Andrew Dancis

Molecular characterization of a copper transport protein in S. cerevisiae: An unexpected role for copper in iron transport

We report the identification and characterization of CTR1, a gene in the yeast S. cerevisiae that encodes a multispanning plasma membrane protein specifically required for high affinity copper transport into the cell. The predicted protein contains a methionine- and serine-rich domain that includes 11 examples of the sequence Met-X2-Met, a motif noted in proteins involved in bacterial copper metabolism. CTR1 mutants and deletion strains have profound deficiency in ferrous iron uptake, thus revealing a requirement for copper in mediating ferrous transport into the cell. Genetic evidence suggests that the target for this requirement is the FET3 gene (detailed in a companion study), predicted to encode a copper-containing protein that acts as a cytosolic ferro-oxidase. These findings provide an unexpected mechanistic link between the uptake of copper and iron.

A Permease-Oxidase Complex Involved in High-Affinity Iron Uptake in Yeast

Iron must cross biological membranes to reach essential intracellular enzymes. Two proteins in the plasma membrane of yeast—a multicopper oxidase, encoded by the FET3 gene, and a permease, encoded by the FTR1 gene—were shown to mediate high-affinity iron uptake. FET3 expression was required for FTR1 protein to be transported to the plasma membrane. FTR1 expression was required for apo-FET3 protein to be loaded with copper and thus acquire oxidase activity. FTR1 protein also played a direct role in iron transport. Mutations in a conserved sequence motif of FTR1 specifically blocked iron transport.

RESPONSIVE-TO-ANTAGONIST1, a Menkes/Wilson Disease–Related Copper Transporter, Is Required for Ethylene Signaling in Arabidopsis

Ethylene is an important regulator of plant growth. We identified an Arabidopsis mutant, responsive-to-antagonist1 (ran1), that shows ethylene phenotypes in response to treatment with trans-cyclooctene, a potent receptor antagonist. Genetic epistasis studies revealed an early requirement for RAN1 in the ethylene pathway. RAN1 was cloned and found to encode a protein with similarity to copper-transporting P-type ATPases, including the human Menkes/Wilson proteins and yeast Ccc2p. Expression of RAN1 complemented the defects of a ccc2delta mutant, demonstrating its function as a copper transporter. Transgenic CaMV 35S::RAN1 plants showed constitutive expression of ethylene responses, due to cosuppression of RAN1. These results provide an in planta demonstration that ethylene signaling requires copper and reveal that RAN1 acts by delivering copper to create functional hormone receptors.

AFT1: a mediator of iron regulated transcriptional control in Saccharomyces cerevisiae.

Using a scheme for selecting mutants of Saccharomyces cerevisiae with abnormalities of iron metabolism, we have identified a gene, AFT1, that mediates the control of iron uptake. AFT1 encodes a 78 kDa protein with a highly basic amino terminal domain and a glutamine‐rich C‐terminal domain, reminiscent of transcriptional activators. The protein also contains an amino terminal and a C‐terminal region with 10% His residues. A dominant mutant allele of this gene, termed AFT1‐1up, results in high levels of ferric reductase and ferrous iron uptake that are not repressed by exogenous iron. The increased iron uptake is associated with enhanced susceptibility to iron toxicity. These effects may be explained by the failure of iron to repress transcription of FRE1, FRE2 and FET3. FRE1 and FRE2 encode plasma membrane ferric reductases, obligatory for ferric iron assimilation, and FET3 encodes a copper‐dependent membrane‐associated oxidase required for ferrous iron uptake. Conversely, a strain with interruption of the AFT1 gene manifests low ferric reductase and ferrous iron uptake and is susceptible to iron deprivation, because of deficient expression of FRE1 and negligible expression of FRE2 and FET3. Thus, AFT1 functions to activate transcription of target genes in response to iron deprivation and thereby plays a central role in iron homeostasis.

Iron-regulated DNA binding by the AFT1 protein controls the iron regulon in yeast

Iron deprivation of Saccharomyces cerevisiae induces transcription of genes required for high‐affinity iron uptake. AFT1 mediates this transcriptional control. In this report, the 5′‐flanking region of FET3, which encodes a copper‐dependent oxidase required for iron transport, was analyzed and found to contain a DNA sequence responsible for AFT1‐regulated gene expression. AFT1 was capable of interacting specifically with this DNA sequence. A core element within this DNA sequence necessary for the binding of AFT1 was also determined. In vivo footprinting demonstrated occupancy of the AFT1 binding site in cells deprived of iron and not in cells grown in the presence of iron. Thus, the environmental signal resulting from iron deprivation was transduced through the regulated binding of AFT1 to the FET3 promoter, followed by the activation of transcription. A regulon of genes under the control of AFT1 could be defined. AFT1 was able to bind to a consensus binding site (PyPuCACCCPu) in the 5′ region of FRE1, FRE2, FTR1, FTH1 and CCC2.

Genetic evidence that ferric reductase is required for iron uptake in Saccharomyces cerevisiae

The requirement for a reduction step in cellular iron uptake has been postulated, and the existence of plasma membrane ferric reductase activity has been described in both procaryotic and eucaryotic cells. In the yeast Saccharomyces cerevisiae, there is an externally directed reductase activity that is regulated by the concentration of iron in the growth medium; maximal activity is induced by iron starvation. We report here the isolation of a mutant of S. cerevisiae lacking the reductase activity. This mutant is deficient in the uptake of ferric iron and is extremely sensitive to iron deprivation. Genetic analysis of the mutant demonstrates that the reductase and ferric uptake deficiencies are due to a single mutation that we designate fre1-1. Both phenotypes cosegregate in meiosis, corevert with a frequency of 10(-7), and are complemented by a 3.5-kilobase fragment of genomic DNA from wild-type S. cerevisiae. This fragment contains FRE1, the wild-type allele of the mutant gene. The level of the gene transcript is regulated by iron in the same was as the reductase activity. The ferrous ion product of the reductase must traverse the plasma membrane. A high-affinity (Km = 5 microM) ferrous uptake system is present in both wild-type and mutant cells. Thus, iron uptake in S. cerevisiae is mediated by two plasma membrane components, a reductase and a ferrous transport system.

Copper-dependent degradation of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p in the apparent absence of endocytosis.

The cell surface protein repertoire needs to be regulated in response to changes in the extracellular environment. In this study, we investigate protein turnover of the Saccharomyces cerevisiae plasma membrane copper transporter Ctr1p, in response to a change in extra‐cellular copper levels. As Ctr1p mediates high affinity uptake of copper into the cell, modulation of its expression is expected to be involved in copper homeostasis. We demonstrate that Ctr1p is a stable protein when cells are grown in low concentrations of copper, but that exposure of cells to high concentrations of copper (10 microM) triggers degradation of cell surface Ctr1p. This degradation appears to be specific for Ctr1p and does not occur with another yeast plasma membrane protein tested. Internalization of some Ctr1p can be seen when cells are exposed to copper. However, yeast mutant strains defective in endocytosis (end3, end4 and chc1‐ts) and vacuolar degradation (pep4) exhibit copper‐dependent Ctr1p degradation, indicating that internalization and delivery to the vacuole is not the principal mechanism responsible for degradation. In addition, a variant Ctr1p with a deletion in the cytosolic tail is not internalized upon exposure of cells to copper, but is nevertheless degraded. These observations indicate that proteolysis at the plasma membrane most likely explains copper‐dependent turnover of Ctr1p and point to the existence of a novel pathway in yeast for plasma membrane protein turnover.

Yeast Mitochondrial Protein, Nfs1p, Coordinately Regulates Iron-Sulfur Cluster Proteins, Cellular Iron Uptake, and Iron Distribution

Nfs1p is the yeast homolog of the bacterial proteins NifS and IscS, enzymes that release sulfur from cysteine for iron-sulfur cluster assembly. Here we show that the yeast mitochondrial protein Nfs1p regulates cellular and mitochondrial iron homeostasis. A strain of Saccharomyces cerevisiae, MA14, with a missenseNFS1 allele (I191S) was isolated in a screen for altered iron-dependent gene regulation. This mutant exhibited constitutive up-regulation of the genes of the cellular iron uptake system, mediated through effects on the Aft1p iron-regulatory protein. Iron accumulating in the mutant cells was retained in the mitochondrial matrix while, at the same time, iron-sulfur proteins were deficient. In this work, the yeast protein was localized to mitochondria, and the gene was shown to be essential for viability. Furthermore, Nfs1p in the MA14 mutant was found to be markedly decreased, suggesting that this low protein level produced the observed regulatory effects. This hypothesis was confirmed by experiments in which expression of wild-type Nfs1p from a regulated galactose-induced promoter was turned off, leading to recapitulation of the iron regulatory phenotypes characteristic of the MA14 mutant. These phenotypes include decreases in iron-sulfur protein activities coordinated with increases in cellular iron uptake and iron distribution to mitochondria.

Intramembrane Bis-Heme Motif for Transmembrane Electron Transport Conserved in a Yeast Iron Reductase and the Human NADPH Oxidase

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91phox protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.

Mt-Hsp70 Homolog, Ssc2p, Required for Maturation of Yeast Frataxin and Mitochondrial Iron Homeostasis

Here we show that the yeast mitochondrial chaperone Ssc2p, a homolog of mt-Hsp70, plays a critical role in mitochondrial iron homeostasis. Yeast withssc2-1 mutations were identified by a screen for altered iron-dependent gene regulation and mitochondrial dysfunction. These mutants exhibit increased cellular iron uptake, and the iron accumulates exclusively within mitochondria. Yfh1p is homologous to frataxin, the human protein implicated in the neurodegenerative disease, Friedreich’s ataxia. Like mutants ofyfh1, ssc2-1 mutants accumulate vast quantities of iron in mitochondria. Furthermore, using import studies with isolated mitochondria, we demonstrate a specific role for Ssc2p in the maturation of Yfh1p within this organelle. This function for a mitochondrial Hsp70 chaperone is likely to be conserved, implying that a human homolog of Ssc2p may be involved in iron homeostasis and in neurodegenerative disease.