Emmanuel Lesuisse

We have studied the intracellular trafficking of Sit1 [ferrioxamine B (FOB) transporter] and Enb1 (enterobactin transporter) in Saccharomyces cerevisiae using green fluorescent protein (GFP) fusion proteins. Enb1 was constitutively targeted to the plasma membrane. Sit1 was essentially targeted to the vacuolar degradation pathway when synthesized in the absence of substrate. Massive plasma membrane sorting of Sit1 was induced by various siderophore substrates of Sit1, and by coprogen, which is not a substrate of Sit1. Thus, different siderophore transporters use different regulated trafficking processes. We also studied the fate of Sit1‐mediated internalized siderophores. Ferrioxamine B was recovered in isolated vacuolar fractions, where it could be detected spectrophotometrically. Ferrioxamine B coupled to an inhibitor of mitochondrial protoporphyrinogen oxidase (acifluorfen) could not reach its target unless the cells were disrupted, confirming the tight compartmentalization of siderophores within cells. Ferrioxamine B coupled to a fluorescent moiety, FOB‐nitrobenz‐2‐oxa‐1,3‐diazole, used as a Sit1‐dependent iron source, accumulated in the vacuolar lumen even in mutants displaying a steady‐state accumulation of Sit1 at the plasma membrane or in endosomal compartments. Thus, the fates of siderophore transporters and siderophores diverge early in the trafficking process.

We screened a collection of 4847 haploid knockout strains (EUROSCARF collection) of Saccharomyces cerevisiae for iron uptake from the siderophore ferrioxamine B (FOB). A large number of mutants showed altered uptake activities, and a few turned yellow when grown on agar plates with added FOB, indicating increased intracellular accumulation of undissociated siderophores. A subset consisting of 197 knockouts with altered uptake was examined further for regulated activities that mediate cellular uptake of iron from other siderophores or from iron salts. Hierarchical clustering analysis grouped the data according to iron sources and according to mutant categories. In the first analysis, siderophores grouped together with the exception of enterobactin, which grouped with iron salts, suggesting a reductive pathway of iron uptake for this siderophore. Mutant groupings included three categories: (i) high-FOB uptake, high reductase, low-ferrous transport; (ii) isolated high- or low-FOB transport; and (iii) induction of all activities. Mutants with statistically altered uptake activities included genes encoding proteins with predominant localization in the secretory pathway, nucleus, and mitochondria. Measurements of different iron-uptake activities in the yeast knockout collection make possible distinctions between genes with general effects on iron metabolism and those with pathway-specific effects.

The ability of the parasitic flagellate Tritrichomonas foetus to use various iron sources for its physiological requirements was studied. The siderophores ferrioxamine B, ferrichrome, triacetylfusarinine, coprogen, enterobactin and pyoverdine sustained growth of the cells under iron-limited conditions, and siderophore iron was incorporated into the major iron protein of T. foetus, ferredoxin. The kinetics of siderophore uptake by the cells indicated that a non-saturable transport is involved, unlike the uptake of a ferrous salt. Siderophore uptake by the cells did not involve extracellular reductive dissociation of the ferric chelates, although T. foetus cells had some ferrireductase activity on ferric citrate. Fluorescent analogues of siderophores were used to show that the siderophores taken up by the cells were in small intracellular vesicles. The fluorescence emission maximum of pyoverdine in these intracellular vesicles shifted from 460 nm to 530 nm, indicating a very acidic environment. The results suggest that a wide range of chemically unrelated siderophores can be taken up non-specifically and efficiently used by T. foetus; the mechanism involved may be pinocytosis and removal of the iron from the siderophores in acidic intracellular vesicles. Haemin also sustained the growth of T. foetus cells under iron-limited conditions. The use of haemin iron by the cells probably involves haem oxygenase, since traces of biliverdin were found in the medium when haemin was the iron source. The iron uptake and ferrireductase activities of the cells do not seem to be regulated by the amounts of iron and copper in the growth medium.

Summary: We cloned the CaYFH1 gene that encodes the yeast frataxin homologue in Candida albicans. CaYFH1 was expressed in Δyfh1 Saccharomyces cerevisiae cells, where it compensated for all the phenotypes tested except for the lack of cytochromes. Double ΔCayfh1/ΔCayfh1 mutant had severe defective growth, accumulated iron in their mitochondria, lacked aconitase and succinate dehydrogenase activity and had defective respiration. The reductive, siderophore and haem uptake systems were constitutively induced and the cells excreted flavins, thus behaving like iron‐deprived wild‐type cells. Mutant cells accumulated reactive oxygen species and were hypersensitive to oxidative stress, but not to iron. Cytochromes were less abundant in mutants than in wild‐type cells, but this did not result from defective haem synthesis. The low cytochrome concentration in mutant cells was comparable to that of iron‐deprived wild‐type cells. Mitochondrial iron was still available for haem synthesis in ΔCayfh1/ΔCayfh1 cells, in contrast to S. cerevisaeΔyfh1 cells. CaYFH1 transcription was strongly induced by iron, which is consistent with a role of CaYfh1 in iron storage. Iron also regulated transcription of CaHEM14 (encoding protoporphyrinogen oxidase) but not that of CaHEM15 (encoding ferrochelatase). There are thus profound differences between S. cerevisiae and C. albicans in terms of haem synthesis, cytochrome turnover and the role of frataxin in these processes.

Analysis of Saccharomyces cerevisiae cell transcriptome revealed that iron deprivation/supplementation affects genes other than those of the iron regulon (controlled by Aft proteins). Several genes regulated by zinc (induced by zinc deprivation) were induced by iron. Cells lacking the yeast frataxin homologue Yfh1 accumulate large amounts of iron in their mitochondria. We have shown that the zinc metabolism of these cells is also impaired: zinc uptake and zinc accumulation were both much lower in Δyfh1 cells than in wild-type cells. Excess zinc in the growth medium also influenced the phenotypes of Δyfh1 cells. It prevented the accumulation of iron in the mitochondria of Δyfh1 cells and increased the growth rate of these cells and their resistance to oxidative stress. However, zinc did not restore the deficiency of Fe–S and haem proteins of Δyfh1 cells. Zinc inhibited mitochondrial respiration and protected Yah1p, the mitochondrial ferredoxin. These results suggest that zinc nutrition may be important in the aetiology of Friedreich's ataxia.

The YFH1 gene is the yeast homologue of the human FRDA gene, which encodes the frataxin protein. Saccharomyces cerevisiae cells lacking the YFH1 gene showed very low cytochrome content. In Δyfh1 strains, the level of ferrochelatase (Hem15p) was very low, as a result of transcriptional repression of HEM15. However, the low amount of Hem15p was not the cause of haeme deficiency in Δyfh1 cells. Ferrochelatase, a mitochondrial protein, able to mediate insertion of iron or zinc into the porphyrin precursor, made primarily the zinc protoporphyrin product. Zinc protoporphyrin instead of haeme accumulated during growth of Δyfh1 mutant cells and, furthermore, preferential formation of zinc protoporphyrin was observed in real time. The method for these studies involved direct presentation of porphyrin to mitochondria and to ferrochelatase of permeabilized cells with intact architecture, thereby specifically testing the iron delivery portion of the haeme biosynthetic pathway. The studies showed that Δyfh1 mutant cells are defective in iron use by ferrochelatase. Mössbauer spectroscopic analysis showed that iron was present as amorphous nano-particles of ferric phosphate in Δyfh1 mitochondria, which could explain the unavailability of iron for haeme synthesis. A high frequency of suppressor mutations was observed, and the phenotype of such mutants was characterized by restoration of haeme synthesis in the absence of Yfh1p. Suppressor strains showed a normal cytochrome content, normal respiration, but remained defective in Fe–S proteins and still accumulated iron into mitochondria although to a lesser extent. Yfh1p and Hem15p were shown to interact in vitro by Biacore studies. Our results suggest that Yfh1 mediates iron use by ferrochelatase.

Candida albicans, unlike Saccharomyces cerevisiae, was able to use extracellular haemin as an iron source. Haemin uptake kinetics by C. albicans cells showed two phases: a rapid phase of haemin binding (with a K d of about 0·2 μM) followed by a slower uptake phase. Both phases were strongly induced in iron-deficient cells compared to iron-rich cells. Haemin uptake did not depend on the previously characterized reductive iron uptake system and siderophore uptake system. CaHMX1, encoding a putative haem oxygenase, was shown to be required for iron assimilation from haemin. A double ΔCahmx1 mutant was constructed. This mutant could not grow with haemin as the sole iron source, although haemin uptake was not affected. The three different iron uptake systems (reductive, siderophore and haemin) were regulated independently and in a complex manner. CaHMX1 expression was induced by iron deprivation, by haemin and by a shift of temperature from 30 to 37 °C. CaHMX1 expression was strongly deregulated in a Δefg1 mutant but not in a Δtup1 mutant. C. albicans colonies forming on agar plates with haemin as the sole iron source showed a very unusual morphology. Colonies were made up of tubular structures that were organized into a complex network. The effect of haemin on filamentation was increased in the double ΔCahmx1 mutant. This study provides the first experimental evidence that haem oxygenase is required for iron assimilation from haem by a pathogenic fungus.

The non-reductive uptake of several siderophores (ferrioxamine B, ferrichrome, triacetylfusarinine C and ferricrocin) by various strains of Saccharomyces cerevisiae was studied. Several aspects of siderophore transport were examined, including specificity of transport, regulation of transport and intracellular localization of the ferri-siderophores. Ferrioxamine B was taken up preferentially via the products of the SIT1 gene and triacetylfusarinine C by the TAF1 gene product, but the specificity was not absolute. Ferrichrome and ferricrocin uptake was not dependent on a single major facilitator superfamily (MFS) gene product. The apparent specificity of transport was strongly dependent on the genetic background of the cells. Non-reductive uptake of siderophores was induced under more stringent conditions (of iron deprivation) than was the reductive uptake of ferric citrate. Regulation of transport depended on the transcriptional factors Aft1 and Tup1/Ssn6. Cells disrupted for the TUP1 or SSN6 genes were constitutively derepressed for the uptake of ferrichrome, ferricrocin or ferrioxamine B, but not for the uptake of triacetylfusarinine C. Cells bearing the AFT1 up mutation accumulated large amounts of ferric siderophores. Intracellular decomplexation of the siderophores occurred when transcription of the AFT1 up gene was repressed. Ferrioxamine B and ferrichrome seemed to accumulate in an endosomal compartment, as shown by biochemical studies and by confocal microscopy study of cells loaded with a fluorescent derivative of ferrichrome. Endocytosis was, however, not involved in the non-reductive uptake of siderophores.

Summary: Uptake of iron from various siderophores by a Δfet3Δfet4 strain of Saccharomyces cerevisiae was investigated. The catecholate enterobactin and the hydroxamate coprogen were taken up by the cells by passive diffusion, whereas the hydroxamates ferrioxamine B (FOB) and ferricrocin (FC) were taken up via a high-affinity energy-dependent mechanism. The kinetics of FOB and FC uptake showed reciprocal competitive inhibition. The transport was regulated by iron availability, but was independent of the Aft1p and Mac1p transcriptional activators. Mutants affected in the transport of FOB were isolated. The transport of FC was not impaired in these mutants. Functional complementation of one mutant allowed the identification of the SIT1 gene (Siderophore iron Transport) encoding a putative permease belonging to the major facilitator superfamily. The Sit1 protein is probably a permease specific for the transport of ferrioxamine-type siderophores. The evidence suggests that the uptake of ferrichrome-type siderophores like FC involves other specific permease(s), although there seems to be a common handling of FOB and FC following their internalization by the cell.