A Specific Role of the Yeast Mitochondrial Carriers Mrs3/4p in Mitochondrial Iron Acquisition under Iron-limiting Conditions

Abstract

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...

A Mitochondrial-Vacuolar Signaling Pathway in Yeast That Affects Iron and Copper Metabolism

“…Foury and Rogati (1) show that deletion of these genes limited mitochondrial iron accumulation, leading to the hypothesis that these genes encode mitochondrial iron transporters. This hypothesis was supported by Muhlenhoff et al (2), who assayed mitochondrial heme and Fe-S synthesis in both ⌬mrs3⌬mrs4 cells and in cells FIG. 8.…”

“…Indeed, Muhlenhoff et al (2) show that overexpression of MRS3 or MRS4 stimulated iron-sulfur cluster synthesis and that this stimulation could not be accounted for by increased iron transport. Their explanation for this stimulatory activity was that Mrs3p or Mrs4p may transport molecules other than ferrous iron.…”

“…In particular, ⌬mrs3⌬mrs4 cells show increased cellular iron acquisition due to up-regulation of the iron regulon (1,2). Disruption of the MRS3/MRS4 homologue in Cryptococcus neoformans also leads to increased expression of iron transport genes (7).…”

“…⌬yfh1 cells are protected from mitochondrial iron toxicity when MRS3 and MRS4 are deleted (1). Deletion of these genes does not completely abrogate mitochondrial iron uptake, leading to the suggestion that they encode mitochondrial iron transporters whose function only becomes apparent under low iron conditions (2).…”

“…Recent studies have shown that deletion of both MRS3 and MRS4, members of the mitochondrial carrier family, results in decreased mitochondrial iron content. Double deletion strains (⌬mrs3⌬mrs4) demonstrate a low iron growth defect and decreased heme and iron-sulfur cluster synthesis (1,2). Furthermore, Foury and Roganti (1) deleted MRS3 and MRS4 in a strain lacking Yfh1p, the yeast frataxin homologue.…”