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 mM) 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 DCahmx1 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 Defg1 mutant but not in a Dtup1 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 DCahmx1 mutant. This study provides the first experimental evidence that haem oxygenase is required for iron assimilation from haem by a pathogenic fungus. INTRODUCTIONIron is required by most living systems. Iron availability plays a critical role in the host-pathogen relationship and in the virulence of bacteria and fungi [reviewed by Howard (1999) and Payne (1993)]. In the host, iron is predominantly intracellular, occurring as haem, iron-sulphur proteins or ferritin, with smaller quantities in other iron proteins. The limited amounts of extracellular iron are tightly held by proteins such as transferrin in the serum and lactoferrin on body surfaces. The withholding of iron by the host has been shown to block virulent infections by many pathogens, and high-affinity iron acquisition by pathogens often promotes virulent infections (Ratledge & Dover, 2000). Candida albicans is a saprophytic organism for humans; it can be found inhabiting external surfaces of the body, including the skin and the mucosae of the gut and mouth in more than half of the normal population. In some settings C. albicans invades tissue and disseminates systemically. Prolonged neutropenia, following cancer chemotherapy or bone marrow transplantation, is a major risk factor for invasive fungal infections, and Candida species are the most frequently implicated organisms (Rex et al., 1995). Recurrent oropharyngeal and oesophageal candidiasis is encountered in most AIDS patients (Laine & Bonacini, 1994). There has been a rapid rise in the frequency of drug resistance in clinical C. albicans infections (Rex et al., 1995); hence, there is a pressing need to identify new drug targets.The first studies about iron uptake by C. albicans emphasized the possi...

†These authors contributed equally to this study.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.

SummaryWe cloned the CaYFH1 gene that encodes the yeast frataxin homologue in Candida albicans .

The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase (CPO), an oxygen-requiring enzyme catalysing the sixth step of heme biosynthesis. Its transcription is increased 40-50-fold in response to oxygen- or heme-deficiency. We have analyzed CPO activity and HEM13 mRNA levels in a set of isogenic strains carrying single or double deletions of the CYP1 (HAP1), ROX1, SSN6, or TUP1 genes. The cells were grown in the presence or absence of oxygen and under heme-deficiency (hem1 delta background). Both Rox1p and Cyp1p partially repressed HEM13 in aerobic heme-sufficient cells, probably in an independent manner. In the absence of heme, Cyp1p activated HEM13 and strongly repressed ROX1, allowing de-repression of HEM13. Cyp1p had no effect on HEM13 expression in anaerobic cells. Deletions of SSN6 or TUP1 dramatically de-repressed HEM13 in aerobic cells. A series of deletions in the HEM13 promoter identified at least four regulatory regions that are required for HEM13 regulation. Two regions, containing motifs similar to the Rox1p consensus sequences, act as repression sites under aerobic growth. The two other sites act as activation sequences required for full induction under oxygen- or heme-deficiency. Taken together, these results suggest that induction of HEM13 occurs in part through relief of repression exerted by Rox1p and Cyp1p, and in part by activation mediated partly by Cyp1p under heme-deficiency and by unknown factors under oxygen-deficiency.

TRH induces two separate events in pituitary PRL cells. It increases the release of stored PRL and enhances the rate of PRL gene transcription, which results in an increased steady state concentration of PRL messenger RNA (mRNA) and a concomitant augmentation of PRL production. The mechanisms underlying the release process involve the activation of phosphatidylinositol turnover which generates inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. In order to determine whether these intracellular messengers also mediate the stimulation of PRL gene expression by TRH, we have correlated the level of receptor occupancy with the rate of gene transcription and investigated the action of drugs which increase cytosolic calcium or activate protein kinase C. We have determined that sustained stimulation of transcription requires the persistent occupancy of a limited number of TRH receptor sites and that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophores (A23187, ionomycin), and the calcium channel agonist BAY K 8644 enhance PRL gene transcription. However, TPA is less potent and ionomycin requires a low concentration of TPA to fully mimic TRH action, whereas BAY K 8644 alone displays the same potency as TRH. The effects of BAY K 8644 and TRH are not additive and thus suggest that the influx of calcium plays a predominant role in the regulation of PRL gene transcription by TRH.

The Saccharomyces cerevisiae HEM13 gene codes for coproporphyrinogen oxidase, an oxygen-requiring enzyme catalyzing the sixth step of heme biosynthesis. Its transcription has been shown to be induced 40 -50-fold in response to oxygen or heme deficiency, in part through relief of repression exerted by Rox1p and in part by activation mediated by an upstream activation sequence (UAS). This report describes an analysis of HEM13 UAS and of the Rox1p-responsive sites by electrophoretic mobility shift assays, DNase I footprinting, and mutational mapping. HEM13 UAS is composed of two subelements: a 16-base pair sequence binding a constitutive factor acting as a transcriptional activator, and a 5-flanking 20-base pair GC-rich region. Both subelements were required additively for transcription, but each element alone was sufficient for almost normal control by oxygen/heme deficiency. Mutations in both elements decreased the induction ratio 3-4-fold. HEM13 UAS conferred a 2-4-fold oxygen/heme control on a heterologous reporter gene. Two Rox1p-responsive sites, R1 and R3, were identified, which accounted for the 6 -7-fold repression by Rox1p. A factor bound to a sequence close to site R3. This DNA-binding activity was only detected in protein extracts of aerobic heme-sufficient ROX1 TUP1 cells, suggesting a possible role in site R3 function.

The yeast Saccharomyces cerevisiae contains a flavohemoglobin, encoded by the gene YHB1, whose function is unclear. Previous reports presented evidence that its maximal expression requires disruption of mitochondrial respiration and that it plays a role in the response to oxidative stress. We have studied the expression of YHB1 in respiratory deficient cells and in cells exposed to various compounds causing oxidative stress. Several different strains and approaches (spectroscopic detection of the oxygenated form of Yhb1p, ␤-galactosidase activity of a YHB1-lacZ fusion, and Northern blot analysis) were used to demonstrate that YHB1 expression and Yhb1p production are not increased by respiration deficiency. YHB1 expression was unchanged in cells challenged with antimycin A or menadione, while it decreased in cells exposed to H 2 O 2 , diamide, dithiothreitol, and Cu 2؉ . Transcription of YHB1 is not under the control of the transcriptional factor Yap1p. These results do not support a participation of YHB1 in the genetic response to oxidative stress. We also analyzed the growth phenotypes associated with altered Yhb1p production using YHB1-deleted strains and strains that greatly overproduced Yhb1p. Yhb1p appears to protect cells against the damage caused by Cu 2؉ and dithiothreitol, while sensitizing them to H 2 O 2 . Yhb1p overproduction in a glucose-6-phosphate dehydrogenase-deficient mutant decreased its growth rate. These data indicate that there is a complex relationship(s) between Yhb1p function(s) and cell defense reactions against various stresses.

Plasma membrane proteins involved in transport processes play a crucial role in cell physiology. On account of these properties, these molecules are ideal targets for development of new therapeutic and agronomic agents. However, these proteins are of low abundance, which limits their study. Although yeast seems ideal for expressing heterologous transporters, plasma membrane proteins are often retained in intracellular compartments. We tried to find yeast mutants potentially able to improve functional expression of a whole set of heterologous transporters. We focused on Arabidopsis thaliana ureide transporter 1 (AtUPS1), previously cloned by functional complementation in yeast. Tagged versions of AtUPS1 remain mostly trapped in the endoplasmic reticulum and were able to reach slowly the plasma membrane. In contrast, untagged AtUPS1 is rapidly delivered to plasma membrane, where it remains in stable form. Tagged and untagged versions of AtUPS1 were expressed in cells deficient in the ubiquitin ligase Rsp5p, involved in various stages of the intracellular trafficking of membrane‐bound proteins. rsp5 mutants displayed improved steady state amounts of untagged and tagged versions of AtUPS1. rsp5 cells are thus powerful tools to solve the many problems inherent to heterologous expression of membrane proteins in yeast, including ER retention.