrate members are named Zinc Transporter (ZnT) or Solute carrier family 30 (SLC30).
Most CDF family transporters also contain a histidine-rich region, either between TMDs IV and V, or at the N and/or C termini. Such regions are predicted to be cytoplasmic, cis to metal uptake, and could function as potential metal (Zn2+, Co2+, and/or Cd2+) binding domains. However the ER-localised Zrg17, a Zn2+ transporter recently characterised in yeast, displays a histidine-rich loop predicted to reside between TMDs III and IV toward the ER lumen . The plant CDF member ShMTP1 (ShCDF8) isolated from Stylosanthes hamata, is involved in Mn2+ homeostasis and tolerance, shows a predicted 4–5 TMD topology, and does not contain the histidine-rich domain [11, 16].
Some CDF transporters function as homo-oligomeric complexes [23–26] or hetero-oligomeric complexes [22, 24], and may directly interact with other proteins to regulate their activity by means of metal release to these proteins . Indirect evidence that CDF transporters interact with other proteins also comes from studies on the rat ZnT4 .
Research over the past few years, mainly performed on prokaryotic CDF members, has aimed at identifying molecular mechanisms involved in metal binding and transport across the membranes. Key functional amino acid residues, identified by site-directed mutagenesis, reside in the three amphipathic and conserved transmembrane helices II, V and VI, which are supposed to constitute an inner core forming a channel [14, 18, 29, 30]. Indeed recent studies on the CDF transporter EcFieF (YiiP) from Escherichia coli localised a substrate binding site at the dimer interface created by the TMDs II and V .
Here we undertake a phylogenetic analysis of CDF family amino acid sequences based on a set of CDF sequences retrieved from databases. Based on a multiple sequence alignment, we propose a modified signature for the CDF family that takes account of newly characterised members. Coupled with the substrate specificities of some characterised transporters, we are able to classify the family members into three major groups that have different selectivity towards the principally-transported metal (Zn-, Fe/Zn- and Mn-CDF). Functional analyses, based on site-directed mutagenesis, were carried out on the eukaryotic Zn2+ transporter PtdMTP1  to identify key residues important for CDF function. The role in metal selectivity of the group-conserved residue D (for Mn-specific CDF transporters) or H (for Zn- and Fe/Zn-CDF transporters) embedded in TMD V is also discussed.