The CorA Mg2+ Transport Protein of Salmonella typhimurium

Abstract

The CorA transport system is the major Mg 2؉ influx pathway for bacteria and the Archaea. CorA contains three C-terminal transmembrane segments. No conserved charged residues are apparent within the membrane, suggesting that Mg 2؉ influx does not involve electrostatic interactions. We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport. Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity. Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment. , albeit only at extracellular concentrations that are toxic to the cell. The amino acid sequence of CorA lacks homology to other known families of proteins (3). Studies of its phylogenetic distribution (4) and the recent plethora of microbial genome sequences have demonstrated that CorA is virtually ubiquitous in bacteria and the Archaea and likely forms the major Mg 2ϩ influx system for these kingdoms (5). The membrane topology of CorA has been previously studied using C-terminal protein fusions to ␤-lactamase and ␤-galactosidase (3). Like its amino acid sequence, its membrane topology is also unlike that of other known transport proteins. The N-terminal 235 residues reside in the periplasm while the remaining 80 amino acids form three transmembrane segments, including a short 6 residue C-terminal sequence in the cytosol. Three transmembrane segments are unlikely to be sufficient to form a transport channel or pore; thus, CorA probably functions as a homoligomer.The mechanism of ion transport through CorA may also be unique. Unlike many ion transporters and channels, CorA contains only two charged residues, both within the first membrane domain; neither is conserved in other homologs (5). This suggests that the most charge dense of the common biological cations passes through the membrane without involvement of electrostatic bonds. Mg 2ϩ coordinates virtually exclusively with oxygen rather than nitrogen or sulfur (6) which suggests backbone carbonyl groups and hydroxyl bearing residues within the membrane environment would be important. Sequence alignment of the CorA homologs currently available suggest a high degree of conservation of such groups in the second and third transmembrane segments. In this study, conserved residues in the third transmembrane segment (TM3) 1 of CorA were mutated. All but one mutation resulted in stable expression of protein and protein insertion into the membrane. Conserved residues Phe 290 , Tyr 309 , and Phe 310 at the termini of TM3 could be substituted by other aromatic residues without significant change in transport properties suggesting a structural rather than a transport role. In contrast, mutations at Tyr 292 , Met 299 , and Tyr 307 showed large decreases i...

ArabidopsisTransporter MGT6 Mediates Magnesium Uptake and Is Required for Growth under Magnesium Limitation

“…Studies have demonstrated that Mg 2+ and Ni 2+ can use the same transport system with similar kinetics in the cell (Smith et al, 1998;Szegedy and Maguire, 1999). Inhibition of 63 Ni 2+ tracer uptake by nonradioactive Mg 2+ or other cations represents the uptake efficiency of these cations.…”

“…Uptake of 63 Ni 2+ was performed as described previously (Snavely et al, 1989b;Smith et al, 1998;Li et al, 2001). 63 Ni 2+ uptake was assayed using MM281-MGT6 for MGT6, mutant strain MM281 as a negative control, and MM281-MGT1 for MGT1 as a positive control.…”

Escherichia coli CorA Periplasmic Domain Functions as a Homotetramer to Bind Substrate

“…It not only uptakes Mg 2ϩ , Co 2ϩ , and Ni 2ϩ but also mediates Mg 2ϩ efflux when extracellular magnesium concentration is higher than 1 mM (25). Site-directed mutagenesis revealed that three conserved residues on the ␣-helices of TM2 and TM3 participate directly in the transporting process and may be involved in substrate binding and formation of a specific Mg 2ϩ -permeable pore (26,27). Cobalt(III) hexa-amine, an analog of hydrated Mg 2ϩ , can inhibit CorA specifically, which implies that it is hydrated Mg 2ϩ that binds to the N-terminal periplasmic domain in the first step of transporting Mg 2ϩ (28).…”

Cation Hexaammines Are Selective and Potent Inhibitors of the CorA Magnesium Transport System

“…Site-directed mutagenesis of amino acids in the third transmembrane domain revealed the importance of three specific residues that line a single face of an ␣-helix. Likewise, mutagenesis of the second transmembrane segment indicated that a single face of its ␣-helix is important (15,16 (9). MgtB-dependent 63 Ni 2ϩ uptake was assayed using strain MM1111, which is MM281 transformed with the mgtB-containing plasmid pSPH39 (10).…”

“…All other reagents were obtained from Sigma, unless otherwise specified. Cultures were grown with shaking at 37°C, with appropriate antibiotics, in Luria-Bertani (LB) broth (18) or supplemented N-minimal medium, which is N-minimal salts plus 0.4% (w/v) glucose, 0.1% (w/v) casamino acids, and 1 mM leucine (15,19). Ampicillin was used at 60 g/ml, chloramphenicol at 170 g/ml, and kanamycin at 50 g/ml (20).…”