1.A.35 The CorA Metal Ion Transporter (MIT) Family
The MIT family, also called the CorA family, is a large and diverse family with sequenced members in Gram-positive and Gram-negative bacteria, blue-green bacteria, archaea, plants, animals, yeast, slime molds, Guillardia theta, and Plasmodium. Functionally characterized proteins include the Mg2+-Co2+-Ni2+ CorA permeases of Salmonella typhimurium and E. coli, a Zn2+-Cd2+ effluxing system in S. typhimurium, a divalent cation transporting CorA homologue in Methanococcus janaschii, aluminum-resistance Mg2+ transport permeases (AlR1p and AlR2p) of Saccharomyces cerevisiae and a putative manganese-resistance magnesium transport permease (MnR2p) of S. cerevisiae. Most members of the MIT family are between 300 and 400 amino acyl residues in length and possess two (or three) putative transmembrane α-helical spanners (TMSs). It seems likely that some homologues have two and others have three TMSs. The greatest degree of conservation between homologues is found in TMSs 1 and 2 of the Thermotoga maritima protein. The yeast metal resistance proteins, which are 850-900 amino acyl residues in length, also exhibit two or three putative TMSs. Overexpression of the yeast proteins, AlR1p and MnR2p, overcomes toxicity to aluminum and manganese, respectively.
The crystal structure of the CorA homologue from Thermotoga maritima has been solved at 3.9 Å resolution for the full-length protein and at 1.85 Å resolution for the cytoplasmic domain (Lunin et al., 2006). It is a funnel-shaped homopentamer with 2 TMSs per monomer. The channel is formed by an inner group of five helices and putatively gated by bulky hydrophobic residues. The large cytoplasmic domain forms a funnel whose wide mouth points into the cell and whose walls are formed by five long helices that are extensions of the transmembrane helices. The cytoplasmic neck of the pore is surrounded, on the outside of the funnel, by a ring of highly conserved positively charged residues. Two negatively charged helices in the cytoplasmic domain extend back towards the membrane on the outside of the funnel and abut the ring of positive charge. An apparent Mg2+ ion was bound between monomers at a conserved site in the cytoplasmic domain suggesting a mechanism to link gating of the pore to the intracellular concentration of Mg2+. These results contrast with those of Wang et al. (2006) who identified a soluble oligomeric N-terminal domain of the E. coli CorA that appeared to be tetrameric and to bind its substrates with the same affinity of native CorA (Wang et al., 2006).
The intracellular funnel domain of CorA constitutes an allosteric regulatory module that can be engineered to promote an activated or closed state (Payandeh et al., 2008). A periplasmic gate is centered about a proline-induced kink of the pore-lining helix. 'Helix-straightening' mutations result in gain-of-function. The narrowest constriction along the pore is a hydrophobic gate, likely forming an energetic barrier to ion flux. Highly conserved acidic residues found in the short periplasmic loop are not essential for CorA function or Mg2+ selectivity but may be required for proper protein folding and stability. Cation selectivity by the CorA Mg2+ channel requires a fully hydrated cation (Moomaw and Maguire, 2010). The structure, mechanism and regulation of divalent ion transport via MIT family channels have been reviewed (Guskov and Eshaghi 2012; Payandeh et al. 2013).
The CorA permeases of S. typhimurium and E. coli mediate both influx and efflux of Mg2+. They transport Mg2+, Co2+ and Ni2+ but not Fe2+ (Papp and Maguire, 2004). Mg2+ is transported with an apparent KM of 20-30 μM. The archaeal CorA protein is functionally similar to the CorA homologues of enteric bacteria. The yeast proteins appear to exhibit broad specificity transporting a wide range of di- and trivalent metal cations. In this respect, and also with respect to topology, MIT family members resemble channel proteins. The ZntB protein is a very distant homologue showing greatest similarity to the M. janaschii protein but catalyzing efflux of Zn2+ and Ca2+ (Worlock and Smith, 2002).
The CorA proteins of E. coli and S. typhimurium are each 316 amino acyl residues in length. Hydropathy analysis had predicted two transmembrane α-helical spanners (TMSs) in the C-terminal regions of these proteins. Site-specific mutagenesis studies of three conserved residues in TMS 3 suggest that they contribute to the Mg2+ transport pathway.
The transport mode and energy coupling mechanism(s) are poorly understood. A homopentameric structure for several CorA homologues has been established (Lunin et al., 2006), a channel-type mechanism rather than a carrier-type mechanism is operative, based on its high capacity and its 'gating' properties. CorA of S. typhimurium is a high capacity transporter that is constitutively synthesized. Some MIT family members may serve as the major Mg2+ influx systems in several prokaryotes, but others may catalyze divalent cation efflux.
Mrs2 of Saccharomyces cerevisiae is essential for the splicing of group II introns from RNA in mitochondria, and independently, for the maintenance of a functional respiratory system. Mrs2 is an integral protein of the inner mitochondrial membrane. It exhibits two adjacent putative TMSs in its C-terminal half. The large N-terminal domain and a shorter C-terminal domain are probably localized to the mitochondrial matrix. Mrs2 is distantly related to the E. coli CorA protein and other members of the MIT family. Null mutations in the mrs2 gene give mitochondria with low internal Mg2+ concentrations, and overexpression of the E. coli corA gene in yeast partially suppresses the mrs2 phenotype; CorA also partially restores intramitochondrial Mg2+ concentrations. More recently, it has been shown directly that Mrs2 is the electrophoretic mitochondrial Mg2+ uptake system, involved in Mg2+ homeostasis. It catalyzes Mg2+ uptake in the presence of a membrane potential and Mg2+ efflux in its absence (Kolisek et al., 2003). Thus, it functions like a Mg2+ channel as expected for a MIT family member. Mrs2 is demonstrably homologous to another S. cerevisiae ORF (YPL060w), as well as ORFs from other yeast, fungi, protozoans, plants and animals.
The transport reaction probably catalyzed by Mrs2 is:
Mg2+ (cytoplasm) Mg2+ (mitochondrial matrix)
The transport reaction generally catalyzed by MIT family members is:
M2+(in) (or possible M3+(in)) M2+(out) (or possibly M3+(out))
M2+ = a divalent heavy metal ion