Structural and functional characterization of the N-terminal domain of the yeast Mg2+ channel Mrs2.

Mg(2+) translocation across cellular membranes is crucial for a myriad of physiological processes. Eukaryotic Mrs2 transporters are distantly related to the major bacterial Mg(2+) transporter CorA, the structure of which displays a bundle of giant α-helices forming a long pore that extends beyond the membrane before widening into a funnel-shaped cytosolic domain. Here, a functional and structural analysis of the regulatory domain of the eukaryotic Mg(2+) channel Mrs2 from the yeast inner mitochondrial membrane is presented using crystallography, genetics, biochemistry and fluorescence spectroscopy. Surprisingly, the fold of the Mrs2 regulatory domain bears notable differences compared with the related bacterial channel CorA. Nevertheless, structural analysis showed that analogous residues form functionally critical sites, notably the hydrophobic gate and the Mg(2+)-sensing site. Validation of candidate residues was performed by functional studies of mutants in isolated yeast mitochondria. Measurements of the Mg(2+) influx into mitochondria confirmed the involvement of Met309 as the major gating residue in Mrs2, corresponding to Met291 in CorA.

The G-M-N motif determines ion selectivity in the yeast magnesium channel Mrs2p.

The highly conserved G-M-N motif of the CorA-Mrs2-Alr1 family of Mg(2+) channels has been shown to be essential for Mg(2+) transport. We performed random mutagenesis of the G-M-N sequence of Saccharomyces cerevisiae Mrs2p in an unbiased genetic screen. A large number of mutants still capable of Mg(2+) influx, albeit below the wild-type level, were generated. Growth complementation assays, performed in media supplemented with Ca(2+) or Co(2+) or Mn(2+) or Zn(2+) at varying concentrations, lead to identification of mutants with reduced growth in the presence of Mn(2+) and Zn(2+). We hereby conclude that (1) at least two, but predominantly all three amino acids of the G-M-N motif must be replaced by certain combinations of other amino acids to remain functional, (2) replacement of any single amino acid within the G-M-N motif always impairs the function of Mrs2p, and (3) we show that the G-M-N motif determines ion selectivity, likely in concurrence with the negatively charged loop at the entrance of the channel thereby forming the Mrs2p selectivity filter.

Functional analysis of the conserved hydrophobic gate region of the magnesium transporter CorA.

The Leu294 residue in the cytoplasmic neck of Thermotoga maritima CorA is considered to be the main gate for Mg2+ transport. We created three site-directed mutants at this position: in the Leu294Asp and Leu294Gly mutants we observed a defect in closing of the pore, while in the Leu294Arg mutant not only gating, but also the regulation of Mg2+ uptake was affected. Our results confirmed the importance of the Leu294 for gating of Mg2+ transport and in addition revealed the influence of the charge and structural features of the amino acid residues on the gating mechanism.

Lpe10p modulates the activity of the Mrs2p-based yeast mitochondrial Mg2+ channel.

Saccharomyces cerevisiae Lpe10p is a homologue of the Mg(2+)-channel-forming protein Mrs2p in the inner mitochondrial membrane. Deletion of MRS2, LPE10 or both results in a petite phenotype, which exhibits a respiratory growth defect on nonfermentable carbon sources. Only coexpression of MRS2 and LPE10 leads to full complementation of the mrs2Delta/lpe10Delta double disruption, indicating that these two proteins cannot substitute for each other. Here, we show that deletion of LPE10 results in a loss of rapid Mg(2+) influx into mitochondria, as has been reported for MRS2 deletion. Additionally, we found a considerable loss of the mitochondrial membrane potential (DeltaPsi) in the absence of Lpe10p, which was not detected in mrs2Delta cells. Addition of the K(+)/H(+)-exchanger nigericin, which artificially increases DeltaPsi, led to restoration of Mg(2+) influx into mitochondria in lpe10Delta cells, but not in mrs2Delta/lpe10Delta cells. Mutational analysis of Lpe10p and domain swaps between Mrs2p and Lpe10p suggested that the maintenance of DeltaPsi and that of Mg(2+) influx are functionally separated. Cross-linking and Blue native PAGE experiments indicated interaction of Lpe10p with the Mrs2p-containing channel complex. Using the patch clamp technique, we showed that Lpe10p was not able to mediate high-capacity Mg(2+) influx into mitochondrial inner membrane vesicles without the presence of Mrs2p. Instead, coexpression of Lpe10p and Mrs2p yielded a unique, reduced conductance in comparison to that of Mrs2p channels. In summary, the data presented show that the interplay of Lpe10p and Mrs2p is of central significance for the transport of Mg(2+) into mitochondria of S. cerevisiae.

Splice-variant 1 of the ancient domain protein 2 (ACDP2) complements the magnesium-deficient growth phenotype of Salmonella enterica sv. typhimurium strain MM281.

Evidence arguing for the existence of genes encoding for proteins directly involved in the transport of Mg2+ through the cytoplasmic membrane have accumulated over the last few years. Gene ACDP2 (ancient conserved domain protein 2; old name CNNM2, cyclin M2) is one such gene. ACDP2 is a distant homologue of the bacterial gene corC, which is known to be involved in cobalt resistance. We have previously demonstrated that the over-expression of the human Mg2+ carrier SLC41A1 partly complements the Mg2+-dependent growth deficiency of Salmonella strain MM281 (triple disruptant in genes: mgtA, mgtB and corA) cultivated in media containing growth non-permissive [Mg2+]e. We have used the same approach to examine whether over-expressed human ACDP2 has a similar efficacy to complement growth deficiency of the MM281 strain in media containing growth non-permissive [Mg2+]e. Two splicing variants of the ACDP2 gene have been tested. Here, we show that over-expressed isomorph 1 is efficient in restoring growth of the MM281 strain in media containing growth non-permissive [Mg2+]e, whereas isomorph 2 is not. Therefore, we conclude that ACDP2sp.v.1 is a functional Mg2+-transporting entity per se. Our conclusion is supported by the measurable Mg2+ influx seen in MM281 bacteria over-expressing ACDP2sp.v.1 but not in MM281 bacteria over-expressing ACDP2sp.v.2 or in cells transformed with the empty vector.

A root-expressed magnesium transporter of the MRS2/MGT gene family in Arabidopsis thaliana allows for growth in low-Mg2+ environments.

The MRS2/MGT gene family in Arabidopsis thaliana belongs to the superfamily of CorA-MRS2-ALR-type membrane proteins. Proteins of this type are characterized by a GMN tripeptide motif (Gly-Met-Asn) at the end of the first of two C-terminal transmembrane domains and have been characterized as magnesium transporters. Using the recently established mag-fura-2 system allowing direct measurement of Mg(2+) uptake into mitochondria of Saccharomyces cerevisiae, we find that all members of the Arabidopsis family complement the corresponding yeast mrs2 mutant. Highly different patterns of tissue-specific expression were observed for the MRS2/MGT family members in planta. Six of them are expressed in root tissues, indicating a possible involvement in plant magnesium supply and distribution after uptake from the soil substrate. Homozygous T-DNA insertion knockout lines were obtained for four members of the MRS2/MGT gene family. A strong, magnesium-dependent phenotype of growth retardation was found for mrs2-7 when Mg(2+) concentrations were lowered to 50 microM in hydroponic cultures. Ectopic overexpression of MRS2-7 from the cauliflower mosaic virus 35S promoter results in complementation and increased biomass accumulation. Green fluorescent protein reporter gene fusions indicate a location of MRS2-7 in the endomembrane system. Hence, contrary to what is frequently found in analyses of plant gene families, a single gene family member knockout results in a strong, environmentally dependent phenotype.

Mutational analysis of functional domains in Mrs2p, the mitochondrial Mg2+ channel protein of Saccharomyces cerevisiae.

The nuclear gene MRS2 in Saccharomyces cerevisiae encodes an integral protein (Mrs2p) of the inner mitochondrial membrane. It forms an ion channel mediating influx of Mg2+ into mitochondria. Orthologues of Mrs2p have been shown to exist in other lower eukaryotes, in vertebrates and in plants. Characteristic features of the Mrs2 protein family and the distantly related CorA proteins of bacteria are the presence of two adjacent transmembrane domains near the C terminus of Mrs2p one of which ends with a F/Y-G-M-N motif. Two coiled-coil domains and several conserved primary sequence blocks in the central part of Mrs2p are identified here as additional characteristics of the Mrs2p family. Gain-of-function mutations obtained upon random mutagenesis map to these conserved sequence blocks. They lead to moderate increases in mitochondrial Mg2+ concentrations and concomitant positive effects on splicing of mutant group II intron RNA. Site-directed mutations in several conserved sequences reduce Mrs2p-mediated Mg2+ uptake. Mutants with strong effects on mitochondrial Mg2+ concentrations also have decreased group II intron splicing. Deletion of a nonconserved basic region, previously invoked for interaction with mitochondrial introns, lowers intramitochondrial Mg2+ levels as well as group II intron splicing. Data presented support the notion that effects of mutations in Mrs2p on group II intron splicing are a consequence of changes in steady-state mitochondrial Mg2+ concentrations.

Diverse biomodulatory effects of glucomannan from Candida utilis.

Using four experimental model systems, it was demonstrated that glucomannan (GM) isolated from the cell wall of the industrial yeast Candida utilis revealed a broad range of protective activities. This effect depended on the nature and mode of action of the counteracting genotoxic compound as well as on the experimental model system used. In the Saccharomyces bioprotectivity assay, GM increased resistance towards ofloxacin-induced toxicity in the wild type and recombination repair-deficient yeast strains significantly enhancing survival of the cells. In the chromosomal aberration assay, GM exerted anticlastogenic effect against maleic hydrazide induced clastogenicity in Vicia faba L. In the DNA-topology assay, GM protected plasmid DNA from the breaks induced by Fe(2+) ions, but enhanced damage induced by bleomycin and hydrogen peroxide. In the cell-revitalization assay, it enhanced cytotoxic/cytostatic effect of teniposide applied to mouse leukemia cells. Thus, depending on the experimental model, GM acted as antimutagen, anticlastogen, DNA breaks inhibitor or inducer, and as cytotoxic/cytostatic effect enhancer. Several possible mechanisms of bioprotective action underlying the observed activities are suggested including iron chelation and free radical scavenging. The results imply that GM is a polysaccharide with marked biological activities and suggest its potential biomedical application, especially in combination with other bioactive compounds.

The role of natural biopolymers in genotoxicity of mutagens/carcinogens elimination.

Nowadays naturally occurring compounds with the potential antimutagenic and anticarcinogenic effects are of great importance for their prospective use in cancer chemoprevention and treatment. The new water soluble derivative of microbial polysaccharide beta-D-glucan-carboxymethyl glucan (CMG) belongs to such a category of natural substances. CMG isolated from the cell wall of baker's yeast Saccharomyces cerevisiae is included into the class of biopolymers known as biological response modifiers (BRMs) with a broad range of activities, above all ones interfering with cancer therapy. It was demonstrated on four experimental model systems that biological and consequential medicinal importance of CMG is based on the combined application with another active compound. In the Saccharomyces cerevisiae antimutagenicity assay CMG significantly reduced ofloxacin-induced mutagenicity in the yeast strain D7. CMG exerted bioprotective (anti-toxic and antimutagenic) effect after its simultaneos application with methyl methanesulphonate on the repair-deficient strain uvs10 of the unicellular green alga Chlamydomonas reinhardtii. In the Vicia sativa simultaneous phytotoxicity and anticlastogenicity assay CMG exerted statistically significant anticlastogenic efect against maleic hydrazide-induced clastogenicity in Vicia sativa L. Only in the Salmonella/microsome assay CMG did not exert statistically significant antigenotoxic effect, despite of the fact that it reduced 9-aminoacridine-induced mutagenicity in S. typhimurium TA97, but his(+) revertants decreasing was statistically significant only at the highest CMG concentration used. The data presented unambiguously documented that even biopolysaccharides (e.g., derivatives of beta-glucan) belonging to the most abundant class of natural biopolymers may contribute to cancer prevention and therapy.