Molecular biology and function of eukaryotik Mg2+ transporters

With our ongoing work we have made a major step towards a molecular biology of Mg2+ transporters and thereby led a basis for the understanding of the cellular and organellar homeostasis of this ion. We have identified two related proteins in the yeast mitochondrial inner membrane, Mrs2p and Lpe10p, and their homologues in mammalia and plants as candidate for Mg2+ transporters. Structural and functional data support the notion that these proteins are distantly related to the bacterial CorA Mg2+ transporters. The yeast genome encodes three further proteins, Alr1p, Alr2p and Mnr2p, which are distantly related to CorA as well as to Mrs2p and Lpe10p. We have shown that they are integral proteins of the yeast plasma membrane, essential for Mg2+ absorption and for life of the yeast cell. Here we want to further characterize these yeast proteins with respect to the structure and function of the transporters. First, we will analyze possible homo- or heteromultimeric complexes which these proteins may form to constitute transport membrane channels or carriers. Second, we will develop techniques suitable to determine Mg2+ transport kinetics in yeast and to study Mg2+ transport mechanisms of Alr1p and Mrs2p. Third, we aim at determining sites in the Mrs2/Lpe10 proteins and in the Alr1/Alr2 proteins which play roles in Mg2+ transport. Finally, we will extend our ongoing genome-wide screens for further candidates directly or indirectly involved in Mg2+ transport. This work will not only contribute to our understanding of cellular and organellar Mg2+ influx and efflux, but also of the physiological role of this metal ion which due to the lack of specific transport inhibitors and mutants remained enigmatic.

Magnesium is the second most abundant metal ion in cells. It serves as a cofactor of hundreds of enzymes, stabilizes membranes and folding structures of nucleic acids. Magnesium as well as other metal ions cannot freely penetrate membranes. Its uptake into cells is mediated by transport proteins in membranes. A gene for a magnesium transport protein (CorA) in bacteria had been characterized before the start of our study. We have then identified a gene for a magnesium transport protein in bakers yeast (named MRS2), plants and mammalia which is distantly related to CorA. The Mrs2 protein is located in the inner mitochondrial membrane where it forms a channel for magnesium influx from the cytoplasm into the organelle, driven by the high negative voltage inside the mitochondrion (> -150 mV). Deletion of the MRS2 gene causes a 95% reduction of magnesium flux and a functional defect of mitochondria in yeast, but it is not lethal for these cells because other cation transporters partly can substitute for it. Down-regulation of MRS2 expression in human cells causes a similar reduction in magnesium flux. However, this effect is lethal for human cells. MRS2 is the first gene identified in eukaryotic cells encoding a mitochondrial cation transport protein. Starting from a mrs2 deletion mutant a genetic screen has provided us with further candidate genes for mitochondrial ion transport proteins. Among them is the gene (MKH1) encoding the K+/H+ exchanger, which also is well conserved from yeast to human cells. It has a prominent role in controlling the volume of the organelle and its deletion may cause part of a human disease phenotype (Wolf-Hirschhorn syndrome).