Saccharomyces cerevisiae responds to iron deprivation by increased transcription of the iron regulon, including the high affinity cell-surface transport system encoded by FET3 and FTR1. Here we demonstrate that transcription of these genes does not respond directly to cytosolic iron but rather to the mitochondrial utilization of iron for the synthesis of iron-sulfur (Fe-S) clusters. We took advantage of a mutant form of an iron-dependent enzyme in the sterol pathway (Erg25-2p) to assess cytosolic iron levels. We showed that disruption of mitochondrial Fe-S biosynthesis, which results in excessive mitochondrial iron accumulation, leads to transcription of the iron transport system independent of the cytosolic iron level. There is an inverse correlation between the activity of the mitochondrial Fe-S-containing enzyme aconitase and the induction of FET3. Regulation of transcription by Fe-S biosynthesis represents a mechanism by which cellular iron acquisition is integrated with mitochondrial iron metabolism.High affinity iron uptake in Saccharomyces cerevisiae is mediated by a transport system comprising the multicopper oxidase Fet3p and the transmembrane permease Ftr1p (1). Genes that encode these proteins are part of an iron regulon in which transcription is activated by Aft1p. Aft1p is cytosolic when iron is replete and translocates to the nucleus when iron is limited (2). It has been speculated that Aft1p responds to cytosolic levels of elemental iron, as conditions that lower cytosolic iron result in increased transcription of the iron regulon. Binding of iron to Aft1p, however, has not been demonstrated.Mutations that affect mitochondrial Fe-S biosynthesis or export result in increased cellular and mitochondrial iron as a result of decreased mitochondrial iron efflux (3). It has been thought that the increase in mitochondrial iron occurs at the expense of cytosolic iron, and the decrease in cytosolic iron is responsible for the increase in Aft1p-mediated transcription of the iron regulon (3,4). Herein, we demonstrate that activation of Aft1p does not directly respond to cytosolic iron. Using a novel measure of cytosolic iron, we showed that activation of the iron regulon can occur when cytosolic iron levels are high. We further demonstrated that activation of the iron regulon is controlled by the synthesis of Fe-S clusters, which in yeast is localized within mitochondria. Our studies revealed that cellular iron acquisition is coordinated with the mitochondrial use of iron. MATERIALS AND METHODSYeast Strains-All yeast strains used in this study were derived from a W303 background strain. The following yeast strains have been described previously: DY150 (Mata ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100(oc)), METYFH1 (Mat␣ ade2-1 his3-11 ⌬yfh1::HIS3 leu2-3,112 trp1-1 ura3-52 can1-100(oc), pMET3YFH1 [URA3]) (5), METNFS1 (Mat␣ ade2-1 his3-11 ⌬nfs1::HIS3 leu2-3,112 trp1-1 ura3-52 can1-100(oc), pMET3NFS1[URA3]) (3), and erg25-2 (Mat␣ erg25-2 ade2-1 his3-11 leu2-3,112 trp1-1 ura3-52 can1-100(oc)) (6). DNA trans...

A Saccharomyces cerevisae microarray expression study indicated that an ORF, YER044C, now designated ERG28, was strongly coregulated with ergosterol biosynthesis. Disruption of the ERG28 gene results in slow growth and accumulation of sterol intermediates similar to those observed in erg26 and erg27 null strains, suggesting that the Erg28p may interact with Erg26p and/or Erg27p. In this study, a peptide from human hemagglutinin protein (HA) epitope tag was added to ERG26 and ERG27 genes, and a Myc tag was added to the ERG28 gene to detect interactions between Erg28p and Erg26p/Erg27p. Differential centrifugation showed that Erg26p, Erg27p, and Erg28p are all membrane-associated proteins. Green fluorescent protein-fusion protein localization studies showed that Erg26p, Erg27p, and Erg28p are all located in the endoplasmic reticulum. Solubilized membrane protein coimmunoprecipitation studies using rabbit anti-Erg25p indicated that Erg25p coimmunoprecipitates with both Erg27p and Erg28p. Erg28p was also shown to reciprocally coimmunoprecipitate with Erg27p. However, no coimmunoprecipitation was observed with Erg26p, most likely because of the poor solubilization of this protein. Sucrose gradient ultracentrifugation studies suggested that Erg25p/Erg26p/Erg27p/Erg28p, along with other proteins in sterol biosynthesis, might form a complex between 66 and 200 kDa. Using an anti-HA column with Erg27p-HA and Erg26p-HA as target proteins, a complex containing Erg25p/Erg26p/Erg27p/Erg28p was identified. Thus, we suggest that Erg28p works as a transmembrane scaffold to tether Erg27p and possibly other C-4 demethylation proteins (Erg25p, Erg26p), forming a demethylation complex in the endoplasmic reticulum.

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Yeast Saccharomyces cerevisiae is auxotrophic for ergosterol in the absence of oxygen. We showed that complex changes in esterification of exogenously supplied sterols were also induced by anaerobiosis. Utilization of oleic acid for sterol esterification was significantly impaired in anaerobic cells. Furthermore, anaerobic cells fed different sterols exhibited striking variation in esterification efficiency (high levels of sterol esters for cholesterol and sitosterol, low levels for ergosterol, lanosterol or stigmasterol). Relative activities of two yeast acylCoA:sterol acyltransferases (Are1p and Are2p) changed in response to anaerobiosis: while Are2p was dominant under aerobic conditions, Are1p provided the major activity in the absence of oxygen. Our results indicate that sterol esters may fulfil different roles in aerobic and anaerobic cells. ß

UPC2 and ECM22 belong to a Zn(2)-Cys(6) family of fungal transcription factors and have been implicated in the regulation of sterol synthesis in Saccharomyces cerevisiae and Candida albicans. Previous reports suggest that double deletion of these genes in S. cerevisiae is lethal depending on the genetic background of the strain. In this investigation we demonstrate that lethality of upc2D ecm22D in the S288c genetic background is attributable to a mutation in the HAP1 transcription factor. In addition we demonstrate that strains containing upc2D ecm22D are also inviable when carrying deletions of ERG6 and ERG28 but not when carrying deletions of ERG3, ERG4, or ERG5. It has previously been demonstrated that UPC2 and ECM22 regulate S. cerevisiae ERG2 and ERG3 and that the erg2D upc2D ecm22D triple mutant is also synthetically lethal. We used transposon mutagenesis to isolate viable suppressors of hap1D, erg2D, erg6D, and erg28D in the upc2D ecm22D genetic background. Mutations in two genes (YND1 and GDA1) encoding apyrases were found to suppress the synthetic lethality of three of these triple mutants but not erg2D upc2D ecm22D. We show that deletion of YND1, like deletion of GDA1, alters the sphingolipid profiles, suggesting that changes in sphingolipids compensate for lethality produced by changes in sterol composition and abundance.

All fungal genomes harbour numerous ABC (ATP-binding cassette) proteins located in various cellular compartments such as the plasma membrane, vacuoles, peroxisomes and mitochondria. Most of them have initially been discovered through their ability to confer resistance to a multitude of drugs, a phenomenon called PDR (pleiotropic drug resistance) or MDR (multidrug resistance). Studying the mechanisms underlying PDR/MDR in yeast is of importance in two ways: first, ABC proteins can confer drug resistance on pathogenic fungi such as Candida spp., Aspergillus spp. or Cryptococcus neoformans; secondly, the well-established genetic, biochemical and cell biological tractability of Saccharomyces cerevisiae makes it an ideal tool to study basic mechanisms of drug transport by ABC proteins. In the past, knowledge from yeast has complemented work on human ABC transporters involved in anticancer drug resistance or genetic diseases. Interestingly, increasing evidence available from yeast and other organisms suggests that ABC proteins play a physiological role in membrane homoeostasis and lipid distribution, although this is being intensely debated in the literature.

Yeast Saccharomyces cerevisiae is auxotrophic for ergosterol in the absence of oxygen. We showed that complex changes in esterification of exogenously supplied sterols were also induced by anaerobiosis. Utilization of oleic acid for sterol esterification was significantly impaired in anaerobic cells. Furthermore, anaerobic cells fed different sterols exhibited striking variation in esterification efficiency (high levels of sterol esters for cholesterol and sitosterol, low levels for ergosterol, lanosterol or stigmasterol). Relative activities of two yeast acylCoA:sterol acyltransferases (Are1p and Are2p) changed in response to anaerobiosis: while Are2p was dominant under aerobic conditions, Are1p provided the major activity in the absence of oxygen. Our results indicate that sterol esters may fulfil different roles in aerobic and anaerobic cells.