3 Yeast Metabolism

Metabolism refers to the biochemical assimilation (in anabolic pathways) and dissimilation (in catabolic
pathways) of nutrients by a cell. Like in other organisms, in yeast these processes are mediated by
enzymic reactions, and regulation of the underlying pathways have been studied in great detail in
yeast. Anabolic pathways include reductive processes leading to the production of new cellular
material, while catabolic pathways are oxidative processes which remove electrons from substrates
or intermediates that are used to generate energy. Preferably, these processes use NADP or NAD,
respectively, as co-factors.

Although all yeasts are microorganisms that derive their chemical energy, in the from of ATP, from the
breakdown of organic compounds, there is metabolic diversity in how these organisms generate and
consume energy from these substrates. Knowledge of the underlying regulatory mechanisms is not
only valuable in the understanding of general principles of regulation but also of great importance in
biotechnology, if new metabolic capabilities of particular yeasts have to be exploited.

It is now well established that most yeasts employ sugars as their main carbon and hence energy
source, but there are particular yeasts which can utilize non-conventional carbon sources. With regard
to nitrogen metabolism, most yeasts are capable of assimilating simple nitrogenous sources to
biosynthesize amino acids and proteins (Table 3-1). Aspects of phosphorus and sulphur metabolism
as well as aspects of metabolism of other inorganic compounds have been studied in some detail,
predominantly in the yeast, Saccharomyces cerevisiae.

Table 3-1: Nutrients for growth of yeast (S. cerevisiae) cells.

Substrate Intermediates Enzymes Products

Saccharose Invertase Glucose + Fructose
Maltose Maltase Glucose
Melibiose Melibiase Glucose + Galactose
Glucose Products of Glycolysis
Ethanol Acetaldehayde > Alcohol-Dehydrogenase Glucose by gluconeogenesis
Acetyl-CoA>
Oxaloacetate>
Lactate Pyruvate> Lactate-Dehydrogenase Glucose by gluconeogenesis
Glycerol Glycerol-3- Glucose by gluconeogenesis
phosphate>
Dihydroxyaceton-
phosphate
Amino acids
Glutamate
Ammonium

3.1 Sugar Catabolism in Yeast

3.1.1 Principal Pathways

The major source for energy production in the yeast, Saccharomyces cerevisiae, is glucose and
glycolysis is the general pathway for conversion of glucose to pyruvate, whereby production of energy
in form of ATP is coupled to the generation of intermediates and reducing power in form of NADH for
biosynthetic pathways.

Two principal modes of the use of pyruvate in further energy production can be distinguished:
respiration and fermentation (Figure 3-1). In the presence of oxygen and absence of repression,
pyruvate enters the mitochondrial matrix where it is oxidatively decarboxylated to acetyl CoA by the
pyruvate dehydrogenase multi enzyme complex. This reaction links glycolysis to the citric acid cycle,
in which the acetyl CoA is completely oxidized to give two molecules of CO2 and reductive equivalents
in form of NADH and FADH2. However, the citric acid cycle is an amphibolic pathway, since it
combines both catabolic and anabolic functions. The latter results, for example, from the production of
intermediates for the synthesis of amino acids and nucleotides. Replenishment of compounds
necessary to drive the citric acid cycle, such as oxaloacetate and -ketoglutarate, are (i) the fixation of
CO2 to pyruvate by the actions of the enzymes pyruvate carboxylase (ATP-dependent) and
phosphoenolpyruvate carboxykinase and (ii) the glyoxalate cycle (a shortcut across the citric acid
cycle), which is important when yeasts are grown on two-carbon sources, such as acetate or ethanol.

Figure 3-1: Metabolism in yeast under aerobic and anaerobic conditions.

During alcoholic fermentation of sugars, yeasts re-oxidize NADH to NAD in a two-step reaction from
pyruvate, which is first decarboxylated by pyruvate decarboxylase followed by the reduction of
acetaldehyde, catalyzed by alcohol dehydrogenase (ADH). Concomitantly, glycerol is generated from
dihydroyacetone phosphate to ensure production of this important compound.

An alternative mode of glucose oxidation is the hexose phosphate pathway also known as the
pentose phosphate cycle, which provides the cell with pentose sugars and cytosolic NADPH,
necessary for biosynthetic reactions, such as the production of fatty acids, amino acids and sugar
alcohols. The first step in this pathway is the dehydrogenation of glucose-6-phosphate to 6-
phosphogluconolactone and generation of one mole of NADPH (by glucose-6-phosphate
dehydrogenase). Subsequently, 6-phosphogluconate is decarboxlated by the action of
phosphogluconate dehydrogenase to give ribulose-5-phosphate and a second mole of NADPH. Thus,
besides generating NADPH, the other major function of this pathway is the production of ribose sugars
which serve in the biosynthesis of nucleic acid precursors and nucleotide coenzymes.

The redox carriers, NAD and FAD, which become reduced during the breakdown of sugars to NADH
and FADH2, respectively, are reoxidized in the respiratory (electron transport) chain located in the
inner mitochondrial membrane. The energy released during the transfer of electrons is coupled to the
process of oxidative phosphorylation, which is effected by ATP synthase, an enzyme complex which is
also located in the inner mitochondrial membrane and designed to synthesize ATP from ADP and
inorganic phosphate. These pathways will be considered separately in chapter 9: Transport.

3.1.2 Regulation of Biochemical Pathways

Biochemical pathways in yeasts are regulated at various levels:

(i) Enzyme synthesis - induction, repression and derepression of gene expression;
(ii) Enzyme activity - allosteric activation, inhibition, or interconversion of isoenzymes;
(iii) Cellular compartmentalization - localization of particular pathways to the cytosol, mitochondria,
peroxisomes, or the vacuole;
(iv) Transport mechanisms - internalization, secretion, trafficking of compounds between the various
cellular compartments.

Like in the studies of many biochemical aspects, yeast as a versatile system has contributed
significantly to decipher a number of important regulatory circuits, which in many instances have been
conserved among all eukaryotes investigated thus far. Examples will be presented in chapters
Transport and Regulation.

3.1.3 Respiration versus Fermentation

Yeasts can be catagorized in several groups according to their modes of energy production, utilizing
respiration or fermentation (Table 3-2). It is important to note that these processes are mainly
regulated by environmental factors, the best documented being the availability of glucose and oxygen.
Thus yeasts can adapt to varying growth environments, and even within a single species, the
prevailing pathways will depend on the actual growth conditions. For example, glucose can be utilized
in several different ways by S. cerevisiae, depending on the presence of oxygen and other carbon
sources.

Table 3-2: Principal modes of respiration in yeasts.

Types Examples Respiration Fermentation Anaerobic

growth
Obligate respirers Rhodotorula spp. YES NO NO
Cryptococcus spp.
Anaerobic respirers Candida spp. YES Anaerobic in NO
Kluyveromyces spp. pregrown cells
Pichia spp.
Aerobic fermenters S. pombe Limited Aerobic and NO
anaerobic
Facultative aerobic S. cerevisiae Limited Aerobic and Facultative
fermenters anaerobic
Obligate fermenters Torulopsis NO Anaerobic YES

Figure 3-2: Sugar metabolism in different yeasts.

Catabolite repression occurs when glucose or an initial product of glucose metabolism represses the
synthesis of various respiratory and gluconeogenic enzymes. Catabolite inactivation results in the
rapid disappearance of such enzymes on addition of glucose. In catabolite repression, enzyme activity
is lost by dilution with cell growth. Although enzymes are still present, they are no longer synthesized
due to gene repression by signals derived from glucose or other sugars. However, the nature of the
signal(s) is not clear at present.

Glucose repression in yeast describes a long-term regulatory adaptation to degrade glucose

exclusively to ethanol and CO2. Therefore, when S. cerevisiae is grown aerobically on high
concentrations of glucose, fermentation will account for the bulk of glucose consumption. In batch
culutures, when the levels of glucose decline, cells become gradually derepressed, resulting in the
induction of respiratory enzyme synthesis. This in turn results in oxidative consumption of ethanol,
when cells enter a second phase of growth known as the diauxic shift.

Catabolite inactivation is more rapid than repression and is thought to be due to deactivation by
glucose of a limited number of key enzymes, such as fructose 1,6-bisphosphatase. Inacivation occurs
primarily by enzyme phosphorylation, followed by slower vacular degradation of the enzyme. It has
been established that cAMP as a second messenger plays a central role in regulating catabolite
repression and inactivation in S. cerevisiae.

3.1.4 Other Sugars - Galactose

Galactose is a 'non-conventional' nutrient for yeast, which however can be used as a sole carbon
source when glucose is absent from the medium. In yeast cells supplied with glucose, the GAL genes
are repressed. They are activated a thousand fold in cells that are starved for glucose, and this one of
the few pathways in yeast which is regulated in a nearly 'all-or-nothing' mode. The three enzymes
involved are depicted in Figure 3-2.

Figure 3-2: Metabolism of galactose.

3.1.5 Metabolism of Non-Hexose Carbon Sources

In addition to hexose sugars, yeasts can utilize a number of 'non-conventional' carbon sources,
such as biopolymers, pentoses, alcohols, polyols, hydrocarbons, fatty acids and organic acids. This is
of particular interest for biotechnological processes, the most prominent being the use of S. cerevisiae
in fermentation. One should also remember that free glucose is scarce in natural environments or in
natural products used to feed yeast cells.

For example, disaccharides, such as maltose, sucrose, melibiose, lactose or cellobiose can easily be
accepted as nutrients by the action of corresponding hydrolases which break these disaccharides
down into their constituent monosaccharides (Table 3-3). Notably, hydrolysis is coupled to transport of
either the disaccharide or the resulting monosaccharides.

Table 3-3: Disaccharides as substrates in yeasts.

Disaccharide Extracellular Intracellular Products Organism

hydrolysis hydrolysis
Maltose Maltase 2 Glucose S. cerevisiae
Sucrose Invertase Glucose + Fructose S. cerevisiae
Melibiose galactopyranosidase Glucose + S. carlsbergensis
Galactose
Lactose -galactosidase Glucose + Kluyveromyces
Galactose
Cellobiose -glucosidase 2 Glucose Brettanomyces

Other saccharide biopolymers, like starch, inulin, cellulose, hemicellulose, or pectin, can be
metabolized by some specialized yeasts directly, while for the use of carbon sources to other species
they have to be hydrolyzed by non-yeast enzymes before utilization.

Pentose sugars can be fermented to ethanol by only very few yeast species, although many yeasts
can grow aerobically on pentoses. The inability of S. cerevisiae to ferment xylose (e.g. derived from
hemicellulose) could be circumvented by introducing genes for xylose reductase and xylitol
dehydrogenase from xylose-fermenting species (Pichia) by recombinant DNA technology. However,
the efficiency of xylose fermentation remains low.

Many yeasts have the capability of metabolizing ethanol (Table 3-4) or methanol, an approach used
in biomass production of yeasts of biotechnological interest. Methanol-utilizing (methylotropic) yeasts
are found, for example, in Hansenula polymorpha, Pichia pastoris, several Candida species, and
Torulopsis sonorensis. In these organisms, methanol is first metabolized by an O2-dependent oxidase
to formaldehyde, which is then converted into dihydroxy acetone (DAH) by a DAH synthase. DHA and
GAP can be utilized to synthesize fructose-6-phosphate.

Glycerol functions as a compatible solute in osmoregulation in osmotolerant yeasts that are capable
of growing in high sugar or salt environments. Many yeasts can grow on glycerol as a sole carbon
source under aerobic conditions, but glycerol is a non-fermentable carbon source for many yeast
species, including S. cerevisiae. To serve as a carbon source, glycerol after internalization has to
converted by glycerol kinase to glycerol-3-phosphate, which is then transformed into DAH phosphate
by glycerol-3-phosphate dehdrogenase that is a substrate in gluconeogenesis.
Table 3-4: Use of unusual nutrients in yeasts.

Carbon source Metabolites Examples

Starch Glucose Candida spp.; Pichia spp.
Cellulose Glucose
Hemicellulose Glucose, Xylose Candida spp. ; Pichia spp.
Pectin Galacturonic acid Candida spp. ; Kluyveromyces
Inulin Fructose Candida spp. ; Kluyveromyces
Xylose Pyruvate > Ethanol Candida; Pichia; Kluyveromyces
Organic acids Acetyl-CoA Many yeasts
Protein Amino acids Candida spp. ; Kluyveromyces spp.; S.
cerevisiae
Lipids Fatty acids + glycerol Candida spp.; Pichia spp.; Yarrowia lipolytica
Alkanes Fatty acids Candida; Pichia; Yarrowia lipolytica
Methanol GAP + DAP Hansenula; Pichia pastoris; Candida

Nitrogen source Metabolites Examples

Urea Ammonium (urea ammonium Many yeasts
hydrolase)
Nitrate > Nitrite > Ammonium Candida spp.; Hansenula spp.

3.2 Gluconeogenesis and Carbohydrate Biosynthesis

The growth of yeast on non-carbohydrate substrates as sole carbon sources necessitates the
synthesis of sugars required for macromolecular biosynthesis, especially that of complex
polysaccharides. Like in other organisms, gluconeogenesis, the conversion of pyruvate to glucose is
dependent on ATP as an energy donor and NADH as a reducing power.

Structural polysaccharide synthesis in yeast is associated with the cell and the spore wall and
include mannans, glucans and chitin. Like in other organisms, all sugar polymerization reactions
employ sugar nucleotides as substrates, which are formed via activation by UTP or GTP, depending
on the substrate.

A major activity in yeast is the synthesis of storage carbohydrates: glycogen and trehalose. Like in
other organisms, glycogen is formed by sequential addition of glucose units from UDP-glucose,
employing glycogen synthase for the linear -1,4-linkage of the backbone chain, and branching
enzyme in the formation of -1,6 branches. Degradation of glycogen to glucoese-1-phosphate is
effected by glycogen phosphorylase. cAMP is known to be involved in the regulation of glycogen
metabolism.

An unconventional storage disaccharide found in yeast is trehalose (,-1,1-diglucose), present in

particularly high concentrations in resting and in stressed cells. Trehalsoe-phosphate is synthesized in
yeast from glucose-6-phosphate and UDP-glucose by trehalose-6-phosphate synthase and converted
to terhalose by a phosphatase. The breakdown of trehalose to glucose is mediated by trehalase. Both
synthesis and degradation are regulated via cAMP.

3.3 Fatty Acid and Lipid Metabolism

Fatty acids available to yeasts for catabolism include those derived from microsomal alkane oxidation
or extracellular lipolysis of fats or those exogenously supplied in the growth medium. The fatty acids
are catabolized by -oxidation in peroxisomes, which differs from the system in the mitochondria in the
involvement of catalase in re-oxidizing FADH2 and in the mechanism of re-oxidizing NADH (Figure 3-
3).

Figure 3-3: Fatty acid utilization.

The series of reactions leading to the synthesis of long-chain fatty acids, starting from acetyl-CoA is
achieved by a multi-enzyme complex, the fatty acid synthase. The subsequent formation of
unsaturated fatty acids, which are needed for membrane integrity, involves an oxidative desaturation
by a fatty acid desaturase.

Synthesis of lipids is similar to the reactions known in other organisms, starting from glycerol-
phosphate and fatty acids. Breakdown of lipids effected by a yeast lipase that generates long-chain
fatty acids and glycerol, which latter is catabolized in the glycolytic pathway.

The metabolic pathways of glycerophospholipids in yeast are depicted in Figure 3-4.

Figure 3-4: Metabolic pathways of glycerophospholipids in yeast [Kohlwein et al., 1996].

Pink box: synthesis and activation of fatty acids; blue box: de nova pathway of phospholipid synthesis, synthesis of bulk
membrane lipids; yellow box: phospholipid degradation and recycling of amino-alcohol head groups (salvage pathway); green
box: phospholipid remodelling, deacylation and reacylation of phospholipids (fatty acid specificity in sn-1 and sn-2 positions); red
box: phosphatidylinositol (Ptdlns) phosphorylation, signalling and membrane vesicle fusion.
Precursors and lipids: CDP-DAC, cytidine diphosphate-diacylglycerol; Cho, choline; Cho-CDP, cytidine diphosphate-choline;
Cho-P, choline phosphate; CL, cardiolipin; DAG, diacylglycerol; DAC-PP, diacylglycerol pyrophosphate; Etn, ethanolamine; Etn-
CDP, cytidine diphosphate-ethanolamine; Etn-P, ethanolamine phosphate; FFA, free fatty acid; Clc-6-P, glucose 6-phosphate;
00-3-P, glycerol 3-phosphate; Ins, inositol; Ins-l-P, inositol 1 -phosphate; PtdCho, phosphatidylcholine; PtdDMEtn,
phosphatidyldimethylethanolamine; PtdEtn, phosphatidylethanolamine; PtdGro, phosphatidylglycerol; PtdGro-P,
phosphatidylglycerol-phosphate; Ptdlns, phosphatidylinositol; PtdMMEtn, phosphatidylmonomethylethanolamine; PtdOH,
phosphatidic acid; PtdSer, phosphatidylserine; TAG, triacylglycerol.
Enzymes and genes (italic): ACCI, acetyl-CoA carboxylase; CCTI, CWcholinephosphate cytidylyltransferase; CDSI, CDP-
diacylglycerol synthase; CHO7, phosphatidylserine synthase; Cff02, phosphatidylethanolamine N-methyltransferase; CKII,
choline kinase; CPTl, cholinephosphotransferase; CTR7, choline transporter; ECT?, ClFethanolaminephosphate
cytidylyltransferase; EPT7, ethanolaminephosphotransferase; FAA7-4, acyl-CoA synthetases l-4; FAS1,2, fatty acid synthase
subunits p and cl; CAT, glycerol-3-phosphate acyltransferase; IN07, inositol-1 -phosphate synthase; NMJ?, myristoyl-CoA
protein N-myristoyltransferase; O/I?, acyl-CoA desaturase; OP13, phospholipid-N-methyltransferase; PAP, phosphatidate
phosphatase; /X7, phosphatidylinositol synthase; PLB7, phospholipase B; PLC7, Ptdlns-specific phospholipase C; PSD7,2,
phosphatidylserine decarboxylase; PLD7/SPO74, phospholipase D.

3.4 Nitrogen Metabolism

3.4.1 Catabolic Pathways

Yeasts are capable of utilizing a range of different inorganic and organic sources of nitrogen for
incorporation into the structural and functional nitrogenous components of the cell, such as amino
acids (and consequently peptides and proteins), polyamines, nucleic acids and vitamins. Growth
media are often supplemented with complex mixtures of amino acids. However, yeasts can also live
on ammonium ions as a sole nitrogen source, because they posses a whole repertoire of genes
encoding enzymes to the biosynthesis of all amino acids.
Ammonium ions, either supplied as nutrient or derived from the catabolism of other nitrogenous
compounds, can be directly assimilated into a couple of amino acids, notably glutamate and
glutamine, which can then serve as donors of the amino group in other amino acids. The major route
for assimilation of ammonium is the reaction of the NADPH-dependent glutamate dehydrogenase
(GDH) which forms glutamate from -ketoglutarate and ammonium. Whenever ammonium ion
concentration is low, but also as a prerequisite for the synthesis of many nitrogenous compounds
glutamine synthase is activated, which forms glutamine from ketoglutarate and ammonium in an
ATP-dependent reaction. Glutamine is absolutely required as a prominent precursor in several
important pathways, such as the synthesis of asparagine, tryptophane, histidine, arginine, carbamoyl
phosphate, CTP, AMP, GMP, glucosamine, and NAD.

Whereas S. cerevisiae is incapable of utilizing nitrate as a nitrogen source, there are a couple of other
yeast species that have this capability. Nitrate assimilation occurs by the action of NADPH-dependent
nitrate reductase, forming nitrite. Subsequently, nitrite is reduced to ammonium by NADPH-dependent
nitrite reductase.

Urea is widely used by yeasts as a nitrogen source. In urease-negative S. cerevisiae, urea

aminohydrolase (ATP-dependent urea carboxylase plus allophanate hydrolase), hydrolyses urea to
ammonium and carbonate.

Figure 3-5: Scheme of amino acid biosynthesis in yeast.

3.4.2 Amino Acid Biosynthesis Pathways

All of these pathways and their regulation in yeast have been studied in great detail. For example, the
metabolism of methionine and S-adenosyl methionine is mediated by nearly 20 different enzymes.
Because of their complexity, we will not summarize the pathways in this brief overview. Figure 3-5 just
summarizes the major reactions.

3.4.3 Protein Biosynthesis

Protein biosynthesis has been studied in yeast as one of the first eukaryotic model organisms. Many
basal findings on the structure and function of tRNAs, tRNA synthetases, 80S ribosomes, and the
initiation, elongation and termination factors mediating translation have been identified in yeast and
studied in great detail (see chapter Expression).

3.5 Phosphate Metabolism

Figure 3-6: Phosphate acquisition and storage system in yeast.

Phosphorus requirements of yeast cells are met by the uptake of inorganic phosphate from the
nutrient media (Figure 3-6). The phosphate taken up can be utilized for incorporation of major cell
constituents, such as phospholipids, nucleic acids and proteins, and is needed for the many
transphosphorylation reactions in intermediary metabolism. The intracellular concentration of free
phosphate is generally maintained at very low levels. Only when yeast cells switch from respiratory to
fermentative metabolism following a glucose pulse, dynamic fluctuations in cellular phosphate have
been observed.

The bulk of phosphate in yeast is in organic linkage and in the form of polyphosphates. These latter
are linear polymeres of orthophosphate in anhydrous linkage. As high concentrations of
polyphosphates are accumulated and their hydrolysis yields the same amount of free energy as the
hydrolysis of ATP to ADP and Pi, they are important for both phosphorus and energy supply in the cell.

In addition to membrane-associated ATPases, yeast cells contain many important enzymes involved
in phosphorylation and dephosphorylation: kinases and phosphatases are crucial in governing a
multitude of cellular processes, like in other eukaryotes. A peculiarity of yeast is the presence of
alkaline (PHO 8) and acid phosphatases (PHO3, PHO5, PHO10, PHO11) in the periplasm which act
non-specifically on several phosphate esters of sugars, alcohols and nucleosides, to supplement
phosphorus supply. One of the acidic phosphatases genes, PHO3, is constituvely expressed, while
the PHO5 gene is highly regulated and turned on at low phosphate concentrations (see chapter
Regulation).

3.6 Sulphur Metabolism

The sulphur requirement of yeast can be met by the uptake of sulphates, which can be assimilated
through reduction into sulphur amino acids.

3.7 Transition Metals

All eukaryotes and most prokaryotes require transition metals, such as iron, copper, zinc, and
manganese. These metals have to be acquired by cells via specific transport systems that mediate
uptake across the plasma membrane. Much of this understanding has resulted from genetic and
biochemical studies in yeast, and the regulation has been defined at both the transcriptional and
posttranscriptional level. These aspects will be dealt with in chapters 8 (Transport) and 13 (Signalling
and Regulation).

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