Caroline C. Philpott

A Cytosolic Iron Chaperone That Delivers Iron to Ferritin

Ferritins are the main iron storage proteins found in animals, plants, and bacteria. The capacity to store iron in ferritin is essential for life in mammals, but the mechanism by which cytosolic iron is delivered to ferritin is unknown. Human ferritins expressed in yeast contain little iron. Human poly (rC)–binding protein 1 (PCBP1) increased the amount of iron loaded into ferritin when expressed in yeast. PCBP1 bound to ferritin in vivo and bound iron and facilitated iron loading into ferritin in vitro. Depletion of PCBP1 in human cells inhibited ferritin iron loading and increased cytosolic iron pools. Thus, PCBP1 can function as a cytosolic iron chaperone in the delivery of iron to ferritin.

Response to Iron Deprivation in Saccharomyces cerevisiae

Iron is an essential nutrient for almost every organism because iron cofactors, such as heme and iron-sulfur clusters (ISC), are required for the activity of numerous cellular enzymes involved in a wide range of cellular processes. Although iron is a very abundant metal in the earths crust, it has

Regulation of Cation Balance in Saccharomyces cerevisiae

All living organisms require nutrient minerals for growth and have developed mechanisms to acquire, utilize, and store nutrient minerals effectively. In the aqueous cellular environment, these elements exist as charged ions that, together with protons and hydroxide ions, facilitate biochemical reactions and establish the electrochemical gradients across membranes that drive cellular processes such as transport and ATP synthesis. Metal ions serve as essential enzyme cofactors and perform both structural and signaling roles within cells. However, because these ions can also be toxic, cells have developed sophisticated homeostatic mechanisms to regulate their levels and avoid toxicity. Studies in Saccharomyces cerevisiae have characterized many of the gene products and processes responsible for acquiring, utilizing, storing, and regulating levels of these ions. Findings in this model organism have often allowed the corresponding machinery in humans to be identified and have provided insights into diseases that result from defects in ion homeostasis. This review summarizes our current understanding of how cation balance is achieved and modulated in baker’s yeast. Control of intracellular pH is discussed, as well as uptake, storage, and efflux mechanisms for the alkali metal cations, Na+ and K+, the divalent cations, Ca2+ and Mg2+, and the trace metal ions, Fe2+, Zn2+, Cu2+, and Mn2+. Signal transduction pathways that are regulated by pH and Ca2+ are reviewed, as well as the mechanisms that allow cells to maintain appropriate intracellular cation concentrations when challenged by extreme conditions, i.e., either limited availability or toxic levels in the environment.

Metabolic response to iron deficiency in Saccharomyces cerevisiae.

Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways had blocks at points where heme and diiron enzymes function, whereas Ole1, the essential fatty acid desaturase, was resistant to iron depletion. Iron-deficient cells exhibited depletion of most iron enzyme activities, but loss of activity during iron deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid synthesis, altering the properties and functions of cellular membranes.

Coming into view: eukaryotic iron chaperones and intracellular iron delivery.

Eukaryotic cells contain hundreds of metalloproteins, and ensuring that each protein receives the correct metal ion is a critical task for cells. Recent work in budding yeast and mammalian cells has uncovered a system of iron delivery operating in the cytosolic compartment that involves monothiol glutaredoxins, which bind iron in the form of iron-sulfur clusters, and poly(rC)-binding proteins, which bind Fe(II) directly. In yeast cells, cytosolic monothiol glutaredoxins are required for the formation of heme and iron-sulfur clusters and the metallation of some non-heme iron enzymes. Poly(rC)-binding proteins can act as iron chaperones, delivering iron to target non-heme enzymes through direct protein-protein interactions. Although the molecular details have yet to be explored, these proteins, acting independently or together, may represent the basic cellular machinery for intracellular iron delivery.

An iron-sulfur cluster plays a novel regulatory role in the iron-responsive element binding protein

Post-transcriptional regulation of genes important in iron metabolism, ferritin and the transferrin receptor (TfR), is achieved through regulated binding of a cytosolic protein, the iron-responsive element binding protein (IRE-BP), to RNA stem-loop motifs known as iron-responsive elements (IREs). Binding of the IRE-BP respresses ferritin translation and represses degradation of the TfR mRNA. The IRE-BP senses iron levels and accordingly modifies binding to IREs through a novel sensing mechanism. An iron-sulfur cluster of the IRE-BP reversibly binds iron; when cytosolic iron levels are depleted, the cluster becomes depleted of iron and the IRE-BP acquires the capacity to bind IREs. When cytosolic iron levels are replete, the IRE-BP loses RNA binding capacity, but acquires enzymatic activity as a functional aconitase. RNA binding and aconitase activity are mutually exclusive activities of the IRE-BP, and the state of the iron-sulfur cluster determines how the IRE-BP will function.

Each member of the poly-r(C)-binding protein 1 (PCBP) family exhibits iron chaperone activity toward ferritin.

Background: Some iron-dependent enzymes acquire their cofactor from iron chaperones, such as PCBP1. Results: PCBP2, and PCBP3 interact with iron and ferritin in yeast and human cells. Conclusion: All PCBP family members may function as iron chaperones. Significance: The PCBP family members are multifunctional adaptors, mediating interactions between iron or nucleic acids and proteins that act on these molecules. The mechanisms through which iron-dependent enzymes receive their metal cofactors are largely unknown. Poly r(C)-binding protein 1 (PCBP1) is an iron chaperone for ferritin; both PCBP1 and its paralog PCBP2 are required for iron delivery to the prolyl hydroxylase that regulates HIF1. Here we show that PCBP2 is also an iron chaperone for ferritin. Co-expression of PCBP2 and human ferritins in yeast activated the iron deficiency response and increased iron deposition into ferritin. Depletion of PCBP2 in Huh7 cells diminished iron incorporation into ferritin. Both PCBP1 and PCBP2 were co-immunoprecipitated with ferritin in HEK293 cells, and expression of both PCBPs was required for ferritin complex formation in cells. PCBP1 and -2 exhibited high affinity binding to ferritin in vitro. Mammalian genomes encode 4 PCBPs, including the minimally expressed PCBPs 3 and 4. Expression of PCBP3 and -4 in yeast activated the iron deficiency response, but only PCBP3 exhibited strong interactions with ferritin. Expression of PCBP1 and ferritin in an iron-sensitive, ccc1 yeast strain intensified the toxic effects of iron, whereas expression of PCBP4 protected the cells from iron toxicity. Thus, PCBP1 and -2 form a complex for iron delivery to ferritin, and all PCBPs may share iron chaperone activity.

Identification of a Candida albicans ferrichrome transporter and its characterization by expression in Saccharomyces cerevisiae.

Saccharomyces cerevisiaecan accumulate iron through the uptake of siderophore-iron. Siderophore-iron uptake can occur through the reduction of the complex and the subsequent uptake of iron by the high affinity iron transporter Fet3p/Ftr1p. Alternatively, specific siderophore transporters can take up the siderophore-iron complex. The pathogenic fungus Candida albicans can also take up siderophore-iron. Here we identify aC. albicans siderophore transporter, CaArn1p, and characterize its activity. CaARN1 is transcriptionally regulated in response to iron. Through expression studies in S. cerevisiae strains lacking endogenous siderophore transporters, we demonstrate that CaArn1p specifically mediates the uptake of ferrichrome-iron. Iron-ferrichrome and gallium-ferrichrome, but not desferri-ferrichrome, could competitively inhibit the uptake of iron from ferrichrome. Uptake of siderophore-iron resulting from expression of CaARN1 under the control of theMET25-promoter in S. cerevisiae was independent of the iron status of the cells and of Aft1p, the iron-sensing transcription factor. These studies demonstrate that the expression of CaArn1p is both necessary and sufficient for the nonreductive uptake of ferrichrome-iron and suggests that the transporter may be the only required component of the siderophore uptake system that is regulated by iron and Aft1p.

Cell‐cycle arrest and inhibition of G1 cyclin translation by iron in AFT1‐1up yeast

Although iron is an essential nutrient, it is also a potent cellular toxin, and the acquisition of iron is a highly regulated process in eukaryotes. In yeast, iron uptake is homeostatically regulated by the transcription factor encoded by AFT1. Expression of AFT1‐1up, a dominant mutant allele, results in inappropriately high rates of iron uptake, and AFT1‐1up mutants grow slowly in the presence of high concentrations of iron. We present evidence that when Aft1‐1up mutants are exposed to iron, they arrest the cell division cycle at the G1 regulatory point Start. This arrest is dependent on high‐affinity iron uptake and does not require the activation of the DNA damage checkpoint governed by RAD9. The iron‐induced arrest is bypassed by overexpression of a mutant G1 cyclin, cln3‐2, and expression of the G1‐specific cyclins Cln1 and Cln2 is reduced when yeast are exposed to increasing amounts of iron, which may account for the arrest. This reduction is not due to changes in transcription of CLN1 or CLN2, nor is it due to accelerated degradation of the protein. Instead, this reduction occurs at the level of Cln2 translation, a recently recognized locus of cell‐cycle control in yeast.

Metabolic remodeling in iron-deficient fungi ☆

Eukaryotic cells contain dozens, perhaps hundreds, of iron-dependent proteins, which perform critical functions in nearly every major cellular process. Nutritional iron is frequently available to cells in only limited amounts; thus, unicellular and higher eukaryotes have evolved mechanisms to cope with iron scarcity. These mechanisms have been studied at the molecular level in the model eukaryotes Saccharomyces cerevisiae and Schizosaccharomyces pombe, as well as in some pathogenic fungi. Each of these fungal species exhibits metabolic adaptations to iron deficiency that serve to reduce the cells reliance on iron. However, the regulatory mechanisms that accomplish these adaptations differ greatly between fungal species. This article is part of a Special Issue entitled: Cell Biology of Metals.