Ferric reductase of Saccharomyces cerevisiae: molecular characterization, role in iron uptake, and transcriptional control by iron.

Abstract

The principal iron uptake system of Saccharomyces cerevisiae utilizes a reductase activity that acts on ferric iron chelates external to the cell. The FREI gene product is required for this activity. Iron is an essential nutrient that is required by many enzymes for cellular functions such as DNA synthesis and respiration. Ferric iron forms insoluble ferric hydroxide complexes in the presence of oxygen and water, making iron availability a biological problem under these conditions. Ferrous iron is much more soluble but is rapidly converted to ferric iron in the presence of oxygen (1). Cells have developed two major solutions to the problem of assimilation offerric iron from the environment. In Escherichia coli, specific ferric binding compounds termed siderophores are secreted that bind and solubilize environmental iron and deliver it to specific receptors for transport across the outer and inner membranes (2). In the yeast Saccharomyces cerevisiae, the major iron uptake system under aerobic conditions depends on a transplasma membrane electron transport system (3, 4) that reduces ferric iron external to the cell. Multicellular eukaryotes may also use an externally directed ferric reductase prior to transmembrane movement of ferrous iron (5-8). Our previous results indicated that the FREI gene product in S. cerevisiae is required for external ferric reduction and for ferric iron utilization, thus linking these two processes (9). However, a mutation in FREI did not affect the uptake offerrous iron (9), suggesting that the ferrous transport system is an independent component of the iron utilization apparatus in yeast. Many organisms control their rate of iron assimilation by ascertaining their requirement for iron and expressing a limiting component of the uptake system accordingly (10). Our previous results suggest that during aerobic growth of S. cerevisiae, changes in iron availability are reflected in alterations in the levels of FREI mRNA and of ferric iron uptake (9). In this paper we present the structure of the FREI gene and identify DNA sequences that mediate its regulation by ironA MATERIALS AND METHODSYeast Strains. The following yeast strains were derived from the wild-type strain F113 (MATa, can), inol-13, ura3-52): W103 (MATa, can], inol-13, ura3-52,frel-1) was derived by chemical mutagenesis of F113 and selection for a reductase-negative phenotype (9); W126 was derived by transformation of F113 with the plasmid pWDC20, which carries the FREI gene on the high-copy-number vector YEp24 (11). We used one-step gene disruption (12) to create strain N1 (MATa, can), inol-13, Afrel:: URA3), in which an 800-base-pair (bp) Xho I fragment internal to the FREI coding region was replaced with a URA3 marker gene, and strain N2 (MATa, can, inol-13, Afrel:: URA3), in which a 2.7-kilobase (kb) Cla I fragment containing the entire FREI coding region was similarly replaced. Disruption/deletion strains of the FREI locus were also constructed in the wild-type strain H1085, yielding strain W218 (MATa, leu2-3,112, ...

Six Arabidopsis thaliana homologues of the human respiratory burst oxidase (gp91phox)

“…Figure 2(a) shows the similarity/identity of these proteins and a phylogenetic tree. The Atrboh proteins show 59.8-62.3% similarity to gp91 phox , whereas other related flavoproteins such as the yeast ferric reductase FRE1 (Dancis et al, 1992), show only 45.9% similarity. The analysis of the hydropathy profiles of these proteins reveals many coincidences with the profile of gp91 phox ( Figure 2b).…”

“…The Rboh proteins are similar to a family of low potential plasma membrane β cytochromes that includes gp91 phox and some related ferric reductases from yeast such as FRE1 (Dancis et al, 1992). The four critical histidines postulated to bind the two hemes required for transfer of electrons from a cytosolic donor across the membrane to an extracellular acceptor (Finegold et al, 1996) are absolutely conserved (H-459, 473, 561 and 574 in AtrbohD; Figure 1).…”

Siderophore uptake by Candida albicans: effect of serum treatment and comparison with Saccharomyces cerevisiae

“…S. cerevisiae can use two different high-affinity mechanisms, reductive and non-reductive, to take up iron from the extracellular medium (De Luca and Wood, 2000). The reductive mechanism (Dancis et al, 1990;Lesuisse et al, 1987) involves the release of extracellular ferric chelates by reduction at the cell surface by the inducible plasma membrane reductases, Fre1p and Fre2p (Dancis et al, 1992;Georgatsou and Alexandraki, 1994). A permease-oxidase complex, Ftr1p and Fet3p, is than involved in translocating iron through the plasma membrane (Askwith et al, 1994;Stearman et al, 1996).…”

Iron: Nutritious, Noxious, and Not Readily Available

“…Sequence analysis of the yeast FREl gene (which was identified by complementation of the jrel-1 mutant) revealed a single long open reading frame of 686 amino acids with a predicted molecular mass of 78.8 kD (Dancis et al, 1992). The open reading frame has limited homology (17.9% identity and 62.2% similarity over the carboxy-terminal 402 amino acids of FRE1) to the large subunit of human Cyt b5,, (gp91-phox), the protein affected in the X-linked form of chronic granulomatous disease (Dancis et al, 1992). Cyt b558 is a component of the respiratory burst oxidase of neutrophils, a membrane-bound enzyme complex that catalyzes the one-electron reduction of oxygen to 02-.…”