Genetic Dissection of a Mitochondria-Vacuole Signaling Pathway in Yeast Reveals a Link between Chronic Oxidative Stress and Vacuolar Iron Transport

Abstract

Deletion of two homologous genes, MRS3 and MRS4, that encode mitochondrial iron transporters affects the activity of the vacuolar iron importer Ccc1. Ccc1 levels are decreased in ⌬mrs3⌬mrs4 cells, but the activity of the transporter is increased, resulting is reduced cytosolic iron. Overexpression of CCC1 in ⌬mrs3⌬mrs4 cells results in a severe growth defect due to decreased cytosolic iron, referred to as the mitochondriavacuole signaling (MVS) phenotype. Mutants were identified that suppress the MVS growth defect, and FRA1 was identified as a gene that suppresses the MVS phenotype. Overexpression of FRA1 suppresses altered transition metal metabolism in ⌬mrs3⌬mrs4 cells, whereas deletion of FRA1 is synthetically lethal with ⌬mrs3⌬mrs4. Fra1 binds to Tsa1, which encodes a thioredoxin-dependent peroxidase. Deletion of TSA1 or TRR1 is synthetically lethal in ⌬mrs3⌬mrs4 cells, suggesting that ⌬mrs3⌬mrs4 cells generate reactive oxygen metabolites. The generation of reactive oxygen metabolites in ⌬mrs3⌬mrs4 cells was confirmed by use of the reporter molecule 2,7-dichlorodihydrofluorescein diacetate. These results suggest that mitochondria-induced oxidant damage is responsible for activating Ccc1 and that Fra1 and Tsa1 can reduce oxidant damage.Mitochondria are well recognized as the site for energy production and biosynthetic pathways. Mitochondria are central for a variety of downstream signaling pathways initiated by perturbations in mitochondrial activities. Most notably, in many eukaryotes decreased mitochondrial function results in apoptosis. The budding yeast Saccharomyces cerevisiae has been useful in dissecting the genetics and biochemistry of mitochondrial signaling events. Loss of mitochondrial heme synthesis leads to changes in the transcription of a wide variety of genes mediated by the Hap family of transcription factors (1). Decreased mitochondrial respiration, independent of effects on heme synthesis, affects the transcription of nuclear genes that result in a reconfiguration of energy metabolism, referred to as the retrograde response (2). Finally, alteration in mitochondrial iron-sulfur cluster synthesis can induce expression of genes involved in iron metabolism (3).We showed that deletion of two nuclear genes MRS3 and MRS4, which encode homologous mitochondrial iron transporters, had effects on cellular metal metabolism (4). Deletion of MRS3 or MRS4 individually had no phenotype, but a double deletion showed impaired mitochondrial iron metabolism when cells were grown on low iron medium (4 -6). Mitochondrial iron-consuming processes such as heme and iron-sulfur cluster synthesis appeared normal when ⌬mrs3⌬mrs4 cells were grown in iron-sufficient medium, suggesting that low affinity iron transport systems can supply iron to the mitochondria. In iron-sufficient medium, however, ⌬mrs3⌬mrs4 cells showed abnormalities in iron homeostasis as well as alterations in response to other transition metals (4). We showed that many of the phenotypes in the double deletion could be suppressed by deleting CCC1. The...

Yap5 Protein-regulated Transcription of the TYW1 Gene Protects Yeast from High Iron Toxicity

“…porter Ccc1, both leading to decreased cytosolic iron (13). To determine whether overexpression of Tyw1 results in defective iron transport activity, we assayed cellular iron content by inductively coupled plasma optical emission spectrometry.…”

“…In contrast, deletion of TYW1 did not alter transition metal sensitivity. Copper sensitivity and cobalt resistance often result from induction of the iron regulon (12,13). We determined that overexpression of TYW1 increased the activity of the FET3-lacZ reporter construct, FET3 being a member of the iron regulon (Fig.…”

Expression of the Yeast Cation Diffusion Facilitators Mmt1 and Mmt2 Affects Mitochondrial and Cellular Iron Homeostasis

“…Other Procedures-The production of reactive oxygen species was measured using 2Ј,7Ј-dichlorodihydrofluorescein diacetate to DCF conversion (Invitrogen) as described (9). Western blot analysis was performed as described (19).…”

“…Induction of the Iron Regulon by MMT1&2 Overexpression Is Dependent on the Presence of CCC1-Decreased mitochondrial iron accumulation resulting from deletion of MRS3/MRS4 induced the iron regulon via increasing the activity of the vacuolar iron transporter Ccc1 (3,9). This conclusion was based on the fact that the level of Ccc1 does not increase in ⌬mrs3⌬mrs4 cells, but deletion of CCC1 FIGURE 4.…”

“…Homologues of these genes, termed mitoferrins, are found in all eukaryotes and mutations in these genes result in defective mitochondrial iron homeostasis in a wide range of species, including Oryza (5) Drosophila (6), Danio rerio (7), and Mus musculus (8). Studies in S. cerevisiae also identified the mitochondrial carrier family member Rim2 as an iron transporter (9,10). Rim2 was also identified as a pyrimidine exchanger; however, the relationship between iron transport and pyrimidine exchange is unclear (11).…”