Daniel J. Kosman

Molecular mechanisms of iron uptake in fungi

Fungi, like all free‐living organisms, are in competition for limiting nutrients. In accumulating iron, fungi are faced also with a trace metal whose aqueous and redox chemistry make it both relatively bio‐unavailable and strongly cytotoxic. Successful adaptation to this environmental context has provided fungi with an iron uptake strategy that has three features: it relies on redox cycling to enhance iron bio‐availability and reduce iron cytotoxicity; it includes both high‐ and low‐affinity pathways that are mechanistically distinct; and it is autoregulating so as to maintain intracellular iron homeostasis. Using Saccharomyces cerevisiae as a paradigm, this review summarizes current knowledge about the four pathways by which this yeast accumulates iron. These four pathways include: siderophore iron accumulation; high affinity iron uptake via an iron permease; and two lower affinity uptake pathways through relatively non‐specific divalent metal ion transporters. All of these four pathways are directly or indirectly dependent on the activity of metalloreductase activity expressed extracellularly on the plasma membrane. A variety of experimental and genomics data indicate that this resourcefulness is shared by many, if not most, fungi. On the other hand, while the autoregulation of iron metabolism in Bakers yeast is well‐understood, little is known about the apparent homeostatic mechanisms in these other yeasts and fungi. The integration of these multiple uptake mechanisms and their regulation into over‐all iron homeostasis in yeast concludes this brief review.

The yeast copper/zinc superoxide dismutase and the pentose phosphate pathway play overlapping roles in oxidative stress protection.

In Saccharomyces cerevisiae, loss of cytosolic superoxide dismutase (Sod1) results in several air-dependent mutant phenotypes, including methionine auxotrophy and oxygen sensitivity. Here we report that these two sod1Δ phenotypes were specifically suppressed by elevated expression of the TKL1 gene, encoding transketolase of the pentose phosphate pathway. The apparent connection between Sod1 and the pentose phosphate pathway prompted an investigation of mutants defective in glucose-6-phosphate dehydrogenase (Zwf1), which catalyzes the rate-limiting NADPH-producing step of this pathway. We confirmed that zwf1Δ mutants are methionine auxotrophs and report that they also are oxygen-sensitive. We determined that a functional ZWF1 gene product was required for TKL1 to suppress sod1Δ, leading us to propose that increased flux through the oxidative reactions of the pentose phosphate pathway can rescue sod1 methionine auxotrophy. To better understand this methionine growth requirement, we examined the sulfur compound requirements of sod1Δ and zwf1Δ mutants, and noted that these mutants exhibit the same apparent defect in sulfur assimilation. Our studies suggest that this defect results from the impaired redox status of aerobically grown sod1 and zwf1 mutants, implicating Sod1 and the pentose phosphate pathway as being critical for maintenance of the cellular redox state.

HOMEOSTATIC REGULATION OF COPPER UPTAKE IN YEAST VIA DIRECT BINDING OF MAC1 PROTEIN TO UPSTREAM REGULATORY SEQUENCES OF FRE1 AND CTR1

Copper deprivation of Saccharomyces cerevisiae induces transcription of the FRE1 andCTR1 genes. FRE1 encodes a surface reductase capable of reducing and mobilizing copper chelates outside the cell, and CTR1 encodes a protein mediating copper uptake at the plasma membrane. In this paper, the protein encoded by MAC1is identified as the factor mediating this homeostatic control. A novel dominant allele of MAC1, MAC1 up2 , is mutated in a Cys-rich domain that may function in copper sensing (a G to A change of nucleotide 812 resulting in a Cys-271 to Tyr substitution). This mutant is functionally similar to theMAC1 up1 allele in which His-279 in the same domain has been replaced by Gln. Both mutations confer constitutive copper-independent expression of FRE1 and CTR1. A sequence including the palindrome TTTGCTCA … TGAGCAAA, appearing within the 5′-flanking region of the CTR1promoter, is necessary and sufficient for the copper- andMAC1-dependent CTR1 transcriptional regulation. An identical sequence appears as a direct repeat in theFRE1 promoter. The data indicate that the signal resulting from copper deprivation is transduced via the Cys-rich motif of MAC1 encompassing residues 264–279. MAC1 then binds directly and specifically to the CTR1 and FRE1 promoter elements, inducing transcription of those target genes. This model defines the homeostatic mechanism by which yeast regulates the cell acquisition of copper in response to copper scarcity or excess.

The copper-iron connection in biology : structure of the metallo-oxidase Fet3p

Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(II) to Fe(III). This ferrous iron oxidation is coupled to the reduction of O2 to H2O and is termed the ferroxidase reaction. Fet3p-produced Fe(III) is transferred to the permease Ftr1p for import into the cytosol. The posttranslational insertion of four copper ions into Fet3p is essential for its activity, thus linking copper and iron homeostasis. The mammalian ferroxidases ceruloplasmin and hephaestin are homologs of Fet3p. Loss of the Fe(II) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death. Here, we present the structure of the Fet3p extracellular ferroxidase domain and compare it with that of human ceruloplasmin and other multicopper oxidases that are devoid of ferroxidase activity. The Fet3p structure delineates features that underlie the unique reactivity of this and homologous multicopper oxidases that support the essential trafficking of iron in diverse eukaryotic organisms. The findings are correlated with biochemical and physiological data to cross-validate the elements of Fet3p that define it as both a ferroxidase and cuprous oxidase.

Molecular genetics of superoxide dismutases in yeasts and related fungi.

Publisher Summary This chapter discusses the molecular genetics of superoxide dismutases in yeasts and related fungi. The chapter provides information on various enzymes that are responsible for the establishing themselves as first component of defense mechanism, such as the superoxide dismutases. These enzymes catalyze the disproportionation of O2-, to H2O2 and O2. As discussed in the chapter, eukaryotes contain at least two superoxide dismutases that are catalytically equivalent but evolutionarily, genetically, and structurally distinct. Superoxide can be produced in a wide variety of cellular redox processes. The simple type of reaction that can generate O2- is auto-oxidation. In Saccharomyces cerevisiae, the dominant source of O2- appears to be leakage from the mitochondrial electron transport chain. These enzymes are also functionally distinct because these are found in different cell compartments. The regulation of expression of these enzymes is also discussed in the chapter along with the discussion of their physiologic functions in light of the phenotypes of strains of yeast and fungi that lack either or both activities. In addition to these enzymes, an extracellular Cu,ZnSOD is characterized in eukaryotes. The human enzyme, designated ECSOD1, is a secreted glycoprotein containing Cu and Zn; the gene for this SOD has been cloned. Although ECSOD and the intracellular SODl differ in primary sequence, they share a number of structural homologies with respect to the metal centers. Some evidences for this type of dismutase in S. cerevisiae and N. crassa are also presented in the chapter.

Multicopper oxidases: a workshop on copper coordination chemistry, electron transfer, and metallophysiology

Multicopper oxidases (MCOs) are unique among copper proteins in that they contain at least one each of the three types of biologic copper sites, type 1, type 2, and the binuclear type 3. MCOs are descended from the family of small blue copper proteins (cupredoxins) that likely arose as a complement to the heme-iron-based cytochromes involved in electron transport; this event corresponded to the aerobiosis of the biosphere that resulted in the conversion of Fe(II) to Fe(III) as the predominant redox state of this essential metal and the solubilization of copper from Cu2S to Cu(H2O)n2+. MCOs are encoded in genomes in all three kingdoms and play essential roles in the physiology of essentially all aerobes. With four redox-active copper centers, MCOs share with terminal copper-heme oxidases the ability to catalyze the four-electron reduction of O2 to two molecules of water. The electron transfers associated with this reaction are both outer and inner sphere in nature and their mechanisms have been fairly well established. A subset of MCO proteins exhibit specificity for Fe2+, Cu+, and/or Mn2+ as reducing substrates and have been designated as metallooxidases. These enzymes, in particular the ferroxidases found in all fungi and metazoans, play critical roles in the metal metabolism of the expressing organism.

Iron Source Preference and Regulation of Iron Uptake in Cryptococcus neoformans

The level of available iron in the mammalian host is extremely low, and pathogenic microbes must compete with host proteins such as transferrin for iron. Iron regulation of gene expression, including genes encoding iron uptake functions and virulence factors, is critical for the pathogenesis of the fungus Cryptococcus neoformans. In this study, we characterized the roles of the CFT1 and CFT2 genes that encode C. neoformans orthologs of the Saccharomyces cerevisiae high-affinity iron permease FTR1. Deletion of CFT1 reduced growth and iron uptake with ferric chloride and holo-transferrin as the in vitro iron sources, and the cft1 mutant was attenuated for virulence in a mouse model of infection. A reduction in the fungal burden in the brains of mice infected with the cft1 mutant was observed, thus suggesting a requirement for reductive iron acquisition during cryptococcal meningitis. CFT2 played no apparent role in iron acquisition but did influence virulence. The expression of both CFT1 and CFT2 was influenced by cAMP-dependent protein kinase, and the iron-regulatory transcription factor Cir1 positively regulated CFT1 and negatively regulated CFT2. Overall, these results indicate that C. neoformans utilizes iron sources within the host (e.g., holo-transferrin) that require Cft1 and a reductive iron uptake system.

Cuprous oxidase activity of yeast Fet3p and human ceruloplasmin: implication for function

The Fet3 protein in Saccharomyces cerevisiae and mammalian ceruloplasmin are multicopper oxidases (MCO) that are required for iron homeostasis via their catalysis of the ferroxidase reaction, 4Fe2++O2+4H+→4Fe3++2H2O. The enzymes may play an essential role in copper homeostasis since they exhibit a strikingly similar kinetic activity towards Cu1+ as substrate. In contrast, laccase, an MCO that exhibits weak activity towards Fe2+, exhibits a similarly weak activity towards Cu1+. Kinetic analyses of the Fet3p reaction demonstrate that the ferroxidase and cuprous oxidase activities are due to the same electron transfer site on the enzyme. These two ferroxidases are fully competent kinetically to play a major role in maintaining the cuprous–cupric redox balance in aerobic organisms.

Spectral and kinetic properties of the Fet3 protein from Saccharomyces cerevisiae, a multinuclear copper ferroxidase enzyme

High affinity iron uptake in Saccharomyces cerevisiae requires Fet3p. Fet3p is proposed to facilitate iron uptake by catalyzing the oxidation of Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1. Here, a recombinant Fet3p has been produced in yeast that, lacking the C-terminal membrane-spanning domain, is secreted directly into the growth medium. Solutions of this Fet3p at >1 mg/ml have the characteristic blue color of a type 1 Cu(II)-containing protein, consistent with the sequence homology that placed this protein in the class of multinuclear copper oxidases that includes ceruloplasmin. Fet3p has an intense absorption at 607 nm (ε = 5500 m −1 cm−1) due to this type 1 Cu(II) and a shoulder in the near UV at 330 nm (ε = 5000m −1 cm−1) characteristic of a type 3 binuclear Cu(II) cluster. The EPR spectrum of this Fet3p showed the presence of one type 1 Cu(II) and one type 2 Cu(II) (A∥ = 91 and 190 × 10−4cm−1, respectively). Copper analysis showed this protein to have 3.85 g atom copper/mol, consistent with the presence of one each of the three types of Cu(II) sites found in multinuclear copper oxidases. N-terminal analysis demonstrated that cleavage of a signal peptide occurred after Ala-21 in the primary translation product. Mass spectral and carbohydrate analysis of the protein following Endo H treatment indicated that the preparation was still 15% (w/w) carbohydrate, probably O-linked. Kinetic analysis of the in vitro ferroxidase reaction catalyzed by this soluble Fet3p yielded precise kinetic constants. TheK m values for Fe(II) and O2 were 4.8 and 1.3 μm, respectively, whilek cat values for Fe(II) and O2turnover were 9.5 and 2.3 min−1, consistent with an Fe(II):O2 reaction stoichiometry of 4:1.

Redox Cycling in Iron Uptake, Efflux, and Trafficking

Aerobic organisms are faced with a dilemma. Environmental iron is found primarily in the relatively inert Fe(III) form, whereas the more metabolically active ferrous form is a strong pro-oxidant. This conundrum is solved by the redox cycling of iron between Fe(III) and Fe(II) at every step in the iron metabolic pathway. As a transition metal ion, iron can be “metabolized” only by this redox cycling, which is catalyzed in aerobes by the coupled activities of ferric iron reductases (ferrireductases) and ferrous iron oxidases (ferroxidases).