Download Role of the non-respiratory pathways in the utilization of molecular

Yeast
Yeast 2003; 20: 1115–1144.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1026
Review
Role of the non-respiratory pathways in the utilization
of molecular oxygen by Saccharomyces cerevisiae
Eric Rosenfeld1 * and Bertrand Beauvoit2
1 Laboratoire de Génie Protéique et Cellulaire, Bâtiment Marie Curie, Pôle Sciences et Technologies, Université de La Rochelle, Avenue Michel
Crépeau, 17042 La Rochelle Cedex 1, France
2 Institut de Biochimie et Génétique cellulaires du CNRS, Université Victor Segalen, Bordeaux II, 1 Rue Camille Saint-Saëns, 33077 Bordeaux
Cedex, France
*Correspondence to:
Eric Rosenfeld, Laboratoire de
Génie Protéique et Cellulaire,
Bâtiment Marie Curie, Pôle
Sciences et Technologies,
Université de La Rochelle,
Avenue Michel Crépeau, 17042
La Rochelle Cedex 1, France.
E-mail: [email protected]
Received: 24 January 2003
Accepted: 10 June 2003
Abstract
Saccharomyces cerevisiae is a facultative anaerobe devoid of mitochondrial alternative
oxidase. In this yeast, the structure and biogenesis of the respiratory chain, on the one
hand, and the functional interactions of oxidative phosphorylation with the cellular
energetic metabolism, on the other, are well documented. However, to our knowledge,
the molecular aspects and the physiological roles of the non-respiratory pathways
that utilize molecular oxygen have not yet been reviewed. In this paper, we review
the various non-respiratory pathways in a global context of utilization of molecular
oxygen in S. cerevisiae. The roles of these pathways are examined as a function of
environmental conditions, using either physiological, biochemical or molecular data.
Special attention is paid to the characterization of the so-called ‘cyanide-resistant
respiration’ that is induced by respiratory deficiency, catabolic repression and oxygen
limitation during growth. Finally, several aspects of oxygen sensing are discussed.
Copyright  2003 John Wiley & Sons, Ltd.
Keywords: yeast; Saccharomyces cerevisiae; oxygen; aerobiosis; anaerobiosis;
cyanide-resistant respiration; oxygen sensing
Contents
Introduction
Overall oxygen fluxes in S. cerevisiae
Structural oxygen requirements of
S. cerevisiae
Characterization and physiological role
of so-called ‘cyanide-resistant
respiration’
Oxygen sensing in S. cerevisiae: current
knowledge and perspectives
Conclusion
References
Introduction
Oxygen utilization by eukaryotic cells
Since the increase in atmospheric oxygenation over
the past 3.5 billion years, prokaryotes and then
Copyright  2003 John Wiley & Sons, Ltd.
eukaryotes have developed multiple processes to
optimize the utilization of oxygen (O2 ). In eukaryotic cells, most of which are obligate aerobes or
facultative anaerobes, oxygen is metabolized to fulfil both catabolic and anabolic functions. The classical mitochondrial respiratory chain is a part of
a catabolic pathway involved in oxidative phosphorylation. Indeed, the oxidation of substrates,
the transport of electrons by respiratory complexes
(I, II, III) and the terminal oxygen reduction into
water by cytochrome c oxidase (complex IV) result
in the synthesis of ATP through a chemo-osmotic
coupling.136 On the other hand, oxygen can be
consumed by other mitochondrial pathways. First,
electron leakage from the respiratory chain during
oxidative metabolism can partially reduce molecular oxygen into reactive oxygen species (ROS;
e.g. hydrogen peroxide and the superoxide anion)
at the level of flavoproteins and/or the ubiquinol
1116
pool.33,67,137 According to Cadenas,32 this oxygen reduction mechanism represents 1–2% of the
total respiratory oxygen flux in mammals. However, this value may be artificially increased in the
presence of antimycin A, a specific mitochondrial
inhibitor of complex III that blocks electron flow
through the ubiquinone–cytochrome b cycle.137,143
Besides the classical antimycin A- and cyanidesensitive respiratory chain, other types of mitochondrial respiratory pathways have been reported
in numerous eukaryotic cells. In plants, fungi and
protozoans, the alternative respiration pathway is
antimycin A-resistant and is generally caused by
a single cyanide-resistant and salicyl hydroxamate
(SHAM)-sensitive alternative ubiquinol oxidase
(AOX), located on the matrix side of the inner
mitochondrial membrane and encoded by a member of the AOX gene family.179 The constitutive
or inducible AOX bypasses the cytochrome chain
by directly transferring electrons from ubiquinol to
oxygen. The alternative respiration is also called
‘ubiquinol pathway’. It may have several physiological roles: (a) to regenerate reducing equivalents
for the glycolysis and the tricarboxylic acid cycle
when the cytochrome pathway activity is limited;
(b) to prevent the generation of ROS by limiting
the auto-oxidation of ubiquinol; and (c) to produce heat (e.g. in Aracea plants).91,179 The existence of these processes, and the fact that numerous plants and fungi possess mitochondrial alternative (rotenone insensitive) NADH dehydrogenases
not coupled to proton translocation, support the
idea that alternative pathways are invariably nonphosphorylating in plants and fungi.91 However, it
was recently shown in yeasts and fungi that whenever cyanide-resistant AOX is present, complex
I is also present.91,200 – 202 Hence, the organization of alternative components within the electron
transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally
coupled to proton translocation through at least
one site.91 Moreover, cyanide-resistant respiratory
activity is stimulated by purine nucleotides (e.g.
ATP in plants; AMP, ADP, dAMP and GMP in
yeasts and fungi).91,201 For these organisms this
phenomenon, whose mechanism of action is yet to
be determined, may constitute a means to adapt
the overall efficiency of the respiratory chain as a
function of cellular energy state.
Other types of alternative respiratory pathways
have also been reported in other organisms, e.g. the
Copyright  2003 John Wiley & Sons, Ltd.
E. Rosenfeld and B. Beauvoit
obligate aerobe yeast Candida parapsilosis contains both the classical respiratory chain, an alternative ubiquinol oxidase and a parallel respiratory chain containing an alternative cytochrome c
oxidase.34,71,133 On the other hand, the residual
molecular oxygen that is not reduced by the mitochondrial (classical or alternative) respiration is
used by numerous anabolic pathways. Indeed, lipid,
amino acid, vitamin, iron, haem and ubiquinone
metabolisms involve oxygen-dependent steps. The
corresponding reactions depend on flavoproteins,
haemoproteins and other metalloproteins, and consequently are potential sources of ROS. However,
the respective contribution of these pathways in the
overall oxygen consumption or in ROS production
is still poorly documented, owing to the very low
oxygen quantities involved.
Levels of oxygen and classification of yeast
species
Yeasts are routinely cultivated under three distinct levels of oxygen: (a) anaerobic conditions
(anaerobiosis), for which virtually no oxygen
is present, are commonly acquired by saturating the medium with argon or nitrogen gas;
(b) oxygen-limited conditions (oxygen limitation),
for which the growth rate is limited by oxygen
concentration; (c) aerobic conditions (aerobiosis),
for which oxygen is present in excess and the
growth rate is not limited by oxygen concentration. The maximal specific rate of oxygen consumption is then reached (qO2 = qO2 max ) for a
given strain and an experimental condition tested.
On the basis of their metabolic behaviour, yeasts
are classified as: (a) obligate aerobes (e.g. Trichosporon, Rhodotorula spp., Lipomyces, Cryptococcus), with exclusively oxidative metabolism
(respiration); and (b) facultative anaerobes, which
also display the ability to metabolize glucose in
an oxido-reductive manner (fermentation).64,167 In
this case, since both routes may function simultaneously, a mixed (respiro-fermentative) pattern
of energy metabolism is observed.64 The facultative anaerobes group may be subdivided into
two subgroups, depending on their ability to perform aerobic fermentation. Thus, the facultative
anaerobe yeasts can be subdivided into ‘Crabtreenegative’ yeasts (e.g. most Candida and Pichia,
Kluyveromyces), unable to ferment on glucose
under strict aerobic conditions but which produce ethanol under oxygen-limited conditions, and
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
‘Crabtree-positive’ yeasts, which perform alcoholic fermentation on glucose under aerobic conditions (e.g. Saccharomyces, Schizosaccharomyces
spp. and Brettanomyces).64,161,197,198
The diversity among facultatively fermentative
yeasts with respect to the regulation of glucose fermentation by oxygen is illustrated by the
phenomena called ‘Custers effect’ and ‘Pasteur
effect’. These represent regulatory mechanisms
that affect the balance between fermentation and
respiration.64,161 The Custers effect is a characteristic of the yeast species of the genus Brettanomyces,
which ferment glucose faster under aerobic conditions than in the absence of oxygen. The explanation advanced for this effect is that regeneration
of NAD+ by glycerol-phosphate dehydrogenase is
not fast enough under anaerobic conditions.64 The
Pasteur effect (inhibition of glycolysis by oxidative phosphorylation or stimulation of glycolysis by
anaerobiosis) is observed in all Crabtree-negative
(facultatively fermentative) yeasts. It results from
kinetic regulations along the glycolytic pathway
and thermodynamic constraints due to the competition between glycolysis and oxidative phosphorylation for ADP and inorganic phosphate (Pi) and
for reducing equivalents.21,64,161
Oxygen utilization by S. cerevisiae:
characteristics
The facultative anaerobe and Crabtree-positive
yeast S. cerevisiae exhibits a relatively low Pasteur effect that is only evident at low glycolytic
fluxes (e.g. in slowly growing cells).64,161 This
phenomenon may play a crucial role in the balance between fermentation and respiration when
cells are grown under oxygen-limited conditions.
For S. cerevisiae, the critical oxygen concentration
at which limited growth begins has been reported
in the range 2–4 µM (approximately 0.8–1.6%
of air saturation).13,76 In contrast to most of the
obligate aerobes and to Crabtree-negative yeasts
that harbour a typical cyanide-resistant respiration, S. cerevisiae is devoid of both rotenonesensitive NADH : coenzyme Q oxidoreductase (i.e.
complex I) and alternative oxidase.201,202 When
S. cerevisiae is grown under aerobic conditions,
most of the oxygen is used by the classical respiratory chain. Because S. cerevisiae is a fermentative yeast, it does not require an alternative respiratory pathway for growth on sugars when the
Copyright  2003 John Wiley & Sons, Ltd.
1117
classical respiratory chain is blocked by inhibitors
or by genetic alterations (i.e. respiratory-deficient
mutants such as PET, rho − and rho0 ). However, a
so-called ‘cyanide-resistant respiration’ (CNR) has
been identified in this species. As in many other
yeasts, the cyanide-resistant respiration pathway is
favoured when the activity of the classical respiratory chain is limited,3 e.g. CNR was easily elicited
in anaerobically-grown glucose-repressed cells during the early stages of oxygen adaptation. Nevertheless, the corresponding oxygen consumption
fluxes remain quite low in comparison to classical or alternative respiratory oxygen uptake. This is
one reason why cyanide-resistant oxygen consumption capacity has not yet been well characterized in
S. cerevisiae.
Scope of this review
The respiratory chain and its functional interaction with energetic and glucose metabolism, as
well as with oxidative stress, have been extensively reviewed in S. cerevisiae.15,21,64,84,153,161,197
In contrast, to our knowledge, the respective contributions of the numerous non-respiratory pathways
to the overall molecular oxygen consumption have
not yet been reviewed. Moreover, the activity, regulation and physiological roles of non-respiratory
oxygen utilization pathways remain often unclear.
In this paper, we review the non-respiratory pathways that may contribute to the utilization of
molecular oxygen in S. cerevisiae. Their respective
roles are examined under several environmental
conditions using either physiological, biochemical
or molecular data. Special attention is paid to anaerobically grown cells in which ‘cyanide-resistant
respiration’ is known to be induced. Finally, the
oxygen signal transduction pathways that allow
S. cerevisiae cells to shift their metabolism upon
aerobic, oxygen-limited or anaerobic environments
are discussed.
Overall oxygen fluxes in S. cerevisiae
The respiratory chain
In S. cerevisiae, as in most eukaryotic cells, the
mitochondrial respiratory chain (Figure 1) represents the main oxygen utilization pathway in
aerobic cells. Only cytochrome c oxidase can
act as mitochondrial terminal oxidase. Therefore,
Yeast 2003; 20: 1115–1144.
1118
E. Rosenfeld and B. Beauvoit
nH+
Glycerol-3-P
NAD+
NADH
External
NADH
dehydrogenase
Lactate
DHAP
Glycerol-3P
dehydrogenase FADH2
GUT2
2eFADH2
2e2e
NDE1, 2
FADH2
Internal
NADH
dehydrogenase
Complex III
Q6
2e-
Cyt
b + c1
Pyruvate
D, L-Lactate
1edehydrogenases
(LDH)
n’H+
Cyt
c
1e-
1e-
-
FADH2
2e-
FeS
Com
plex
(cyt IV
oxid c
ase)
Cu
I;
a ; a Cu II
3
Complex II :
Succinate
dehydrogenase
(SDH)
NDI1
Succinate
NADH
Fumarate
NAD+
½ O2
+ 2H+
2 H 2O
Figure 1. The respiratory chain of S. cerevisiae. Only the inner mitochondrial membrane is represented. Cytochrome b,
subunits I, II and III of cytochrome c oxidase are respiratory components encoded by mitochondrial genes (as are subunits
VI, VIII and IX of Fo-ATPase, not represented here).69 The two types of D- and L-lactate dehydrogenases are not detailed.46
Q6, ubiquinone 6; Cyt, cytochrome
S. cerevisiae differs from many eukaryotes (plants,
fungi and other yeasts) whose mitochondrial alternative respiration has been quite well characterized (Table 1). Unlike plants and several fungi,
S. cerevisiae mitochondria do not contain an
NADPH : ubiquinone oxido-reductase (on the external face of the inner membrane). Moreover, in
contrast to complex I-type NADH dehydrogenase,
the internal NADH dehydrogenase is neither inhibited by rotenone nor coupled to the generation of proton-motive force. This contributes to
the lower ATP yield of oxidative phosphorylation in S. cerevisiae as compared to other kinds
of mitochondria.56,162 Like plants, but in contrast to mammalian mitochondria, yeast mitochondria possess external NADH : ubiquinone oxidoreductases (NDE1 and/or NDE2) localized in the
inner mitochondrial membrane. These isozymes
are rotenone-insensitive and do not pump proton. They are responsible for the direct oxidation of cytosolic NADH. This property gives
one explanation for the low (or perhaps the
absence of) activity of the malate–aspartate shuttle
in S. cerevisiae.15 Alternatively, cytosolic NADH
can be oxidized by the respiratory chain via
the glycerol-3-phosphate shuttle, which consists
of cytosolic NADH-linked glycerol-3-phosphate
dehydrogenase and a membrane-bound glycerol-3phosphate : ubiquinone oxido-reductase.150 FurtherCopyright  2003 John Wiley & Sons, Ltd.
more, aerobic growth with D- and L-lactate as carbon source is also permitted via the inducible D,Llactate dehydrogenases localized on the external
face of the inner mitochondrial membrane.46
Respiratory oxygen fluxes
The potential cellular respiration rate (qO2 max ;
equal to the actual respiration rate, qO2 , under
aerobic conditions) is an effective indicator of mitochondrial respiratory development that may vary
noticeably, depending on the growth conditions,
the growth rate and the yeast strain used. Since all
the respiratory complexes of S. cerevisiae are subjected to catabolic repression,63 qO2 max strongly
depends on the carbon source used for growth.
Indeed, qO2 max values of aerobic glucose-repressed
wild-type cells are about 10-fold lower than those
of fully derepressed cells (Table 2). In such cells,
the considerable contribution made by respiration
can be potentiated by the supplementation of either
respiratory substrates or protonophoric uncouplers.
In both aerobic cells and isolated mitochondria
(coupled or uncoupled), inhibitors of complex III
(antimycin A, myxothiazol) and/or inhibitors of
cytochrome c oxidase [KCN, NaN3 (sodium azide)
and CO] almost fully inhibit oxygen consumption.
However, despite the detection constraints of low
oxygen fluxes, residual antimycin A- or cyanideresistant oxygen uptake has been elicited. It may
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
1119
Table 1. Characteristics of mitochondrial alternative respirations of some eukaryotes
Organism
Sensitivity toward
Resistance
toward
References
Plantsa
Fungib
SHAM
SHAM
KCN, anti A
KCN, anti A
[179]
[91,179]
Filamentous fungi (obligate aerobe)b
Neurospora crassa
Gaeumannomyces graminis
Aspergillus niger
NaN3
anti A, SHAM
anti A, SHAM
KCN, SHAM, anti A
KCN
KCN
Yeasts (obligate aerobe)b
Pichia membranifaciens
Yarrowia lipolytica
Cryptococcus albidus (several strains)
SHAM
SHAM
SHAM
KCN
KCN
KCN
[200,202]
[201]
[201]
Yeasts (Crabtree-negative)b
Hansenula anomala (Pichia anomala)
Candida parapsilosis
Candida parapsilosis
Candida albicans
Debaryomyces hansenii
Debaryomyces occidentalis (Schwanniomyces castellii)
Debaryomyces occidentalis (Schwanniomyces castellii)
SHAM (3 mM)
SHAM
SHAM, BHAM
KCN (5–10 mM), SHAM, BHAM
anti A, SHAM
SHAM
SHAM
NaN3
KCN, anti A, NaN3
KCN, anti A
anti A, KCN
anti A
KCN
KCN, anti A
NaN3 , anti A
SHAM, anti A
[66]
[135]
[71,133]
[71,133]
[79]
[202]
[157]
[157]
[51]
[90]
[98]
SHAM, salicyl hydroxamate; BHAM, benzohydroxamate; anti A, antimycin A.
a Respiration involving a homodimeric alternative terminal oxidase (AOX).
b The monomeric or dimerization state of AOX is still questionable.
represent from 0.1 to a few percent of the overall
oxygen consumption.42,168,169,205,213 Electron leakage from the reduced ubiquinone pool to produce
ROS may partially explain such oxygen fluxes.25
However, in view of the properties of myxothiazol,
which blocks ubiquinone auto-oxidation at the bc1
level,207 this hypothesis seems unlikely, since no
difference could be detected between antimycin Aand myxothiazol-resistant oxygen fluxes in either
whole cells or isolated mitochondria.168,170 On the
other hand, a significant ROS production from
mitochondrial dehydrogenases cannot be excluded.
Despite the discovery of ROS-sensitive probes to
measure ROS, the in vivo quantification of mitochondrial ROS production is considerably complicated by: (a) the presence of H2 O2 and O2 ž− detoxifying enzymes (such as catalases, glutathione peroxidases and superoxide dismutases); (b) the eventual co-localization of such enzymes and the ROS
production site; (c) the differential behaviour of
cellular membranes towards H2 O2 and O2 ž− (permeability and impermeability, respectively); (d) the
localization of the probes, which may differ from
the ROS production site; and (e) the eventual redox
recycling of the probes, which can perturb both the
quantification and the biological oxygen reduction
Copyright  2003 John Wiley & Sons, Ltd.
mechanisms.119,186 For all these reasons, in vivo
ROS production levels in aerobic S. cerevisiae cells
(treated or not with antimycin A) have not yet been
well quantified.
Oxygen fluxes during fermentative metabolism
Oxygen consumption capacities (qO2 max ) of
S. cerevisiae cells are rather low (but not negligible) during strict fermentative metabolism
(Table 2). In anaerobic wild-type cells, the presence of the respiratory chain has been investigated recently under enological conditions,68,170
and previously under classical laboratory conditions.40,42,165 In such cells, a cyanide-sensitive
‘respiratory’ pathway was identified but it was
not related to the ubiquinone or cytochrome c
oxidase content.94,126,205 Moreover, the inhibition
extent of oxygen flux by cyanide was strictly
similar for both anaerobic rho + and rho0 cells
grown under classical laboratory or enological
conditions.81,168,170 Thus, KCN has cellular targets other than cytochrome c oxidase (e.g. sterol
synthesis; see section on Structural oxygen requirements). Taken together, these data indicate that
a functional respiratory chain is not present in
Yeast 2003; 20: 1115–1144.
1120
E. Rosenfeld and B. Beauvoit
Table 2. Oxygen consumption capacities of wild-type and respiratory-deficient S. cerevisiae cells as a function of growth
conditions
Growth
conditions
Carbon source
Strain
qO2 max #
References
Aerobic
Lactate
Glycerol
Glycerol + ethanol
Galactose
Glucose (derepression)
Glucose (repression)
Glucose (Enol cond, strong repression)
Glucose (repression or derepression)
Lab strains (WT)
Ind strain (YF)
Lab strain (WT)
Lab strain (WT)
Lab strain (WT)
Lab strains (WT)
Lab strains (WT)
Lab strain (WT)
Lab strains (rho− , rho0 mutants)
80–320
150
100
50
100–110
30–150
5–12
5–15
0.3–3.6
[45,151]
[21]
[168]
[27]
[62]
[14,81,126,127,166,204,205]
[1,27,165,168,213]
[168]
[66,81,183,213]
Oxygen-limiteda
Glucose (repression, DOT 0.05 µM)
Glucose (repression, DOT 0.25 µM)
Glucose (repression, DOT 0.5 µM)
Glucose (derepression, DOT 0.5 µM)
Glucose (repression, DOT 1 µM)
Glucose (repression)
Glucose (repression)
Glucose (Enol cond, strong repression)
Glucose (Enol cond, strong repression)
Lab strain (WT)
Lab strain (WT)
Lab strain (WT)
Lab strain (WT)
Lab strain (WT)
Lab strain (WT)
Lab strain (rho− mutant)
Lab strain (WT)
Lab strain (rho0 mutant)
0.5
2–3
4–5
12–13
7–8
5–7
7–9
1–3
1–3
[166]
[165]
[165,166]
[166]
[165]
[81]
[81]
[168]
[168]
Anaerobicb
Glucose (‘derepression’)
Glucose (repression)
Glucose (repression)
Glucose (Enol cond, strong repression)
Glucose (Enol cond, strong repression)
Lab strain (WT)
Lab strain (WT)
Ind strain (YF)
Lab strain (WT)
Lab strain (rho0 mutant)
6–10
0.5–4
2–4
1–3
1–3
[126,204,205]
[81,165]
[111]
[168]
[168]
WT, wild-type; YF, yeast foam; Lab, laboratory; Ind, industrial; DOT, dissolved oxygen tension; Enol cond, enological conditions (which are
normally anaerobic). # Expressed as nmol O2 /min/mg dry yeast mass. Oxygen consumption capacities (qO2 max ) were determined at saturating
oxygen concentrations on growing or non-growing cells in the presence of the carbon source used for growth.
a Under oxygen-limited conditions, the development of cellular respiration is very sensitive to DOT, and the actual respiration rate (qO )
2
measured in situ can be significantly lower than qO2 max .166
b Some discrepancies were noted in the literature for qO max of anaerobic glucose-repressed wild-type cells, but values were systematically at
2
least four-fold lower than those of aerobic glucose-repressed cells.
anaerobic cells, and that the term ‘cyanide-resistant
respiration’ is thus no longer valid. In contrast,
resistance to antimycin A and/or myxothiazol is
very suitable for quantifying non-respiratory oxygen fluxes. Thus, the antimycin A-resistant oxygen
flux (estimated under aerobiosis) of both wild-type
and respiratory-deficient cells grown under classical laboratory and enological conditions depends
on the oxygen tension during growth: the less oxygen available, the higher the antimycin A-resistant
oxygen uptake.81,168 Among all the non-respiratory
oxygen utilization pathways required for growth
(see section on Structural oxygen requirements),
only a few of them may significantly contribute
to the oxygen consumption capacity retained by
S. cerevisiae cells during fermentative metabolism
(see section on ‘Cyanide-resistant respiration’).
Copyright  2003 John Wiley & Sons, Ltd.
Structural oxygen requirements
of S. cerevisiae
Haem biosynthesis and haemoproteins
Regulation of haem synthesis
Haem (or haem b, protohaem (IX), Fe2+ -protoporphyrin IX), and haems a and c (both originating from protoporphyrin), are prosthetic groups of
haemoproteins. Their biosynthesis requires molecular oxygen and involves several mitochondrial
and cytosolic biochemical steps (Figure 2). The
two oxygen-dependent steps involve the cytosolic coproporphrinogen III oxidase (encoded by
the HEM13 gene) and protoporphyrinogen oxidase (encoded by the HEM14 gene). The oxygen availability also influences the regulation
of the haem pathway. In anaerobic cells, all
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
1121
MITOCHONDRIA
δ-ALA
HEM2
δ-ALA
HEM1
Krebs
cycle
PBG
HEM 3
Glycine
Preurogen
SuccinylCoA
MET1
Urogen III
HEM12
+Fe++ Proto
Heme-independent
and
oxygen-dependent
pathways
HEME
HEM14
+Fe++
Apoproteins
NUCLEAR
GENE
REGULATION
HEM15
O2
Coprogen III
O2
HEM13
Protogen IX
Heme c, a
Respiratory cytochromes
Redox
state ?
Oxidative stress
HEMOPROTEINS
Lipid synthesis
Iron uptake
Other functions
Figure 2. Biosynthesis and roles of haem in S. cerevisiae. Abbreviations: δ-ALA, 5-aminolevulenate; PBG,
porphobilinogen; Preurogen, hydroxymethylbilane; Urogen, uroporphyrinogen; Coprogen, coproporphyrinogen; Protogen,
protoporphyrinogen; Proto, protoporphyrin. Enzymes: HEM1, δ-ALA synthase; HEM2, PBG synthase or δ-ALA dehydratase;
HEM3, PBG desaminase; MET1, urogen III synthase; HEM12, urogen decarboxylase; HEM13, coprogen III oxidase; HEM14,
protogen oxidase; HEM15, ferrochelatase. References 6,27,103,109,159,171,206
enzymes of the haem pathway are present.109 Thus,
enhancement of haem synthesis solely requires
the addition of oxygen. In such cells, HEM13
is likely the controlling step of haem biosynthesis. Under aerobic conditions, regulation of
haem synthesis is still poorly understood.109 However, a negative feed-back regulation by oxidized haem (i.e. hemin) has been hypothesized
on the mitochondrial δ-5-aminolevulinate synthase (encoded by the HEM1 gene), the first
step of the haem pathway.206 Alternatively, porphobilinogen synthase (the HEM2 gene product) has also been considered to be a controlling step, since δ-5-aminolevulinate accumulates
in aerobic cells.109 On the other hand, the compartmented haem pathway has to be coordinated
with mitochondrial membrane biogenesis and the
Copyright  2003 John Wiley & Sons, Ltd.
synthesis of respiratory cytochromes (encoded by
nuclear or mitochondrial genes). Indeed, haem
and lipid synthesis pathways interact with each
other, especially via transcriptional regulations,
e.g. HEM13 (and other haem-responsive genes)
expression is increased when sterol synthesis is
inhibited.16 Inversely, an increase in δ-5-aminolevulinate intracellular concentration derepresses the
activity
of
3-hydroxyl-3-methylglutaryl-CoA
(HMG-CoA) reductase, a key step in sterol synthesis.124
Transport and degradation of haem
Cellular haem contents are poorly documented in
S. cerevisiae because the free haem pool is quite
difficult to measure. However, data in Table 3
show that bound and free haem contents are
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E. Rosenfeld and B. Beauvoit
Table 3. Haem and porphyrin contents in (wild-type) S. cerevisiae cells
Growth
conditions
Carbon
source
Strain
Bound and free haem
(nmol/g dry yeast mass)
Free porphyrins
(nmol/g dry yeast mass)
References
Aerobic
Ethanol
Glycerol
Glucose
Lab and Ind strains
Lab and Ind strains
Lab and Ind strains
150–300
200–210
60–130
ND
ND
4–8
[27,109]
[27,109]
[27,106,109]
Anaerobic
Glucose
Lab and Ind strains
25–60
4
[27,106,109]
Lab, laboratory; Ind, industrial; ND, no data.
significantly affected by glucose repression and
oxygen limitation. Interestingly, the haem content
in anaerobically grown cells is not negligible,
and free porphyrin (including haem) contents in
glucose-repressed cells remain quite low upon
oxygenation. This indicates that free haem and free
porphyrin contents are considerably lower than the
haemoprotein content in both aerobic and anaerobic
cells.
The mechanisms of haem translocation from
mitochondria to the other cellular compartments, as
well as the nature (free or bound) of the transported
molecule, remain uncharacterized in S. cerevisiae.
Haem degradation is generally thought to be
absent in both aerobic and anaerobic S. cerevisiae
cells. However, an aerobic gene (YLR205c)103,191
exhibits a quite strong similarity to the haem oxygenase of other eukaryotes.57
It has been shown that anaerobiosis leads to
coproporphyrin(ogen) excretion into the culture
medium and to the accumulation of haem precursors and derived compounds, such as protoporpho(di)methene (503 nm pigment; P503).106,107,110
Protoporphyrin and Zn-protoporphyrin also accumulate in anaerobic cells. Their detection has been
explained by the presence of contaminating traces
of oxygen in ‘anaerobic’ cultures, which are practically inevitable under the conditions used routinely
in laboratories. However, it is rather difficult to
understand how typical haemoproteins were more
recently detected in anaerobic cells.27,109 It has
been suggested that the haem released by the degradation of respiratory cytochromes may be recycled in the cytoplasm. Mdl1p, a putative mitochondrial haem carrier of the ABC class which
is upregulated under anaerobic conditions, may
transport haem from the mitochondrial matrix to
the cytoplasm to synthesize haemoproteins (b-type
cytochromes, globins, catalases) that remain under
anaerobiosis.36,105
Copyright  2003 John Wiley & Sons, Ltd.
Haemoproteins of S. cerevisiae
Haemoproteins of S. cerevisiae include cytochromes involved in respiration, lipid synthesis and
iron uptake, on the one hand, and proteins involved
in oxidative stress or still unknown functions, on
the other (Figure 2). Both the HAP1 transcription
factor and the proteins involved in the transformation or the insertion of haem into apoproteins bind
haem and thus may be regarded as haemoproteins.
However, we have only listed the haemoproteins
involved in oxido-reduction reactions (Table 4).
Haemoproteins of aerobic cells Mitochondrial respiratory cytochromes (a + a3, b(±b2), c and c1)
are the major haemoproteins of wild-type yeast
cells grown in the presence of oxygen. The synthesis of respiratory cytochromes is optimal under
aerobic conditions, and becomes limited at oxygen concentrations below 2–4 µM.31,159,165 Interestingly, this is the range at which oxygen starts to
limit growth.13,76 This suggests that the decrease in
cytochrome synthesis (rather than the sole oxygen)
can limit growth under oxygen-limited conditions.
In aerobic wild-type cells, the abundance and
spectral properties of mitochondrial respiratory
cytochromes often complicate the detection of
other haemoproteins. Such a detection can be performed in aerobic rho0 strains (devoid of respiratory cytochromes), but it is known that the nuclear
gene expression pattern is modified when mitochondrial respiratory functions are impaired.53,121
Alternatively, their detection is made possible by
changing the oxidizing or reducing agent used
for spectral analysis. The presence of the cytosolic flavohaemoglobin (YHB1) in aerobic cells has
been evidenced in this way.27 Moreover, in both
wild-type and respiratory-deficient mutants, b5
(encoded by CYB5 ) and P450 (encoded by ERG5
or ERG11 ) cytochromes could also be detected in
aerobic glucose-repressed cells.80,81,132,164 – 166
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
1123
Table 4. Haemoproteins of S. cerevisiaea
Haemoprotein
Localization
References
Respiratory cytochromes
Cytochrome a + a3 (COX1)b
Cytochrome b (COB)
Cytochrome b2 (CYB2)
Cytochrome c (CYC1, CYC7)
Cytochrome c1 (CYT1)
Inner mitochondrial membrane
Inner mitochondrial membrane
Inner mitochondrial membrane (outer side)
Inner mitochondrial membrane (outer side)
Inner mitochondrial membrane
[171]
[171]
[171]
[171]
[171]
Non-respiratory cytochromes and other haemoproteins
Cytochrome c peroxidase (CCP1)
Catalase A (CTA1)
Catalase T (CTT1)
Cytochrome b5 (CYB5)
Acyl-CoA desaturase (OLE1)
Inositol-phosphorylceramide hydroxylase (SCS7)
Cytochrome P450: C14-demethylase (ERG11 = CYP51)
Cytochrome P450: C22-desaturas (ERG5 = CYP61)
Cytochrome P450: N-formyl tyrosine oxidase (CYP56)
Flavocytochrome b (FRE1)c
Flavocytochrome b (FRE2)c
Flavohaemoglobin (YHB1)
YNL234w
YML131w (leukotriene B4 12-hydroxydehydrogenase)
Mitochondrial intermembrane space
Peroxisome
Cytosol, vacuole
Endoplasmic reticulum
Endoplasmic reticulum
Endoplasmic reticulum
Endoplasmic reticulum
Endoplasmic reticulum
?
Plasma membrane
Plasma membrane
Cytosol
Cytosol (?)
?
[156]
[171]
[171]
[81]
[138]
[50]
[195]
[182]
[26]
[117]
[54]
[214]
[175]
[85]
a The proteins involved in haem metabolism and the transcription factors (e.g. HAP1) that bind haem are not listed. The sirohaem-containing
(oxygen-independent) cytosolic enzymes involved in sulphate assimilation (MET10, ECM17) are also not listed.
b COX1, one of the 12 cytochrome c oxidase subunits, contains haem a, haem a3, and the CuB centre.
c The five homologues to FRE1 and FRE2 in S. cerevisiae exhibit sequence similarity in the regions expected to bind FAD, NADPH and
two haems.
Haemoproteins of anaerobic cells The absence of
functional cytochrome a + a3 in anaerobic cells
is now widely admitted. In many studies, the low
content of cytochrome a + a3 in anaerobic cells
has been justified by the presence of contaminant
traces of oxygen during growth or by an induction
during cell harvest. Recently, Dagsgaard et al.42
detected almost all the cytochrome c oxidase subunits (except subunits IV and VIII) in the promitochondria of anaerobic cells. This result is quite
surprising, since the authors used drastic anaerobic
growth and harvesting conditions, but the absence
of subunits IV and VIII is sufficient to explain
the absence of assembled and active cytochrome c
oxidase in anaerobic cells. In these cells, the presence of functional holocytochrome c is also a matter of discussion. The S. cerevisiae genome contains two genes encoding isoforms of cytochrome
c (viz. CYC1 and CYC7 ) that are differentially
regulated.49 Burke et al.31 have shown that CYC7
is a strict anaerobic gene, since it is repressed under
both aerobic and strongly oxygen-limited conditions. Nevertheless, upon oxygenation, CYC7 is
Copyright  2003 John Wiley & Sons, Ltd.
transiently and strongly induced before the aerobic
isoform, CYC1, reaches its steady-state level of
expression.103 Hence, it has been postulated that
Cyc7p plays a protective role during oxidative
stress, because it may behave as an electron sink for
the partially assembled complex III under conditions of low defence against ROS.29,36,105,160 However, because of the numerous constraints of Cyc7p
detection, there is no conclusive evidence that functional Cyc7p is present in anaerobic cells.
Historically, the so-called ‘cytochrome a1’
(thought to be responsible for the 580–590 nm
α-band observed in reduced minus oxidized difference spectra of whole cells) had been regarded as a
remnant terminal oxidase in anaerobic S. cerevisiae
cells.52,120 Later, the 580–590 nm band was
attributed to promitochondrial cytochrome c
peroxidase,80 to Zn-protoporphyrin (583–585 nm
α-band) and neutral porphyrins.80,106,108 However,
more recently it turned out that there was probably
some confusion between prophyrins and the flavohaemoglobin (encoded by YHB1) in both anaerobic and aerobic cells. Indeed, the absorption band
Yeast 2003; 20: 1115–1144.
1124
near 575–577 nm, obtained with low temperature
spectra of whole cells (reduced with endogenous
substrates) and previously attributed to porphyrins
and Zn-protoporphyrins, may be due in fact to the
oxygenated form of Yhb1p.27
Both microsomal b5 (previously named ‘b1 ’)
and P450 cytochromes are major haemoproteins of
anaerobic cells. This is likely linked to microsomal proliferation under anaerobic conditions.22,132
In some instances, carbon monoxide difference
spectra (reduced + CO minus reduced) performed
for P450 detection have revealed the presence of
‘cytochrome P 420’ in both particulate and soluble fractions.80,148 Denaturation and further solubilization of P450 may be responsible for the
γ -420 nm peak, but the native soluble hemichrome
encoded by the anaerobic YNL234w gene may also
account for this peak.175 Thus, the typical absorption bands (420, 542, 572 nm) of Ynl234wp were,
together with P450, easily detected in stationaryphase cells grown under anaerobic enological conditions which favoured its induction.168 Besides
YNL234w, only a few genes encoding haemoproteins (ERG11, CYC7, OLE1, and SCS7 ) were found
to be upregulated under anaerobic batch culture
conditions in galactose medium.105 In contrast, in
glucose-limited chemostat cultures, such an anaerobic activation was not observed for CYC7, ERG11
and OLE1 genes.191
Sterol biosynthesis
Sterol biosynthesis represents another crucial oxygen-dependent pathway in S. cerevisiae. Many
aspects of its regulation are still unknown because
of the large number of ERG genes and the crosstalk between different environmental signals. However, the following is widely admitted: (a) most
of the ERG genes are regulated by oxygen;
(b) endogenous or exogenous sterols are regulators
of gene expression (including ERG genes) through
still unknown molecular mechanisms; (c) some
sterols can inhibit several enzymatic steps of the
sterol pathway; and (d) the regulatory process may
depend on the sterol requirement for growth and
on the presence of other lipids. The roles and
the intracellular traffic of sterols have been extensively discussed44,105,114,134,155,196,211 (for review,
see ref. 43). Here, we focus mainly on the relationships between oxygen and sterol biosynthesis.
Copyright  2003 John Wiley & Sons, Ltd.
E. Rosenfeld and B. Beauvoit
Aerobic sterol exclusion
Exogenous sterols are required for anaerobic
growth.4 On the other hand, ‘aerobic sterol exclusion’ obligates aerobic cells to synthesize endogenous sterols for growth.163 By studying hem
mutants that can incorporate sterols aerobically,
it has been suggested that haem-dependent sterol
biosynthesis is the primary factor of sterol exclusion. First, some sterols were thought to inhibit
sterol uptake.125,141 In addition, mutations affecting
some putative transcription factors (e.g. SUT1,2,
UPC2 or YLR228c) were shown to enable
the sterol influx in haem-competent aerobic
cells.24,41,142,178 Interestingly, both SUT1 and, to a
lesser extent, UPC2 are upregulated under anaerobic conditions and contain a Rox1 binding site
in their promotor region.24,105,142,191 This reinforces the hypothesis that aerobic sterol exclusion
is due to the transcriptional inhibition of genes
involved in sterol uptake. Recently, it has been
suggested that two UPC2-responsive and anaerobic
genes encoding ATP-binding transporters, AUS1
and PDR11, are required for anaerobic growth and
sterol uptake.210
Oxygen-independent isoprenoid and squalene
synthesis
The initial steps of the sterol pathway (from acetylcoA to squalene) do not require oxygen [but
require NAD(P)H and ATP] and are responsible
for the accumulation of squalene under anaerobic conditions (for review, see ref. 43). The
first step of the linear path of sterol synthesis is the microsomal squalene synthase (Erg9p)
which catalyses the NADPH-dependent condensation of two farnesyl-pyrophosphates into squalene. Like HMG2 (the anaerobic counterpart of
the two hydroxymethylglutaryl-CoA reductase isoforms involved in mevalonate synthesis), ERG9 is
positively regulated by fatty acids and is negatively
regulated by oxygen, haem and sterols.97,105 However, ERG9 activity remains quite high in cells
grown anaerobically with ergosterol and oleate in
excess.128
Oxygen-dependent sterol synthesis
Squalene conversion to ergosterol requires molecular oxygen at the level of six enzymatic steps catalysed by five enzymes: a non-P450 mono-oxygenase
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
(ERG1), two di-ferric and non-heminic hydroxylases/desaturases (ERG25 and ERG3) and two
cytochromes P450 (ERG11 and ERG5). In sum,
12 O2 molecules are required for the conversion of
one squalene to ergosterol (Figure 3 and references
therein). On the other hand, the oxido-reduction
reactions also require NAD(P)H. However, the
in vivo contribution of either NADPH or NADH
as preferential electron donors for sterol synthesis
1125
is still hypothetical. It is admitted that the activity of both P450 cytochromes (ERG11 and ERG5)
and ERG1 depends on the activity of the microsomal NADPH-cytochrome P450 reductase (i.e.
CPR1 or NCP1).43,190 In contrast, ERG25 and
ERG3 are thought to depend on the microsomal
NADH-cytochrome b5 reductase and cytochrome
b5.18,149 In aerobic cpr1 mutant cells, it has been
suggested that NADH-cytochrome b5 reductase
SQUALENE
NAD(P)H
+ 1 O2
ERG1
Allylamines
SQUALENE EPOXIDE
ERG7
LANOSTEROL
3 NAD(P)H
+ 3 O2
ERG11
Azoles
4,4-DIMETHYLCHOLESTA-8,14,24 TRIENOL
ERG24
Morpholines
NAD(P)H
4,4-DIMETHYLZYMOSTEROL
ERG25, 26,27
KCN
3 NAD(P)H
+ 3 O2
4α-METHYLZYMOSTEROL
3 NAD(P)H
+ 3 O2
ERG25, 26,27
KCN
ZYMOSTEROL
S-adenosyl-L-methionine
ERG6
FECOSTEROL
ERG2
Morpholines
EPISTEROL
NAD(P)H
+ 1 O2
ERG3
KCN
(not CO and azoles)
ERGOSTA 5,7,24 (28) TRIENOL
NAD(P)H
+ 1 O2
ERG5
Azoles, CO
(not KCN and NaN3)
ERGOSTA5,7,22,24 (28) TETRAENOL
ERG4
Morpholines
ERGOSTEROL
AT )
AC E1,2
R
(A
er
est
yl lase
r
e
Esterification St dro
y
and storage in lipid h
Fatty acids
particules
Plasmalemma
Inner mitochondrial membrane
Secretory vesicles
Figure 3. The oxygen-dependent steps of sterol synthesis in S. cerevisiae: intermediates, oxygen requirements and
inhibitors. Enzymes: ERG1, squalene epoxidase; ERG7, lanosterol synthase; ERG11 (CYP51), lanosterol C-14 demethylase;
ERG24, sterol C-14 reductase; ERG25, sterol C-4 methyloxydase; ERG26, sterol C-3 dehydrogenase (C4-decarboxylase);
ERG27, sterol C-3 ketoreductase; ERG6, sterol C-24 methyltransferase; ERG2, sterol C-8 isomerase; ERG3, sterol C-5
desaturase; ERG5, sterol C-22 desaturase; ERG4, sterol C-24 reductase; ACAT (ARE1,2), acyl-CoA sterol acyl transferases.
References 7–9,18,19,43,47,59,96,115,149,152,180,196; oxygen (and NADPH) stoichiometries of the reactions catalysed
by ERG1,43 ERG11,7 ERG25,9,18,59,152 ERG3149 and ERG596 ; some alternative routes from lanosterol to sterols have been
described in cells treated with inhibitors and in mutants60,152
Copyright  2003 John Wiley & Sons, Ltd.
Yeast 2003; 20: 1115–1144.
1126
can provide electrons (via cytochrome b5 ) to all
oxygen-dependent steps of the sterol pathway.113
However, by using various physiological modulations of the cellular NADH level during transitions from anaerobic (classical laboratory and enological) to aerobic conditions, it has been shown
that the oxygen uptake due to sterol synthesis
(i.e. terbinafine- and fenpropimorph-sensitive O2
consumption) does not correlate with the cellular NADH redox state168 (Rosenfeld et al., unpublished data).
Regulation of sterol synthesis by oxygen
and glucose
During batch cultivation on glucose medium, ERG1
is upregulated upon sterol limitation128,129 and
is also activated by increasing oxygen availability.82,129 Conversely, ERG2 is activated under
anaerobic conditions.184 Surprisingly, in glucoselimited chemostat cultures, none of the ERG genes
were found to be upregulated under anaerobic
conditions.191 On the other hand, during batch
cultivation on galactose medium (growth phase),
many ERG genes of the oxygen-dependent pathway of sterol synthesis (ERG3, ERG4, ERG6,
ERG24, ERG25, ERG26, ERG11 ) were upregulated under anaerobic conditions.105 This was also
the case for the ERG28 (YER044c) gene, which
is required for normal sterol composition.77 These
discrepancies suggest that the regulation of ERG
genes by oxygen interacts with other factors, such
as carbon source or cultivation mode. Accordingly, cytochrome P450 accumulates both in the
presence of fermentable carbon sources, especially
glucose, and when the oxidative metabolism is
repressed.93,193 Translational and post-translational
regulation by glucose have also been proposed.187
Among the three CYP genes encoding cytochrome
P450 in S. cerevisiae [i.e. ERG11 (CYP51 ) and
ERG5 (CYP61 ) genes involved in ergosterol synthesis; and DIT2 (CYP56 ), likely involved in
spore wall assembly], only ERG11 is a typical anaerobic gene. Thus, Erg11p is likely the
sole cytochrome P450 accumulated in anaerobic
cells. Like cytochrome P450, NADPH-cytochrome
P450 reductase (CPR1), NADH-cytochrome b5
reductase (CBR1) and cytochrome b5 (CYB5)
are also activated under anaerobic conditions
and are repressed when respiratory functions are
induced.22,23,80,81,120,132,187,195
Copyright  2003 John Wiley & Sons, Ltd.
E. Rosenfeld and B. Beauvoit
Quantitative data
Sterol content contributes little (less than 1% w/w)
to the cell dry mass. Only a small amount of ergosterol (about 5 mg/l) is required for optimal anaerobic growth under laboratory, brewing or enological conditions.10,11,169 Nevertheless, sterol (mainly
ergosterol and squalene) contents strongly depend
on the strains used, the lipidic composition of
the culture medium, and the oxygen availability
(Table 5). Aerobic S. cerevisiae cells contain significant amounts of ergosterol (75–90%), lanosterol (5–15%) and squalene (2–15%).44 The less
oxygen available during growth, the lower the total
sterol content and the degree of sterol esterification. However, squalene content increases under
anaerobic conditions, especially in the absence of
exogenous sterols (Table 5 and references therein).
The main role of the anaerobically accumulated
squalene is likely to optimize sterol synthesis when
oxygen is provided. Accordingly, using different
measurement methods (and different laboratory or
enological conditions of cultivation), it has been
shown that the sterol synthesis rate is higher upon
oxygenation of anaerobic (growing or resting) cells
than in aerobic growing cells (Klein et al., 1954,
cited in ref. 152).112,168,169
Unsaturated fatty acid biosynthesis
The Acyl-CoA desaturase (OLE1)
S. cerevisiae cannot synthesize polyunsaturated
fatty acids.176 However, mono-unsaturated fatty
acids (UFAs) are required for growth. Their synthesis plays a critical role in maintaining a correct
ratio of saturated to mono-unsaturated fatty acids
in membranes.43 Since UFA synthesis is oxygendependent, Tween 80 (a source of oleate) is routinely added to the anaerobic culture media.5 Under
aerobic conditions, biosynthesis of the UFAs,
palmitoleate (C16 : 19 ) and/or oleate (C18 : 19 )
is catalysed by the unique microsomal acyl-CoA
desaturase, OLE1. Synthesis of one UFA molecule
requires one NAD(P)H as electron donor and one
O2 as acceptor of two electron pairs (one from
NAD(P)H and the other from the saturated fatty
acyl molecule [palmitoyl(or steroyl)-CoA)]. OLE1
functionality does not require cytochrome b5 but
its cytochrome b5 -like domain is involved in the
desaturation reaction.138 In vivo, the nature of both
the electron donor (NADH or NADPH) and the
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
1127
Table 5. Sterol contents of (wild-type) S. cerevisiae cells as a function of oxygen availability and lipid composition of the
culture medium
Growth conditions
Carbon
source
Strain
Total
sterol#
Aerobic
−AFa
Glucose
Lab strains
11–25
Glucose
Glucose
Malt wort
Malt wort
Lab strain
Lab strain
Ind strain
Ind strain
1.8
9–12
Oxygen-limited
−AF
−AF (DOT = 0.05 µM)
−AF (DOT = 0.5 µM)
Glucose
Glucose
Glucose
Lab strain
Lab strain
Lab strain
0.5–1.0
0.5
4.2
Anaerobic
−AF
−AF
−AF (Brewing)b
−AF (Brewing)
+AF
+AF
+AF
Glucose
Glucose
Malt wort
Malt wort
Glucose
Glucose
Galactose
Lab strain
Lab strain
Ind strain
Ind strain
Lab strain
Lab strain
Lab strain
0.4
−AF
−AF
−AF (Brewing)b
−AF (Brewing)
Ergosterol#
Squalene#
Reference
8–20
[30–80% esterified]
0.4–3.3
[44]
5.7–6.3
2.6
2–3
[75% esterified]
1–3
5.5
3–6
[83]
[88]
[146]
[10]
[83]
[166]
[166]
0.1
1.7
[20% esterified]
0.7
2.0
1.8–2.6
16–22
6.5
6.0
2–4
[83]
[88]
[146]
[10]
[88]
[11]
[88]
AF, anaerobic growth factors [ergosterol and Tween 80 (as source of oleate)]; DOT, dissolved oxygen tension; Lab, laboratory; Ind, industrial.
# Expressed as mg/g dry yeast mass.
a Sterol composition was analysed in several wild-type strains.
b Under classical brewing conditions, oxygen is available only during the early stage of growth.
reductase (NAD(P)H-P450 reductase or NADH-b5
reductase) that transfers electrons to the b5 domain
remains unclear. UFA synthesis depends on the
supply of saturated fatty acids (FA). These may be
either neo-synthesized from acetyl-CoA (an ATPrequiring process) or released (via the steryl ester
hydrolase) from the steryl esters stored in lipid particles. As for the sterol pathway, interactions and
contacts between the cellular compartments likely
coordinate the biosynthesis and transformation processes of FA, UFA and Phospholipids. This coordination is required for a specific lipid homeostasis
within cellular membranes.43
Regulation of OLE1 by oxygen and (U)FAs
It has been shown that OLE1 expression depends
on several cis regulatory sequences, such as STRE
(stress response element), FAR (fatty acid response
element) and LORE (low oxygen response element). OLE1 transcription is slightly activated in
the presence of saturated fatty acids (FA) but
strongly repressed by UFAs.35,58 It has also been
Copyright  2003 John Wiley & Sons, Ltd.
shown that the OLE1 ARNm half-life is affected
by incorporated UFA but not by FA.68 Furthermore, OLE1 is induced by oxygen concentrations below 1 µM.103,159 Both FAR and LORE
elements seem to respond to both oxygen and
UFA. Hence, oxygen and UFA signal transduction pathways likely interact at the OLE1 gene
level.140,199 The Mga2p–LORE interaction seems
to play an important role in OLE1 expression under
aerobic, oxygen-limited and anaerobic conditions,
while Rox1p is likely not involved in its aerobic repression86,140 (see also section on Oxygen
sensing). Free or esterified (Tween 80) oleate is
required for anaerobic growth. Under such conditions, the OLE1 gene is both repressed by oleate
and derepressed by the absence of oxygen. Repression by oleate is likely dominant, since OLE1
transcription is poorly activated under anaerobic
conditions when UFAs are added.140 However, it
should be kept in mind that UFAs may repress
the anaerobiosis-induced complex formation with
LORE box, and thus OLE1 expression, in a dosedependent manner.86
Yeast 2003; 20: 1115–1144.
1128
E. Rosenfeld and B. Beauvoit
Quantitative data
In S. cerevisiae, the acyl chain composition of
lipids is quite simple but depends on the presence
of contaminant lipids in the culture medium. Under
classical aerobic conditions (no lipid supplementation), the major fatty acids are stearate, oleate (both
represent 60–80% of the total fatty acids), palmitate (15%) and palmitoleate (5%). Some traces
of very long chain fatty acids (C26) have also
been detected.37,44 Total fatty acid content is much
(five- to 10-fold) higher than sterol content, and
UFA content is generally three- to four-fold higher
than ergosterol content (Tables 5, 6). However,
ergosterol : UFA, sterol : phospholipid and phospholipid : protein ratios in the sub-cellular compartments depend on growth conditions.61,176 As
shown in Table 6, fatty acid contents are greatly
influenced by changes in oxygenation conditions.
In the absence of exogenous lipids, the strong
increase in fatty acid contents upon oxygenation
is mainly explained by UFA biosynthesis.166 In
cells grown under anaerobic enological conditions
in the presence of excess oleate (0.5 ml/l Tween
80) and ergosterol, only traces of palmitoleate
were detected.168 The relatively high UFA : FA
ratio (about 1.25) was due to oleate incorporation from Tween 80 (which contains 71% moles
of oleic acid88 ). Instead of using Tween 80, the
maximal anaerobic growth under laboratory or enological conditions may be obtained by adding about
15–30 mg/l free oleate.17,169,203
Other hydroxylase/desaturases involved in lipid
synthesis
In addition to ERG25, ERG3 and OLE1, the
S. cerevisiae genome contains two other oxo-
diferric hydroxylase/desaturases, SUR2 and SCS7.
These catalyse NADPH- and oxygen-dependent
hydroxylation reactions and are involved in sphingolipid and phytoceramide metabolisms, respectively.43,50,72 Interestingly, both SUR2 and SCS7
genes are upregulated under anaerobic conditions.105 Moreover, it has been shown that SUR2 is
repressed when sterol synthesis is impaired. Thus,
this evidences functional relationships between
these oxygen-dependent lipid synthesis pathways.16
Another hydroxylase that has been recently identified in S. cerevisiae is the YNL045w gene.
It encodes a leukotriene A4 hydroxylase which
is a zinc metalloenzyme like its mammalian
counterpart.101 Daum et al.44 have shown that the
YNL045w mutation affects cellular sterol contents.
However, the role of YNL045w in the absence of
added leukotriene remains unclear.
The peroxisomal acyl-CoA oxidase (POX1)
In contrast to mammals, fatty acid β-oxidation in
S. cerevisiae does not occur in mitochondria. It
involves several peroxisomal enzymes that have
been detected in cells grown aerobically with
oleate as carbon source.43,102,130 The first (limiting) step of the β-oxidation cycle is catalysed by the acyl-CoA oxidase (POX1) that forms
trans-2,3-dehydroacyl-CoA in the presence of oxygen. During this FAD-dependent reaction, electrons are directly transferred to molecular oxygen to produce H2 O2 , which is further detoxified by the catalase CTA1. Despite the low
flux of peroxisomal β-oxidation, it allows aerobic growth on oleate.43 Moreover, the ORE (oleate
responsive element)-dependent transcription of the
Table 6. Fatty acid contents of (wild-type) S. cerevisiae cells as a function of oxygen availability and lipid composition of the
culture medium
Growth conditions
Carbon
source
Strain
Fatty acids
Content#
References
Aerobic
−AF
Glucose
Lab strains
Phospholipids
25–30
[44]
Glucose
Lab strain
Phospholipids
10
[11]
Glucose
Glucose
Glucose
Glucose
Lab strain
Lab strain
Lab strain
Lab strain
Total fatty acids
Unsaturated fatty acids
Total fatty acids
Unsaturated fatty acids
35
9
108
83
[166]
[166]
[166]
[166]
Anaerobic
+AF
Oxygen-limited
−AF (DOT = 0.05 µM)
−AF (DOT = 0.05 µM)
−AF (DOT = 0.5 µM)
−AF (DOT = 0.5 µM)
AF, anaerobic growth factors; Lab, laboratory; DOT, dissolved oxygen tension. # Expressed as mg/g dry yeast mass.
Copyright  2003 John Wiley & Sons, Ltd.
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
genes involved in fatty β-oxidation (including
POX1 ) is repressed by glucose, even in the presence of both oleate and oxygen.20 This suggests
that the oxygen-dependent fatty acid catabolism has
a low activity during glucose repression.
Nicotinic acid biosynthesis
Two nicotinic acid biosynthesic pathways have
been reported in S. cerevisiae. The one deriving
from aspartate is oxygen-independent, whereas the
other, from tryptophan, is oxygen-dependent. Since
the oxygen-independent pathway does not fulfil
nicotinic acid requirements, nicotinic acid supplementation is required for anaerobic growth.75 Two
gene products catalysing oxygen-dependent steps
potentially involved in nicotinic acid synthesis
were identified: BNA1(HAD1) (3-hydroxyanthranilate-3,4-dioxygenase) and YBL098w [kynurenine3-hydroxylase (NADPH-dependent flavin monoxygenase)].65,171 To our knowledge, none of these
genes are typical oxygen-responsive genes.105,191
Ubiquinone synthesis
Ubiquinone is an important component of the respiratory chain and serves as a lipid-soluble antioxidant and as a regenerator of cellular antioxidants. In S. cerevisiae, ubiquinone-6 (Q6, isoprenylated benzoquinone) is also found in the plasma
membrane.55,173,174 Q6 synthesis is oxygen-dependent but the pathway shares common oxygenindependent steps with sterol synthesis (for review,
see ref. 131). Two related proteins are needed to
fulfil the oxygen-dependent steps of Q6 synthesis.
The COQ7 gene is required for the last monooxygenase step in Q6 biosynthesis, but the amino
acid sequence does not share similarity to any
known mono-oxygenase or hydroxylase proteins.89
In contrast, Coq6p is a flavin-dependent monooxygenase involved in Q6 synthesis.158 While
it is known that high concentrations of glucose
repress Q6 biosynthesis,126,181 little is known about
the possible accumulation of Q6 precursors in
anaerobic cells and Q6 synthesis rates upon oxygenation of such cells. However, it has been
shown that anaerobic cells contain trace amounts
of ubiquinone (<15 µg/g dry weight),126,165 and
that the ubiquinone content of cells shows a linear response to oxygen tension similar to that of
cytochrome c.165 Moreover, the ubiquinone content of aerobic glucose-repressed and derepressed
Copyright  2003 John Wiley & Sons, Ltd.
1129
cells does not exceed 100 and 200 µg/g dry weight,
respectively.126 Therefore, as for the haem pathway, low oxygen quantities are likely to be required
for optimal Q6 synthesis.
Plasma membrane oxidases
The ferrireductase system
In S. cerevisiae, the multicomponent ferrireductase system of the plasma membrane, which is
responsible for the high-affinity uptake of external
Fe3+ , constitutes an additional site of oxygen consumption. It mainly comprises a metalloreductase
(mainly FRE1 and FRE2), a multi-copper ferroxidase, and a metal permease (FTR1). The ferric
reductase FRE1 is a b558-type cytochrome showing similarity with the human neutrophile gp91phox
NADPH oxidase.177 In gp91phox and to a lower
extent in FRE1, molecular oxygen can be used as
opportunist electron acceptor to produce O2 ž− .117
Alternatively, ROS (O2 ž− and/or H2 O2 ) may be
released from the reduced NADPH-P450 reductase (CPR1) which provides electrons to the ferrireductase system.116,123,194 On the other hand,
an obligate oxygen-dependent step is required for
iron (or copper) translocation. It is catalysed by
the ferroxidase FET3 that forms Fe3+ according
to the following reaction: 4Fe2+ + O2 + 4H+ →
4Fe3+ + 2H2 O. The oxidized iron is further incorporated into cells via the permease.73 FET3 has a
high affinity for oxygen (1–3 µM) and is almost
fully inhibited (75–80%) by sodium azide.74 Like
some other genes directly involved in oxygen utilization, the FET3 aerobic gene has an anaerobic
counterpart, FET4, a multisubstrate metal ion transporter. Derepression of FET4 may allow metal
uptake under anaerobic conditions.209 However,
under these conditions, no ferrireductase activity and no high-affinity iron uptake have ever
been measured. Upon oxygenation, iron uptake was
obtained through rapid (5 min) FET3 induction.
Hence, molecular oxygen may directly or indirectly
regulate the Fe3+ : Fe2+ ratio, either as a modulator of gene transcription or as a substrate of the
ferroxidase reaction.73
Other plasma membrane oxidases
Plasma membranes of S. cerevisiae catalyse NADH
oxidation using a variety of artificial electron
acceptors such as ferricyanide, cytochrome c and
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1130
ascorbate free radical.173,174 Reduction of ascorbate
free radical at the plasma membrane is comprised
of two independent electron transport chains. The
first probably involves the NADH-b5 reductase
responsible for the reduction of ubiquinone-6 pool,
while the second is the ferrireductase system. Concerning the latter, several data suggest that both the
NADH-ferricyanide reductase38 and the NADHQ6-iron-dependent ascorbate reductase (described
by Santos-Ocana et al.173,174 ) are potential sites of
oxygen consumption and ROS production.
Potential soluble oxidases
Yeast haemoglobins
Two cytosolic flavohaemoglobin-like proteins,
YHB1 and YNL234w, have been identified in
S. cerevisiae (Table 5). They may be involved in
transport, storage, facilitated diffusion and release
of oxygen at low oxygen tensions, and may act
as oxygen sensors and/or as terminal oxidases. In
contrast to bacterial Hb genes, it is widely acknowledged that YHB1 is an aerobic gene. It is repressed
by either anaerobiosis or haem deficiency.39,213
Nevertheless, Yhb1p could also be detected in
anaerobic cells27 and, surprisingly, the YHB1 gene
was found to be slightly downregulated in aerobic glucose-limited chemostat cultures.191 Whereas
YHB1 binds molecular oxygen reversibly, it probably has no oxidase activity in vivo. Indeed, the
‘cyanide-resistant respiration’ of aerobic rho + and
rho0 cells was not affected by the deletion or the
overexpression of the YHB1 gene.27,213 Whereas
YBH1 is likely not implicated in oxygen sensing (see section on Oxygen sensing), it might be
involved in the response to oxidative stress27,213
and in the protection of NO-induced nitrosylations
under both aerobic and anaerobic conditions.122
However, it cannot be definitely excluded that
YHB1 plays a role in the facilitated diffusion of
oxygen in anaerobic or oxygen-limited cells in the
absence of NO.
Little is known about the haemoglobin
YNL234w or its function. Moreover, the overall
structural organization of Ynl234wp makes this
protein unique among the known haemoglobins.
While not essential for growth, it is induced by
anaerobiosis, glucose repression, nitrogen starvation and osmotic stress.175
Copyright  2003 John Wiley & Sons, Ltd.
E. Rosenfeld and B. Beauvoit
Old yellow enzymes (OYE2,3)
The S. cerevisiae genome contains two genes
(OYE2, OYE3) encoding old yellow enzymes
(NADPH oxido-reductase, EC 1.6.99.1). The
OYE3/OYE2 pair constitutes one aerobic–anaerobic gene pair among those that have been identified
to date.105 Both genes are probably involved in
sterol metabolism188 NADPH is the electron donor
to OYEs but the physiological electron acceptors are still unknown. It has been suggested that
OYE3 forms H2 O2 in the presence of oxygen
and NADPH,144 and that at least one of the two
OYEs may contribute to the oxygen consumption
observed in the cytosolic fraction.87,94 However,
oxygen is now considered to be an opportunist
electron acceptor, as are ferricyanide and methylene blue. Indeed, the physiological electron acceptor (that prevents oxygen reduction) of OYE may
be an intermediate sterol with an α,β-unsaturated
carbonyl residue.188 In vivo, the OYEs may bind
β-oestradiol, and the reaction be involved in the
control of the cell cycle (see ref. 144 and discussion
therein).
Other oxygen-dependent pathways
Other oxidases
Among the additional oxygen-dependent enzymes
identified in the S. cerevisiae genome, some of
them involve oxidases. One is the mitochondrial L-proline oxidase (PUT1) that catalyses
the O2 -dependent transformation of L-proline to
1-pyrroline 5-carboxylase and to glutamate 5semialdehyde. PUT1 is induced by L-proline
and is strongly downregulated under anaerobic
conditions.191,208 However, during anaerobic enological fermentation (strong glucose repression), it
has been shown that L-proline is rapidly metabolized after moderate oxygen addition at the end
of the growth phase.172 This practice is of great
biotechnological importance, since natural musts
often contain limiting amounts of potentially assimilated nitrogen, and since L-proline is one of the
more abundant amino acids in these media. In rich
medium, it cannot be ruled out that promitochondrial PUT1 contributes to the oxygen consumption
capacities retained by anaerobically grown cells.
Another mitochondrial oxidase, D-arabino-1,4lactone oxidase (ALO1), which catalyses the final
oxygen-dependent desaturation step of D-erythroascorbate synthesis has also been identified.78
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
This enzyme may also catalyse the formation of
185
L-ascorbate from L-galactono-1,4-lactone.
Very
low contents of ascorbate-erythroascorbarte have
been reported (about 50–150 µg/g dry weight
in aerobic cells) and the form that predominates is still under controversy (see ref. 185 and
references therein). Moreover, the corresponding
biosynthetic pathways are still poorly characterized
in S. cerevisiae. Nevertheless, if ALO1 operates
as its mammalian counterpart (the L-gulono-1,4lactone oxidase), it potentially constitutes an additional H2 O2 production site.
Other mono-oxygenases and dioxygenases
In addition to the known or putative monooxygenases previously described (ERG1, P450
cytochromes, YBL098w, COQ6), the genome
of S. cerevisiae also contains the so-called
‘yeast flavin-dependent mono-oxygenase’ yFMO
(YHR176w). This NADPH- and oxygen-dependent
enzyme contributes to the reduction of thiols
and, together with reduced glutathione, plays a
crucial role in the intracellular redox balance.189
However, it is easily conceivable that yFMO likely
makes a very poor contribution to the overall
non-respiratory oxygen consumption capacities. On
the other hand, two additional genes, YJR149w
and YLL057c, encoding the putative dioxygenases,
2-nitropropane dioxygenase and α-ketoglutaratedependent sulphonate dioxygenase, respectively,
were identified in the S. cerevisiae genome.65,171
Characterization and physiological roles
of so-called ‘cyanide-resistant respiration’
Many of the oxygen-dependent biochemical pathways previously listed (see section on Structural oxygen requirements) likely contribute to the
‘cyanide-resistant respiration’ (CNR) of S. cerevisiae. However, only a few studies (Table 7)
have been devoted to the characterization of the
molecular protagonists of ‘CNR’. As in many
of these studies, we focus here on anaerobic
cells in which ‘CNR’ is known to be favoured.
In fact, the term ‘non-respiratory (antimycin Aresistant) oxygen consumption’ (NOC) would be
more appropriate,168 since cyanide [like NaN3 ,
TTFA (thenoyltrifluoroacetone) and SHAM] can
inhibit the oxygen uptake measured in the presence
of antimycin A.8,81,149,168
Copyright  2003 John Wiley & Sons, Ltd.
1131
Contribution of haem, sterol and unsaturated
fatty acid synthesis
Both molecular and biochemical data (see section
on Structural oxygen requirements) suggest that
haem, sterol and unsaturated fatty acid de novo
synthesis contribute to the CNR capacity that preexists in anaerobic rho + cells. Haem and lipids
are required for de novo mitochondrial biogenesis
upon oxygenation of such cells. However, the
contribution made by the haem pathway to oxygen
consumption is likely minor. Indeed, yeast requires
small amounts of haem or porphyrin (Table 3)
and only 2.5 oxygen molecules are required for
the synthesis of 1 haem molecule.109 Moreover,
haem oxygenase activity is classically thought to
be absent in S. cerevisiae (see section on Transport
and degradation of haem). Nevertheless, the actual
oxygen uptake linked to the haem pathway could
not be checked in hem − mutants due to the
pleiotropic effects of the hem mutations.124,141,163
On the other hand, several data point to the
strong contribution of de novo lipid synthesis to
the CNR capacity of anaerobic cells. In such cells,
the accumulation of both b5 and P450 cytochromes
led some authors to conclude that sterol and unsaturated fatty acid (UFA) synthesis are the major CNR
systems, but this could not be demonstrated.81,165
In lipid-depleted media, it was further shown that
30–100% of the oxygen required for fermentative growth under brewing and enological conditions accounted for concomitant UFA and sterol
synthesis.99,100,145,146,169 Interestingly, the anaerobic cells in lipid-depleted synthetic musts exhibit
the highest CNR capacity168,169 (also, Rosenfeld
et al., unpublished data). In contrast, in synthetic
musts containing excess oleate and ergosterol (both
classically added to allow anaerobic growth), the
impairment of the desaturase activity (i.e. ole1
mutant) does not affect the CNR capacity. Under
these conditions, only the inhibition of the sterol
pathway (e.g. in the presence of terbinafine and
cyanide) significantly decreases the CNR (about a
25% decrease). This contribution of the sterol pathway in the CNR became almost undetectable when
cells were oxygenated for several hours.168 It has
also been shown that the stimulation of enological
fermentation that is usually observed after an oxygen pulse is abolished when the de novo sterol synthesis is impaired (i.e. erg1 mutant). This particular
role of newly synthesized sterol on fermentation
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1132
E. Rosenfeld and B. Beauvoit
Table 7. Related characteristics of non-respiratory oxygen consumption of wild-type and respiratory-deficient S. cerevisiae
cells
Growth
conditions
Carbon
source
Strain
Localization
Sensitivity
toward
Resistance
toward
Aerobic
Glucose
Glucose
Glucose
Glucose
Lab strain (WT)
Lab strain (rho0 mutant)
Lab strain (rho− mutant)
Lab strain (rho− mutant)
Cellular membranes
?
Light-microsomes
Mitochondria
NaN3 a
CO
SHAM
KCNb
KCN, SHAM, anti Aa
KCNa
KCNa
[1]
[66]
[12]
[12]
Glucose
Cellular membranes
CO, PIC
anti A
[94]
Glucose
Glucose
Lab strains (rho− , rho0
mutants)
Ind strain (Baker’s yeast)
Lab strain (WT)
Membranes + cytosol PCMB
Microsomes
anti A
KCNa
[212]
[165]
Glucose
Glucose
Ind strain
Lab strain (WT)
Cytosol
Cellular membranes
Glucose
Glucose
Glucose
Glucose
(Enol cond)
Glucose
(Enol cond)
Glucose
Lab strain (WT)
Lab strain (WT)
Lab strain (WT)
Lab strain (WT)
Oxygenlimited
Anaerobic
Reference
Cellular membranes
Cellular membranes
Microsomes
Microsomes
KCN
SHAM,
KCN, amytal, anti A, NaN3 ,
Cu2+ chelators CO, rotenone, dicoumarol
KCN, anti A
KCNa
KCNb
KCN
KCNa
anti A, myxothiazolb
DPI, Ketob
[87]
[2]
[81]
[1]
[95]
[168]
Lab strain (rho0 mutant)
Microsomes
DPI, Ketob
anti A, myxothiazolb
[168]
Lab strain (WT)
Microsomes
KCNa
[165]
WT, wild-type; Lab, laboratory; Ind, industrial; Enol cond, enological conditions; SHAM, salicyl hydroxamate; anti A, antimycin A; PIC, phenyl
isocyanate; PCMB, p-chloromercuribenzoate; DPI, diphenylene iodonium; Keto, ketoconazole.
In most cases, poison effects were determined on subcellular fractions.
a Effect determined on whole cells.
b Effect was simultaneously determined on whole cells and subcellular fractions. In all cases, inhibitory effects were not complete. KCN and
SHAM were classically employed at 0.25–1 mM and 0.1–3 mM, respectively. In references81,94,165,212 the involvement of microsomal P450
systems (that comprise CPR1 and/or NADH-b5 reductase, and b5 and P450 cytochromes) in high-affinity oxygen uptake was suggested but
was not quantified in vivo.
capacity undergoes positive effects on cell viability
when ethanol accumulates.169
Contribution of other NAD(P)H-dependent
pathways
Stored carbohydrates and exogenous hexoses seem
to be the sole oxidizable carbon substrates for
the CNR systems that pre-exist in anaerobic
cells grown under laboratory or enological conditions.81,168,183 Neither monocarboxylic acids nor fermentation end-products activate CNR. It has been
hypothesized that both NADH and NADPH are
potential electron donors of oxygen-consuming
systems.2,94,212 However, it was recently shown
(in cells grown under anaerobic enological conditions shifted to aerobic conditions) that CNR
systems (including sterol synthesis) are, both
in vivo and in vitro, more dependent on NADPH
than on NADH.168 The metabolic pathway that
Copyright  2003 John Wiley & Sons, Ltd.
provides NADPH to the CNR systems is still not
characterized.
Most of the numerous NAD(P)H-dependent oxygen utilization pathways previously listed (see
section on Structural oxygen requirements) may
participate in the respiratory adaptation phenomena. Many genes involved in oxygen-dependent
steps are upregulated under anaerobic conditions
or, in contrast, can be rapidly induced upon
oxygenation. However, many of these pathways
require small amounts of oxygen. These include
the oxygen-dependent steps of sphingolipid and
phytoceramide metabolism, and those of nicotinic
acid, ubiquinone and (erythro)ascorbate biosynthesis. The contribution of proline oxidase (a
potential source of H2 O2 ) and other promitochondrial pathways is also likely low because:
(a) CNR are not affected by the absence of
proline in the medium (Table 7 and references
therein); and (b) promitochondria account for less
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Non-respiratory utilization of oxygen by S. cerevisiae
than 1% of the CNR capacities of anaerobically
grown cells.170,204,205 However, uncertainty still
remains about the contribution of the potential NADPH-dependent soluble oxidases, OYE2,
OYE3, YHB1 and YNL234w. Indeed, anaerobic cell fractionation experiments (under classical
laboratory or enological conditions) elicited high
NADPH-dependent oxygen consumption activity
in the cytosolic fractions, whose activity might
rather be due to chemical oxygen reduction.87,94,168
Moreover, as previously discussed (see section on
Yeast haemoglobins), it has been demonstrated that
YHB1 is not involved in CNR.27,213 Nevertheless,
the potential contribution (even low) of OYE(s) and
haemoglobins to CNR after a transition from anaerobic to aerobic conditions remains to be examined
in detail. Also unclear is the contribution of plasma
membrane oxidases listed in the section on Structural oxygen requirements. While many authors
have concluded that CNR is mainly localized in
the microsomes (Table 7), some have suggested
that CNR is localized in the plasmalemma.1 However, it was recently shown under enological conditions that the inhibition extent of cellular CNR by
capsaı̈cine (250 µM) and chloroquine (1 mM), two
specific inhibitors of NAD(P)H oxidases,139,173,174
is quite low.168 Taken together, these observations
suggest that both the NADPH-dependent ferrireductase system and NADH oxidases of the plasmalemma contribute little to CNR. The contribution of the peroxisomal acyl-CoA oxidase (POX1)
is also likely to be small, since the growth conditions that favour CNR (i.e. anaerobiosis and glucose repression) are those that repress POX1.
The major contribution of uncoupled P450
systems
In mammals, it is well known that NADPH-P450
reductase and cytochrome P450 reduce O2 to produce ROS (H2 O2 , O2 ž− ) and ROS plus H2 O,
respectively.70,92,154 This electron leakage is generally high in the microsomes, even when P450
systems are partially coupled to monooxygenations
of lipidic substrates. The contribution of uncoupled microsomal P450 systems to the CNR capacity
of anaerobic S. cerevisiae cells has been hypothesized by some authors but has not been quantified
(Table 7 and references therein). Several reports
raise the question of the fate of molecular oxygen
during non-respiratory oxygen reduction. These
Copyright  2003 John Wiley & Sons, Ltd.
1133
studies were based on in vitro determinations of the
stoichiometry between NADPH oxidation and oxygen consumption (i.e. the NAD(P)H : O2 ratio). An
NADPH : O2 ratio tending towards 2 was obtained
by Kawaguchi et al.,94 suggesting a massive O2
reduction into H2 O. This ratio was estimated at
1 by other authors,2 suggesting that O2 reduction leads either to a massive H2 O2 production
or to a concomitant ROS (H2 O2 , O2 ž− ) and H2 O
(or hydroxylated compounds) production. More
recently, parallel measurements of NADPH, O2
and ROS showed that the NAD(P)H : O2 ratio of
1 was rather due to the concomitant production
of ROS (mainly O2 ž− ) and H2 O. For instance, in
subcellular fractions isolated from anaerobic cells
grown under enological conditions, the total ROS
production reached about half of the total oxygen
reduced by NADPH (e.g. about 4–20% becoming H2 O2 and 17–38% becoming O2 ž− for erg1
mutant).168 However, the respective contribution
of CPR1 and cytochrome P450 (likely ERG11) in
H2 O2 and O2 ž− production is still unknown. Interestingly, the fact that CNR systems could be a
source of ROS in vitro raises the question of the
physiological role of the ROS-detoxifying enzymes
(e.g. catalase, superoxide dismutase, glutathione
peroxidase) that are present in anaerobic cells.36,147
Indeed, the basal level of such enzymes in anaerobic cells may minimize the oxidative damage
caused by CNR systems upon oxygenation. Moreover, the relative level of CPR1, NADH-b5 reductase and cytochrome b5, as well as interactions
between these redox partners, may strongly influence the extent of electron leakage, e.g. cytochrome
b5, via a redox effect, may increase the coupling level at the expense of H2 O2 production.154
Since different CPR1 : b5 : P450 ratio values have
been reported in anaerobic S. cerevisiae cells
grown under classical laboratory or enological
conditions,80,81,87,168,187,212 the contribution of
uncoupled P450 systems might strongly depend on
the yeast strain and on the anaerobic growth conditions used.
In sum, the major enzymatic systems responsible for the non-respiratory oxygen uptake capacity
of anaerobically grown cells are: (a) the oxygenutilizing step of the de novo sterol biosynthetic
pathway; and (b) electron leakage due to P450
system uncoupling. In cells grown under aerobic or oxygen-limited conditions, the contribution of uncoupled P450 systems to the oxygen
Yeast 2003; 20: 1115–1144.
1134
uptake capacity have not yet been quantified. However, several data may suggest their involvement
in glucose-grown respiratory-deficient cells,12,66,94
and in strongly glucose-repressed wild-type cells
(harbouring a low but not negligible respiratory
activity under aerobic conditions).1,165,168,170,212
Future in vitro and in vivo studies of null mutants
affected in the P450 systems (e.g. CPR1, ERG5,
ERG11) will certainly reveal other unknown protagonists of non-respiratory oxygen uptake in
S. cerevisiae.
Oxygen sensing in S. cerevisiae: current
knowledge and perspectives
Expression pattern of aerobic and anaerobic
(‘hypoxic’) genes
In Saccharomyces cerevisiae, it is widely documented that the genes that are only expressed
under aerobic conditions and are repressed under
low oxygen tension include the nuclear and mitochondrial genes encoding for proteins involved
in mitochondrial oxidative phosphorylation (e.g.
cytochromes, subunits of respiratory chain complexes) and in protection against oxidative damage
(e.g. catalases, superoxide dismutases). Conversely,
there are genes (frequently termed ‘hypoxic’ genes)
that are expressed at low levels aerobically and
are induced by anaerobiosis, encoding proteins
involved in haem, sterol and unsaturated fatty
acid synthesis (i.e. ERG11 and CPR1, OLE1
and HEM13 ; extensive reviews are available28,103,159,216 ). Moreover, DNA array analysis has
recently shown that nearly one-sixth of the genome
is differentially expressed with respect to oxygen
tension, the majority of these genes (i.e. more than
65%) being downregulated under anaerobiosis.105
These authors classified the anaerobically induced
genes (i.e. 346 genes) into four major functional
categories: (a) cell wall-related, 42 genes; (b) cell
stress, 35 genes; (c) carbohydrate metabolism, 31
genes; and (d) lipid, fatty acid and isoprenoid
metabolism, 28 genes. For the yeast strain and
under the growth conditions used by the authors,
the latter class contains half of the haem-biosynthetic genes and nearly all of the genes involved in
the sterol biosynthetic pathway downstream from
farnesylpyrophosphate.105
In addition, there are several homologous gene
pairs in which one is expressed aerobically and the
Copyright  2003 John Wiley & Sons, Ltd.
E. Rosenfeld and B. Beauvoit
other under anaerobic conditions. These include
isoenzymes of the sterol biosynthetic enzyme
3-hydroxy-3-methyl-glutaryl-CoA reductase (i.e.
HMG1 and its anaerobic counterpart HMG2), those
of the subunit V of cytochrome oxidase (i.e.
COX5a and COX5b) and those of the ADP/ATP
translocator (i.e. AAC2 and AAC3103,159 ; see ref.
105 for the identification of more gene pairs).
Some of the enzymes of the sterol, haem and
unsaturated fatty acid synthesis that are induced
upon oxygen limitation require oxygen as a substrate. The increase in their levels may presumably maintain the flux of product formation in
their respective pathways. However, under anaerobic conditions, the increased expression of these
genes has no metabolic purpose, so cell growth
under anaerobiosis requires the addition of anaerobic factors in the culture medium. Nevertheless, it has been postulated that the induction of
oxygen-dependent enzymes under anaerobic conditions could be an adaptation of anaerobic cells upon
subsequent oxygenation of the culture medium.
Indeed, the anabolic capacity of anaerobic cells
may somehow trigger oxygen sensing during a
pulse of oxygen or a prolonged aerobic exposure.
However, the amplitude and duration of the ‘signal’
generated by the anabolic reactions upon oxygen
addition is likely to depend on both the pool size
of the intermediary metabolites of each pathway, as
well as on the affinity to oxygen of the respective
oxygen-utilizing enzymes. In this respect, the oxygen affinity of purified enzymes has been shown
to vary from about 0.1 µM [i.e. coproprophyrinogen III oxidase (Hem13p) and protoporphyrin IX
oxidase (Hem14p109 )] to about 4 µM [i.e. squalene epoxidase (Erg1p)82 ]. By extrapolating these
in vitro data to in vivo situations, it may be concluded that the haem- and the sterol-biosynthetic
pathways differentially respond to an increase in
oxygen tension. However, the apparent oxygen
affinity of CNR of anaerobic cells grown under
classical laboratory or enological conditions was
recently measured and no significant difference was
detected between wild-type and various mutants
(e.g. erg1, ole1, rho0 ).168
Role of haem concentration in oxygen sensing
Several features of the haem biosynthetic pathway
indicate why haem is an ideal effector molecule to
sense oxygen. First, in contrast to prokaryotic cells,
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Non-respiratory utilization of oxygen by S. cerevisiae
haem synthesis in eukaryotic cells has an absolute requirement for oxygen for its biosynthesis.
Molecular oxygen serves as an electron acceptor
in two steps (see section on Haem biosynthesis
and haemoproteins). Second, all the enzymes of the
pathway are present in anaerobic cells. Third, the
coproporphyrinogen III oxidase is considered as the
rate-limiting step of the pathway with a high affinity for oxygen.109 Fourth, in mutants with a defect
in their haem synthesis, the haem deficiency mimics the effects of oxygen deprivation on the gene
transcription, i.e. anaerobic genes are upregulated
and aerobic genes downregulated. Fifth, addition
of exogenous haem to anaerobic culture reverses
the effect of low oxygen tension on both aerobic
and anaerobic genes (see ref. 159 and references
therein). Consequently, a simplistic model of haemdependent regulation of aerobic genes has been
established on the assumption that haem levels may
increase during oxygenation of anaerobic cultures
and decrease as oxygen becomes limiting.216
The molecular basis of this regulation model
has been strengthened by the discovery of transcription factors whose activity and expression are
dependent on the intracellular level of haem. Five
genes, HAP1–HAP5, have been found to encode
proteins required for activation of aerobic gene
expression in response to haem. HAP1 encodes a
transcriptional activator that plays a central role in
haem sensing. The regulatory complex consists of
a homodimer of Hap1p that contains haem-binding
and DNA-binding domains. Binding of haem on
Hap1 allows transcriptional activation of specific
aerobic target genes that contain upstream activation sites specific for Hap1 binding. HAP2, HAP3,
HAP4 and HAP5 encode proteins that function as
a heterotetrameric transcriptional complex to activate the transcription of aerobic genes containing
a specific upstream sequence, in the presence of
haem. The mechanisms of Hap-mediated activation of aerobic genes, as well as the genes regulated by these regulatory complexes, have been
extensively detailed.28,103,159,192,216 At least one
additional transcriptional factor, HDS-binding factor, regulates expression of aerobic genes of yeast
in a haem-dependent fashion.159 Another gene,
ROX1, has been described to be involved in the
repression of anaerobic genes under aerobic conditions. ROX1 encodes a repressor protein that binds
to a specific sequence of the anaerobic genes and
represses transcription. It should be pointed out that
Copyright  2003 John Wiley & Sons, Ltd.
1135
ROX1 is an oxygen- and haem-dependent gene,
since its expression is under the control of the
Hap1 transcriptional factor. Thus, the regulation
of aerobic and anaerobic genes by haem content
can be described simplistically through the activity
of these two regulatory proteins: when oxygen is
available and haem is synthesized, it binds to Hap1,
thus inducing aerobic genes, including ROX1,
whose product represses anaerobic genes. When
oxygen becomes limiting, haem levels decrease,
and Hap1 activation of aerobic genes, including
ROX1, decreases, thus permitting the expression
of anaerobic genes. However, such a regulatory
scheme requires the cellular levels of Rox1p to
be strictly controlled. This is achieved by another
regulatory loop that is a repression by Rox1p of
its own synthesis. Moreover, a feedback regulation strictly based on the Hap1p/Rox1p balance
has to take into account the fact that haem, sterol
and fatty acid synthesis are required under aerobic
conditions. Therefore, some anaerobic genes, such
as HEM13, ERG11, CPR1, HMG1-2 and OLE1,
whose functions are required under aerobic conditions, are only partially repressed by Rox1p in the
presence of oxygen. Moreover, DNA array analysis of rox1 null mutant showed that only one-third
of the anaerobically induced genes are regulated by
Rox1p. On the other hand, some genes containing
putative ROX1-binding sites in their promoters are
not always upregulated in rox1 mutants, depending on the experimental design, strain and culture
medium used.105 Therefore, this differential regulation of anaerobic genes requires a further complex
regulatory circuit with additional elements. This is
in agreement with the finding that the repression by
Rox1p is in fact mediated through a general repression complex formed by other regulatory proteins
(e.g. Tup1, Ssn6) whose complex may modulate
the affinity of Rox1 for its binding site on target genes.215 In addition, promoter searches have
shown that nearly one-third of the anaerobically
induced genes contain a consensus binding site
for the Ucp2 transcription factor.105
Role of haemoproteins during
aerobic–anaerobic transitions
Several reports suggest that the haem intracellular level per se is not the only signal involved
in the regulation of gene expression by oxygen.
First, despite the difficulties in measuring its free
Yeast 2003; 20: 1115–1144.
1136
intracellular level, it has been suggested that the
induction of HEM13 gene by oxygen limitation
results in significant haem accumulation, even at
low oxygen tensions.159,165 Second, several genes
are still modulated by oxygen tension in the culture medium at oxygen concentrations well above
the apparent affinity of oxygen-utilizing enzymes.
The data obtained for cytochrome c and COX
genes show that aerobic genes and two anaerobic isoform genes (i.e. COX5b, CYC7 ) are not
regulated collectively at a single oxygen concentration but that they have different thresholds for
activation30,31 (for review, see ref. 159). This pattern of gene expression led the authors to hypothesize that the haem-redox state, in addition to
its absolute intracellular content, could modulate
the expression of oxygen-sensitive genes. Thus,
in yeast, haem may also function as a redoxsensitive component of a haemoprotein oxygen
sensor.
This hypothesis was recently supported by the
finding that carbon monoxide and transition metals, which compromise the ability of haemoproteins to bind O2 , block the induction of a subset of anaerobic genes. For instance, CO blocks
the anaerobic induction of three anaerobic genes
(e.g. CYC7, OLE1 and to some extent COX5b)
but not all of them (e.g. not HEM13, HMG1,
HMG2, CPR1, ERG11 or AAC3 ). Similar results
were found for OLE1 and CYC7 genes by adding
transition metals such as cobalt or nickel.104 More
recently, another report showed that transition metals block the repression of OLE1 under aerobiosis, which led to the identification of a regulatory
sequence involved in the response to anaerobiosis
and cobalt.199
The yeast Saccharomyces cerevisiae has three
major CO binding proteins: cytochrome oxidase,
flavohaemoglobin and cytochrome P450. The use
of respiratory chain inhibitors (e.g. antimycin
A, cyanide) or mutations (e.g. cytochrome b
mutant, Rho◦ strain) showed that functionality
of respiratory chain is required for the anaerobic induction of OLE1 and CYC7. In contrast, neither inhibitors nor mutations blocked
the anaerobic induction of anaerobic genes that
are unaffected by CO (e.g. HEM13, HMG1-2,
ERG11, CPR1 ). Finally, the anaerobic induction of all anaerobic genes was unchanged in
a flavohaemoglobin-deficient mutant.104 Taken
together, these data support the hypothesis that the
Copyright  2003 John Wiley & Sons, Ltd.
E. Rosenfeld and B. Beauvoit
CO-binding haemoprotein involved in gene regulation during the transition from aerobiosis to
anaerobiosis is cytochrome oxidase and not flavohaemoglobin. It is worth noting that the oxygen threshold for the expression of anaerobic
genes is in the range of the apparent Km of
the yeast cytochrome oxidase for oxygen (about
0.25 µM).30,31 Cytochrome oxidase also seems to
be involved in the regulation of anaerobic-hypoxic
genes in a number of mammalian cells, but it is
not clear how cytochrome oxidase ‘senses’ oxygen,
as aerobic cells are shifted to oxygen-limited or
anaerobic conditions, and what signal is transduced
from mitochondria into an effect on nuclear gene
expression (see ref. 159 and references therein).
Recent studies with mammalian cells raised the
possibility that ROS are involved in anaerobichypoxic gene regulation. More recently, it has been
shown in S. cerevisiae that the transition of aerobic cells to anaerobic conditions induces transient
oxidative stress that is considerably diminished in
respiratory-deficient cells. It was suggested that
ROS produced by the respiratory chain and their
action on macromolecules (oxidative damage on
mitochondrial DNA and carbonylations of cytosolic and mitochondrial proteins) would initiate a signalling pathway required for the induction of some
anaerobic nuclear genes (see ref. 48 and references
therein). To our knowledge, the involvement of the
third major CO-binding haemoprotein, cytochrome
P450, in oxygen sensing has never been examined
in S. cerevisiae. Interestingly, P450s have apparent Km values for oxygen in the same range as
cytochrome c oxidase.92,118 Hence, the involvement of both ERG11 (during anaerobic–aerobic
transitions) and ERG5 (during aerobic–anaerobic
transitions) in gene regulation is not excluded, since
these P450 enzymes are differentially expressed in
anaerobiosis vs. aerobiosis.
Role of neo-synthesized sterols and
unsaturated fatty acid during
anaerobic–aerobic transitions
Recently, the adaptation to oxygen of null mutant
cells, erg1 (whose growth requires ergosterol and
the absence of oxygen) and ole1, grown under
anaerobic enological conditions, was analysed. In
contrast to the wild-type and ole1 mutant, a strong
decrease in erg1 cell viability was observed upon
oxygenation (as for the rho0 strain) and no respiratory chain induction (measured by means of the
Yeast 2003; 20: 1115–1144.
Non-respiratory utilization of oxygen by S. cerevisiae
antimycin A sensitivity of oxygen uptake) could be
detected in the remaining viable cells168 (unpublished data). This was observed in the presence
or not of oleate and ergosterol in the incubation
medium. Therefore, de novo sterol synthesis is
required for respiratory chain induction, whereas
de novo UFA synthesis is not. This also suggests
that ergosteryl esters cannot replace de novo sterol
synthesis for this purpose, while stored UFA (from
triacylglycerols or steryl esters) may be used for
mitochondrial biogenesis. Under such conditions,
the primary role of the de novo sterol synthesis is
unclear. It may be hypothesized that the neosynthesized sterols play a role, (a) in the assembly
of respiratory complexes, and/or (b) in the regulation of the expression of oxygen-responsive genes.
Since squalene epoxidase has a high affinity for
oxygen,82 sterol synthesis occurs at very low oxygen tension,83,165,166 so neosynthesized sterols can
be considered as putative oxygen-sensing transducers. To our knowledge, such an aspect of oxygen
sensing had never been analysed, although effects
of sterols on the ERG gene expression, the cell
cycle and the oxidative metabolism have already
been described.44
Conclusion
The thrust of this paper has been the role of
the non-respiratory pathways on the utilization of
molecular oxygen by the yeast S. cerevisiae, a facultative anaerobe devoid of mitochondrial alternative oxidase but possessing a still uncharacterized
‘cyanide-resistant respiration’. In this yeast species,
non-respiratory oxygen consumption is favoured
when respiratory functions are impaired or absent.
Moreover, the systems involved are upregulated
in anaerobic cells. Several reports have shown
that upon oxygenation of such cells, there is a
major contribution of sterol synthesis (unlike haem,
ubiquinone and UFA synthesis) and uncoupled
P450 systems in the overall oxygen uptake. However, uncertainties still remain about the involvement of other NAD(P)H-dependent microsomal
pathways, and of the O2 -dependent pathways of
plasmalemma and cytosol. Upon transient oxygenation and prior to respiratory chain induction,
it is likely that most of the oxygen is dissipated into ROS and H2 O, while a minor part is
Copyright  2003 John Wiley & Sons, Ltd.
1137
used for anabolic purposes. This has to be confirmed in the future by in vivo and in vitro analysis of oxygen fluxes in null mutants affected in
P450 systems and in other (NADPH-) oxygendependent pathways. On the other hand, little is
known about the non-respiratory oxygen uptake
of aerobic cells. The use of respiratory-deficient
cells may be a useful means to quantitate the oxygen utilization by the respective anabolic pathways.
However, this technical advantage vanishes if one
takes into account the fact that the impairment
of respiratory functions, either by genetic alterations or by respiratory inhibitors, has been shown
to exert a retrograde regulation on nuclear gene
expression.53
Despite the tremendous progress made during
the past decade in understanding oxygen-regulated
gene expression, further investigations by DNA
arrays and/or 2-D gels are needed to clarify the regulation of all the genes involved in molecular oxygen utilization. Several anabolic pathways (including haem and sterol synthesis) might be involved in
signal transduction during either anaerobic–aerobic
or aerobic–anaerobic transitions. Analysis of the
genome-wide responses in the corresponding null
mutants on the one hand, and in wild-type cells
treated with specific inhibitors on the other, would
be helpful to elucidate the mechanisms of oxygen sensing. Several data (obtained from aerobic–anaerobic transition experiments) argue for the
involvement of cytochrome c oxidase and ROS
produced by the respiratory chain in a signalling
pathway required for the induction of some anaerobic genes.48,104,159 Interestingly, a possible haemoprotein sensor different from cytochrome c oxidase has been identified in mammals. This is a
multisubunit plasma membrane-bound cytochrome
b NAD(P)H oxidase, capable of reducing oxygen to ROS, that has been proposed to be a
component of a signal transduction pathway (see
ref. 159 and references therein). Since yeast cells
exhibit high apparent affinity for O2 regardless
of the functionality or not of the respiratory
chain or of the sterol biosynthetic pathway168
(unpublished data), it cannot be ruled out that
uncoupled P450 systems and other uncharacterized membrane-bound NADPH-oxidases also participate in a specific signal transduction pathway
in S. cerevisiae. This oxygen-sensing mechanism
based on non-respiratory ROS production has not
yet been investigated in S. cerevisiae.
Yeast 2003; 20: 1115–1144.
1138
E. Rosenfeld and B. Beauvoit
Acknowledgements
The authors thank Dr R. Cooke for his contribution to
editing the manuscript.
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