The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55 Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under ironlimiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition. Proteins belonging to the mitochondrial carrier family (MCF)1 form a large group of structurally related proteins, which exist exclusively in eukaryotes (for reviews, see Refs. 1-3). Typical mitochondrial carrier proteins have a molecular mass of about 35 kDa, contain six membrane-spanning segments, and have a tripartite structure. Each of the three parts is made up of about 100 amino acids and shares sequence homology to the other modules of the proteins. Most members of the MCF are integral proteins of the mitochondrial inner membrane and function in the shuttling of various metabolites and cofactors between the cytosol and mitochondria. The substrates of mitochondrial carrier proteins are rather diverse in size and composition, ranging from protons transported by the uncoupling protein up to large molecules such as ATP and ADP exchanged by the ADP/ATP carrier. Other members of this family are responsible for the transport of phosphate, citrate, fumarate/succinate, carnitine/acylcarnitine, flavine adenine dinucleotide (FAD), and other substrates. One member of the MCF has been located in peroxisomes where it was shown to transport ATP in exchange for AMP (4). The genome of the yeast Saccharomyces cerevisiae contains 35 open reading frames that encode members of the MCF (5-7).The genes can be divided into five subclasses. (i) The functions of the encoded proteins are known, and their transport activities were investigated by expression in Escherichia coli and reconstitution of the purified proteins in liposome vesicles (e.g. Mir1p/PiC, phosphate (8); Arg11p/ORC, o...

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To identify yeast genes involved in cobalt detoxification, we performed RNA expression profiling experiments and followed changes in gene activity upon cobalt stress on a genome-wide scale. We found that cobalt stress specifically results in an immediate and dramatic induction of genes involved in iron uptake. This response is dependent on the Aft1 protein, a transcriptional factor known to regulate a set of genes involved in iron uptake and homeostasis (iron regulon). Like iron starvation, cobalt stress induces accumulation of the Aft1 protein in the nucleus to activate transcription of its target genes. Cells lacking the AFT1 gene (aft1) are hypersensitive to cobalt as well as to other transition metals, whereas expression of the dominant AFT1-1 up allele, which results in up-regulation of AFT1-controlled genes, confers resistance. Cobalt resistance correlates with an increase in intracellular iron in AFT1-1 up cells, and sensitivity of aft1 cells is associated with a lack of iron accumulation. Furthermore, elevated iron levels in the growth medium suppress the cobalt sensitivity of the aft1 mutant cells, even though they increase cellular cobalt. Results presented indicate that yeast cells acquire cobalt tolerance by activating the Aft1p-dependent iron regulon and thereby increasing intracellular iron levels.