Factors involved in the homeostasis of divalent metal ions

Several divalent and trivalent metal ions are nutrients of all organisms because they act as essential cofactors for a wealth of biological processes. They serve also as cofactors in electron transfer systems and in the catalysis of hundreds of proteins. They have equally important roles in maintaining tertiary structures of proteins and RNAs, as well as in catalysis of ribozymes like group I and group II RNA and of the spliceosome. Failure to maintain appropriate levels of metal ions in humans is a feature of several known hereditary and acquired diseases. Major transport systems for calcium and iron have been studied to some extent in mammals, plants and yeast, but the isolation of genes for these transporters is still ongoing. The isolation of factors involved in uptake and homeostasis of other divalent metal ions like Mg 2+, Mn 2+, Ni2+, Zn2+, Co2+, Cu2+ has been started in recent years only and available data on about one dozen of candidate proteins is still rather preliminary. This is mostly due to the facts that eukaryotes have two or more parallel acting transporters and that their ion specificity is low. Moreover, the candidate proteins belong to at least five different gene families. This laboratory has identified four genes in yeast (MRS2, LPE10, MRS3, MRS4) which are likely to be involved in transport of Mg 2+, Ni2+ and Co2+ across the inner mitochondrial membrane. Mrs2p and Lpe10p are related to the bacterial CorA family of Mg 2+ transporters whereas Mrs3p and Mrs4p belong to the large class of eukaryotic, mitochondrial solute carder proteins. Under this project a systematic analysis of proteins with putative functions in metal ion transport will be undertaken. Emphasis will be on the screening of yeast genomic libraries for novel genes involved in uptake and homeostasis of metal ions Mg 2+, Ni2+, Co2+, Mn 2+ and Zn2+. A combined genetic and functional analysis will include multiple gene disruptions, synthetic lethality, co-dominance, cellular location of proteins and the search for partner proteins, measurements of ion concentrations in cellular compartments and ion transport through membranes. In its first phase this study will be based on the genetically amenable yeast Saccharomyces cerevisiae where most of the candidate genes have been isolated previously. In a second phase we will identify and functionally characterize homologues of the yeast genes in plants and mammals, with a particular interest in inherited and acquired disorders in metal ion homeostasis.

Transport and physiological role of divalent metal ions are -with the exception of calcium- widely unknown in molecular terms. We have characterized for the first time a gene product, Alr1p, which transports magnesium (Mg2+) across the plasma membrane of a eukaryotic cell. Moreover, we have used genome-wide expression profiling to molecularly document the cellular response to toxic metal ions, particularly to cobalt. Mg2+ is next to potassium the second abundant metal ion in cells. Its high concentration (0.4 to 0.8 mM free ionized Mg2+) is essential for the function of many cellular components. Studying the Alr1 gene product in budding yeast we could show for the first time that these cells have one major Mg2+ transporter in the plasma membrane (namely Alr1p), that its activity is essential for the live of yeast cells and that both its synthesis and degradation are subjected to a tight control by Mg2+ concentrations. While expression control via transcriptional regulation is a slow process, reducing Alr1p levels during several generations only, endocytosis and vacuolar degradation in response to externally added Mg2+ is a fast and efficient means of controlling Mg2+ transport capacity. This illustrates in a surprising manner how cells react quickly to changing environmental conditions. Cobalt and many other metal ions are toxic for cells and organisms. Only when present in extremely low concentrations they may be used for cellular processes (e.g. cobalt for vitamin B12 synthesis). Their toxic effect is likely to be through competition with essential metal ions like iron or copper for specific ion binding sites. Yet, toxic reactions of metal ions are poorly understood. We have initiated a genome-wide analysis of gene expression using whole genome biochips in order to understand the molecular response of yeast cells to cobalt (and other metal ions). The results are exceptionally straightforward and meaningful: The addition of even subtoxic concentrations of cobalt to growth media upregulates all genes involved in iron uptake into yeast cells (iron regulon under the control of the iron-binding transcription factor Aft1) and increase the intracellular iron concentration. We conclude that more iron in the cells efficiently occupies the respective binding sites and competitively prevents cobalt from binding. This notion is supported by our finding that mutants lacking this increase in iron concentrations (e.g. aft1) are highly sensitive towards cobalt. We regard the control of the iron regulon as the most sensitive response of yeast cells to cobalt. This is likely to be an evolutionary developed genetic program to minimize the toxic effects of cobalt. We have initial data which point to similar reactions of human cells to cobalt treatment. This study documents impressively the great potential of genome-wide expression profiling for the study of toxic effects of metal ions, a field which so far has been widely ignored.