Yeast plasma membrane contains an electron
transport system that maintains ascorbate in its reduced form in the
apoplast. Reduction of ascorbate free radical by this system is
comprised of two activities, one of them dependent on coenzyme
Q6 (CoQ6). Strains with defects in
CoQ6 synthesis exhibit decreased capacity for ascorbate
stabilization compared with wild type or with atp2 or
cor1 respiratory-deficient mutant strains. Both
CoQ6 content in plasma membranes and ascorbate
stabilization were increased during log phase growth. The addition of
exogenous CoQ6 to whole cells resulted in its incorporation
in the plasma membrane, produced levels of CoQ6 in the
coq3 mutant strain that were 2-fold higher than in the wild
type, and increased ascorbate stabilization activity in both strains,
although it was higher in the coq3 mutant than in wild
type. Other antioxidants, such as benzoquinone or
-tocopherol, did
not change ascorbate stabilization.
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INTRODUCTION |
All aerobic organisms are exposed to the toxic effects of reactive
oxygen species (ROS).1 These
are produced during normal metabolism and can also be generated by
exposure to pro-oxidant compounds, an increase in oxygen pressure, or
exposure to ionizing radiation (1). These ROS produce damage to many
cellular components, affecting the function of lipids, proteins, and
nucleic acids. However, in normal conditions, cells have a number of
defense systems to avoid or minimize these problems. A good example is
Saccharomyces cerevisiae, which has at least 14 proteins
that participate in ROS protection (1, 2). The majority of anti-ROS
mechanisms act inside the cell; however, little is known about
mechanisms that protect against oxidative reduction. Some metabolic
reactions involved in metal uptake produce superoxide at the apoplast
(3), such as iron reduction, which is regulated by the presence or
absence of iron in the culture medium (4). These ROS at the plasma
membrane initiate lipid peroxidation and generate a wide array of
oxidation products including shorter fatty-acyl chains. Such products
impair membrane function and structural integrity and increase the
membrane fluidity. The plasma membrane must have a defense system to
scavenge free radicals and repair oxidative damage. A good candidate
may be the redox couple ascorbate-ubiquinone. Ascorbate is a first
order antioxidant and, because it scavenges free radicals in the
aqueous phase of cells, is considered to be the terminal small molecule
antioxidant in biological systems (5). Although ascorbate is a very
efficient inhibitor of the lipid peroxidation process, it cannot
inactivate the free radical effects within the plasma membrane (6).
Recently, we showed that yeast cells have the ability to reduce
ascorbate free radical by an enzymatic mechanism that depends on NADH
as the electron donor and is inhibited by ubiquinone antagonists, such
as chloroquine (7). Ubiquinone is a hydrophobic redox molecule located
in different membranes, including the plasma membrane in animal cells
(8). The redox chemistry of CoQ is crucial for its role in the plasma
membrane electron transport system, where the ubiquinone acts as a
carrier between an internal NADH-dehydrogenase and an external side
final acceptor (9). This NADH deshydrogenase activity is attributed to
a NADH-ubiquinone reductase in the plasma membrane of pig liver
hepatocytes (10, 11). The ubiquinone present in S. cerevisiae is ubiquinone-30 (CoQ6), and yeast mutants
with defects in the COQ genes are being used to characterize
the enzymes involved in CoQ6 synthesis pathway (12-15).
Recently, its importance as an antioxidant was illustrated by the
hypersensitivity of CoQ6-deficient yeast mutants to
oxidative stress induced by treatment with polyunsaturated fatty acids
(16). The present work employs yeast mutants deficient in
CoQ6 synthesis to study the relationship between the
extracellular ascorbate stabilization and CoQ6. The results
of this study suggest that part of the ascorbate stabilization by whole
cells depends on the CoQ6 content of the plasma membrane
and can be increased by the external addition of CoQ6. Both
ascorbate stabilization and CoQ6 content in plasma membrane
can be also restored by transformation with plasmids containing the
COQ3 or COQ7 genes. Ascorbate stabilization activity and plasma membrane CoQ6 content are regulated as
a function of the growth phase. The CoQ6-independent
ascorbate stabilization is not due to CoQ6 biosynthetic
intermediates or other antioxidants but is apparently due to electron
transport by the plasma membrane ferric reductase complex. The
CoQ6-independent ascorbate stabilization is suppressed when
the coq3 mutant strain is cultured in media supplemented
with ferric iron. The results indicate that ascorbate stabilization is
due to two electron transport systems in the yeast plasma membrane, one
dependent on CoQ6 and the other dependent on the
iron-regulated ferric reductase complex.
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EXPERIMENTAL PROCEDURES |
Yeast Strains and Growth Conditions--
The yeast strains used
in this study are described in Table I.
Plasmids pRS12A2-2.5SB (13) and pNMQ71 (14) restored both CoQ
synthesis and growth on nonfermentable carbon sources in strains harboring deletions in the COQ3 and COQ7 genes,
respectively. Cells were grown on YPD medium (2% peptone, 1% yeast
extract, and 2% glucose) incubated at 30 °C with shaking (17).
Yeast harboring the plasmids pRS12A2-2.5SB and pNMQ71 were grown in synthetic complete medium (16). In experiments with iron, 2 mM Fe-EDTA was added to YPD, and the YPD minus iron was
made removing the iron with several washings with 5% hydroxyquinoline
in chloroform, pure chloroform, and ether (18).
In Vivo Assays--
The ascorbate stabilization assay was
described previously (7). Growth was monitored by determining the
A660 nm, and the cultures were collected in
late log phase (A660 nm = 3-3.5). Cells were
washed once in 5 mM EDTA, pH 8, and twice in cold water.
Ascorbate oxidation was followed by the direct reading at 265 nm, with
an extinction coefficient of 14.5 mM
1·cm
1 at pH 7.4 (5). Cells
were resuspended at 107 cells/ml in 0.1 M
Tris-HCl buffer, pH 7.4, with 0.06 µM CuSO4. The addition of ascorbate (final concentration, 0.15 mM) to
the cell suspension initiated the ascorbate oxidation due to the
presence of Cu2+. Cells were removed by centrifugation, and
the supernatants were used to measure the ascorbate oxidation rates.
Ascorbate stabilization is defined as the difference between the
oxidation rate of ascorbate in the presence of cells (and after
treatments as indicated) and the oxidation rate without cells.
Iron reduction was measured using the methods described in Ref. 19.
Cells (0.5 mg of dry weight/ml) were resuspended in reaction buffer (50 mM sodium citrate, pH 6.5, and 5% glucose). After
incubation for 10 min at 30 °C with magnetic shaking, 1 mM bathophenanthroline disulfonic acid and 1 mM
ferric chloride were added. Iron reduction was assayed by the formation
of the complex bathophenanthroline disulfonic acid-Fe(II), as monitored by absorbance readings at 535 nm with an extinction coefficient of 17.5 mM
1·cm
1.
Isolation of Plasma Membranes--
Yeast plasma membranes were
purified by disruption of cells with glass beads followed by a step
sucrose gradient (20). These preparations were used for
CoQ6 determinations. Protein was determined by the
dye-binding method modified for membrane samples with
-globulin as
standard (21).
Biochemical Markers--
Plasma membrane ATPase was measured as
the liberation of inorganic phosphate (22). Cytochrome c
oxidase activity (inner mitochondrial membrane marker) and
NADPH-cytochrome c reductase activity (endoplasmic reticulum
marker) were determined as described (23). IDPase activity (Golgi
marker) was measured as described (24). Outer mitochondrial membrane
contamination was determined measuring the presence of porin in plasma
membrane fractions by means Western blotting. Fractions were analyzed
by SDS-PAGE and subsequent transfer into nitrocelulose membrane
(Millipore). Membranes were blocked in 50 mM Tris-HCl
buffer, pH 7.0, containing 200 mM NaCl, 0.05% Tween 20, and 2% skim milk for 1 h and then incubated for 1 h with
anti-porin (polyclonal antibody, developed in rabbit and kindly
provided by V. Haucke, Biozentrum, University of Basel, Basel,
Switzerland). Membranes were incubated with
alkaline-phosphatase-conjugated anti-rabbit secondary antibody.
Coenzyme Q6 Extraction--
CoQ6
extraction of whole cells was initiated with a saponification of cell
pellets. Yeast samples (about 0.5 g of wet weight) were weighed
out and added to 10 ml of a methanolic potassium hydroxide solution (65 g of potassium hydroxide in 650 ml of 90% methanol in water)
containing 0.81 g of pyrogallol in a 40-ml saponification flask.
The mixture was heated under reflux in a water bath for 30 min and
cooled to 25 °C after leaving the flask on ice. The dark saponified
sample was filtered through a syringe with glass wool and was extracted
three times with hexane (10 ml) (2 min with shaking). The upper phase
of hexane was recovered, pooled, and then evaporated under vacuum in a
Rotavapor (Büchi, Flawil, Switzerland). The residue was dissolved
in 500 µl of ethanol.
Extraction of CoQ6 from plasma membrane samples (500 µl,
0.5-1 mg of protein) was carried out by adding an equal volume of 2%
SDS and vortexing for 1 min; then, 1 ml of 5% isopropanol in ethanol
was added, and samples were vortexed for 1 min. To recover CoQ6, 5 ml of hexane were added, and the mixture was
vortexed at top speed for 1 min and centrifuged at 1000 × g for 5 min. The upper phase was recovered, dried, and
dissolved in 200 µl of ethanol.
Coenzyme Q6 Determination--
Chromatography was
performed with a Beckman high performance liquid chromatography system
composed of two 126-2 pumps and a 168-4 detector. Data were collected
with a System Gold V810 software. The reverse phase column (Ultrasphere
C-18, 5 µm, 4.6 × 250 mm) was equilibrated in 90% methanol and
10% ethanol at 1 ml/min, and after 10 min, the sample was injected.
After data collection (20 min) the percentage of ethanol was increased
to 100% in 5 min, and then the mobile phase was returned to the
initial composition. Quantitation of CoQ6 was made by
injection of external standard of known amounts of commercial
CoQ6 (Sigma). The concentration of standard was determined
using a extinction coefficient measured using the method described
above, and showed a value of 15.33 mM
1·cm
1, in agreement with
previous work (14).
 |
RESULTS |
Measurement of Ascorbate Stabilization in Respiratory-deficient,
CoQ6-deficient, and Wild Type Yeast Strains--
The
stabilization of extracellular ascorbate was determined to be about 32 nmol/107 cells/h in the wild type strain W303.1B (Fig.
1). Mutant strains harboring a deletion
in either coq3, coq7, or coq2, and
hence unable to synthesize CoQ6, showed an ascorbate
stabilization activity that was about 65% that of wild type.

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Fig. 1.
Ascorbate stabilization is reduced in yeast
mutants that lack CoQ6. Cells were harvested during
final log phase to measure the ascorbate stabilization as described
under "Experimental Procedures." Specific activity is shown as the
mean ± S.E. of three separate experiments.
coq3::Q and coq7::Q are
strains coq3 and coq7 transformed with plasmids that carry the wild
type genes COQ3 and COQ7, respectively.
wt, wild type.
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Because coq3, coq2, and coq7 yeast are
unable to respire, the decrease in ascorbate stabilization activity
might result from the respiration-defective phenotype. To test this
possibility, a strain carrying a deletion of the ATP2 gene
(encoding the
-subunit of the mitochondrial F1 ATPase) and a strain
carrying a deletion of the COR1 gene (encoding a protein
subunit of bc1 mitochondrial complex) were studied. As shown
in Fig. 1, the ascorbate stabilization activity in the atp2
null and cor1 mutant strains was not impaired and, in fact,
was slightly higher than in the wild type. Transformation of the
coq3 and coq7 mutants with single copy plasmids
containing the COQ3 and COQ7 yeast genes,
respectively, restored the ascorbate stabilization activity to that of
wild type cells.
All strains displayed a CoQ6-independent ascorbate
stabilization activity. Because decreases in either the pH of the
buffer or the copper concentration could decrease the rate of ascorbate oxidation, these parameters were investigated. The pH was unchanged throughout the assays when run for 4 h. The property to oxidize ascorbate by buffer was abolished when copper was not added (Fig. 2). The incubation of cells for 4 h
in buffer (here named conditioned buffer) did not change its property
to oxidize ascorbate. This conditioned buffer still contained copper
and did not contain any protein released from the cells during
incubation.

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Fig. 2.
Effect of copper on ascorbate oxidation
rates. Ascorbate oxidation was followed in assay buffer with or
without copper. Conditioned buffer was established after the incubation
of 107 cells/ml for 4 h after the cells were
discarded. Also, ascorbate oxidation was stimulated in buffer
containing the CM3262 strain (wild type) and the FTRUNB1 strain, which
lack the copper transporter. Specific activity is shown as the
mean ± S.E. of three separate experiments. Full,
buffer with copper; Cond, conditioned buffer.
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When cells were present, ascorbate oxidation rates were decreased (Fig.
2) as a consequence of ascorbate stabilization at the plasma membrane
(7). Boiled cells lost the ability to prevent ascorbate oxidation.
Because copper is required to oxidize ascorbate and yeast have a high
affinity copper transporter, we checked this activity in the FTRUNB1
strain lacking copper transporter at the plasma membrane (25). This
strain showed the same ascorbate stabilization as the wild type
parental strain (CM3262). These results rule out copper uptake as
responsible for the CoQ6-independent ascorbate
stabilization.
Neither superoxide dismutase nor catalase modified the ascorbate
oxidation rates observed in the presence of cells, indicating that the
ascorbate stabilization by yeast was not due to the production of ROS
during the oxidation of ascorbate.
Determination of CoQ6 Content in Yeast Cells and Plasma
Membrane Fractions--
The concentration of CoQ6 was
measured in both whole cells and plasma membrane purified fractions of
all yeast strains harvested during the final log phase of growth. Yeast
lipid extracts were separated by high performance liquid
chromatography, and CoQ6 was identified based on its
retention time of about 17 min at 20 °C and by the characteristic
spectrum of the quinone. Wild type contained about 18 pmol of
CoQ6/mg of dry weight whole cells (Table
II). This level of CoQ6 was
25% higher than present in the atp2 strain and 33% lower
than in the cor1 strain. CoQ6 was not detected
in the coq3, coq7, or coq2 mutant
strain, but CoQ6 synthesis was restored when these strains
harbored the respective COQ3 or COQ7 genes on a
single copy plasmid.
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Table II
Concentration of coenzyme Q6 in plasma membrane fraction
and whole cells
All strains were grown in the appropriate medium, harvested in final
log phase, and processed to extract and determine CoQ6
concentration. Data (mean ± S.E. from two separate experiments)
are expressed in pmol/mg of protein in plasma membrane and pmol/mg of
dry weight in whole cells.
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The CoQ6 concentration was also determined in yeast plasma
membrane fractions. Wild type yeast atp2 and cor1
mutant strains contained about 150, 195, and 236 pmol
CoQ6/mg protein, respectively. Again, CoQ6 was
not detected in the plasma membrane fraction isolated from the
coq3, coq7, or coq2 mutant.
Different membrane markers were used to check the purity of plasma
membrane fractions (Table III). The
plasma membrane marker ATPase was highly enriched in plasma membrane
fractions compared with total membranes isolated by the sucrose
gradient method. However, endomembrane markers were greatly decreased
in these fractions. Thus, CoQ6 concentrations determined
here represent those extracted from the plasma membrane. We did not
detect porin (a marker of the mitochondria outer membrane) by Western
blotting of plasma membrane fractions with a polyclonal antibody
against yeast porin (data not shown).
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Table III
Biochemical markers of membranes to check the plasma membrane
cross-contamination with other endomembranes
The samples used were obtained using glass beads disruption and a
sucrose step gradient. All activities were measured with 20-30 µg
protein/ml of reaction volume and each assay was performed at 30 °C.
Activity data (mean ± S.E. from two or three experiments) are
expressed in nmols/mg/min. PM, plasma membrane; MF, total cell
membranes; DES, diethylstibestrol-sensitive; GA, Golgi apparatus; ER,
endoplasmic reticulum; IM, mitochondrial inner membrane.
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Measurement of Ascorbate Stabilization and CoQ6 Content
at Different Growth Stages--
Ascorbate stabilization by both wild
type and coq3 mutant strains was determined during log and
stationary phases of growth. Both strains reached stationary phase
between 9 and 12 h, although the wild type culture attained a
higher density than the coq3 mutant (Fig.
3A). Ascorbate stabilization
in wild type cells was increased during log phase and reached a plateau
at the end of log phase (Fig. 3B). Ascorbate stabilization
in the coq3 strain showed a slight increase during the first
6 h but then decayed to the initial level (Fig.
3B).

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Fig. 3.
Ascorbate stabilization by wild type and
coq3 strains during different stages of growth. YPD
cultures (500 ml) were inoculated with both strains (106
cells/ml), and the growth was monitored by absorbance at 660 nm
(A). Every 2 h, an appropriate cell culture volume was
taken to measure the ascorbate stabilization (B). Ascorbate
stabilization activity is shown as the mean ± S.E. of three
determinations. Open circles, wild type strain; closed
circles, coq3 strain.
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CoQ6 content in both total and plasma membrane fractions
increased with culture density in wild type yeast (Fig.
4). The increase in plasma membrane
content was particularly marked and followed apparently the same
pattern as the observed ascorbate stabilization activity (Fig.
3B).

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Fig. 4.
CoQ6 content in whole cells and
plasma membrane during different phases of growth.
CoQ6 concentration is shown as the mean of two
determinations (S.E. 5%) and is expressed as pmol/mg of protein in
plasma membrane fractions and as pmol/mg of dry weight in whole cells.
Open circles, wild type whole cells; closed
circles, wild type plasma membrane fractions. CoQ6 was
not detected in coq3 cells.
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Effect of External CoQ6 Addition on Ascorbate
Stabilization and CoQ6 Content--
Our results suggest
that plasma membrane CoQ6 participates in ascorbate
stabilization. To determine the effect of CoQ6
supplementation on ascorbate stabilization, both wild type and
coq3 mutant yeast were incubated with exogenous
CoQ6 (Table IV). Both wild
type and coq3 strains were cultured and harvested in mid log
phase, resuspended in buffer (108 cells/ml), and incubated
1 h at 30 °C with or without 50 µM
CoQ6. After the incubation, cells were used to determine
the ascorbate stabilization and to measure the CoQ6 content
in plasma membrane purified by sucrose step gradient. Exogenous
CoQ6 significantly increased the content of
CoQ6 in the plasma membrane of the wild type strain and
also increased the rates of ascorbate stabilization (Table IV).
Exogenous CoQ6 was incorporated in coq3 cells
and attained a concentration at the plasma membrane that was almost twice that of wild type (Table IV). Such treatment resulted in a 58%
increase in ascorbate stabilization activity in the coq3 strain.
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Table IV
Effects of exogenus CoQ6 incubation on the ascorbate
stabilization activity and CoQ6 content of plasma membrane
Cells of both strains were cultured and harvested in final log phase,
resuspended in buffer (108 cells/ml) and incubated for 1 h
at 30 °C with or without 50 µM CoQ6 50 µM benzoquinone, and 30 µM -tocopherol.
After the incubation, cells were used to measure the ascorbate
stabilization and to determinate the CoQ6 content previous
plasma membrane purification. The method was described under
"Experimental Procedures." Ascorbate stabilization data (mean ± S.E. from three separate experiments) were expressed in
nmol/107 cells/h, and CoQ6 content data (mean ± S.E. from two separate experiments) were expressed in pmol/mg protein.
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The same incubation experiments were carried out with two well known
antioxidants, benzoquinone and
-tocopherol. Neither of the two
compounds showed a significant effect on ascorbate stabilization
(Table IV).
Ascorbate Stabilization and CoQ6 Contents in Cells
Cultured in the Presence or Absence of Iron--
Ascorbate
stabilization in several strains cultured in media with or without 2 mM iron was measured (Fig.
5A). Wild type, atp2 and cor1 strains displayed high ascorbate
stabilization in iron-deprived media, and this activity was decreased
when iron was present. The ascorbate stabilization in the
coq3 strain also showed an iron-regulated ascorbate
stabilization that was almost abolished in the presence of iron. The
ferric iron reductase, measured under the same conditions as the
ascorbate stabilization, was similar in all strains (Fig.
5B) and was similarly modulated by the presence or absence
of iron. The coq3 strain displayed a higher ability to
reduce ferric iron, twice that of the other respiratory-deficient
mutant strains. The presence or absence of iron in the culture did not
significantly change the CoQ6 content in these strains
(Table V).

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Fig. 5.
Ascorbate stabilization and ferric reduction
in several strains cultured in iron presence or absence. A,
ascorbate stabilization. Cells were harvested during final log phase,
and the activity is shown as the mean ± S.E. of three separate
experiments. B, ferric reduction. Cells were harvested
during final log phase, and the activity is shown as the mean ± S.E. of three separate experiments. In both experiments, + indicates
cells cultured in YPD with the addition of 2 mM FeEDTA,
and indicates cells cultured in YPD without iron (iron was
extracted using a chemical method described under "Experimental
Procedures").
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Table V
CoQ6 contents in several strains cultured in the presence
or absence of iron
All strains were grown in YPD plus 2 mM FeEDTA or YPD with
iron extracted, harvested in final log phase, and processed to extract
and determine CoQ6. Concentration data (mean ± S.E. from
two separate experiments) are expressed in pmol/mg of protein of plasma
membrane.
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DISCUSSION |
Extracellular ascorbate stabilization is an activity present not
only in yeast but in other animal and plant cells (26, 27). In animal
cells, the function is clearly directed to the maintenance of an
optimal redox state and may be related to effects on cell growth and
differentiation (28, 29). In plants, extracellular ascorbate
stabilization plays an important role in cell elongation through
ascorbate peroxidases (30). In yeast, we recently showed that a plasma
membrane electron transport system, which depends on the viability of
intact cells or protoplasts, is responsible for ascorbate
stabilization, indicating the possible participation of plasma membrane
CoQ6 (7).
To determine the functional requirement of CoQ6 in
ascorbate stabilization, we have studied mutant strains with defects in CoQ6 synthesis. No CoQ6 was detected in the
plasma membrane or whole cells of these strains, which also showed a
lower activity of ascorbate stabilization. Wild type yeast
atp2 and cor1 (respiratory-deficient strains)
contained detectable CoQ6, although its distribution inside
the cell was different. Thus, although wild type cells had a higher
content of CoQ6 than did the atp2 mutant, the
latter contained more CoQ6 at the plasma membrane. However,
both the plasma membrane and whole cell CoQ6 content is
higher in cor1 mutant strains than in wild type cells. These
findings may account for the higher ascorbate stabilization activity in
the atp2 and cor1 strains and indicate that
ascorbate stabilization is not dependent on mitochondrial respiratory
function. An explanation of this behavior derives from the observed
increase of trans-plasma membrane electron transport in
mitochondrial-deficient animal cells, which probably functions to
regulate the ratio of cytosolic NAD+/NADH levels (31, 32).
Previous work has shown that the establishment of a
mitochondrial-deficient cell line produced increases in both plasma
membrane CoQ content and the ascorbate stabilization activity (33).
These results are all consistent with the idea that the higher
CoQ6 content in the cor1 and atp2
strains may result from the imposed respiratory deficiency.
In S. cerevisiae, plasma membrane protein represents 1-2%
of total cell protein (34). Considering this percentage, plasma membrane CoQ6 constitutes 8-16% of the total
CoQ6 in the cell. This value was increased in both wild
type and respiratory defective yeast strains after the incubation of
cells in buffer with exogenous CoQ6 (Table IV).
Yeast CoQ6 synthesis and CoQ6 content is
increased during log phase growth and reaches a maximum at stationary
phase (15). Similarly, CoQ6 content in both plasma membrane
and whole cells increased during log phase growth, but the accumulation
of CoQ6 in plasma membrane increased dramatically as
compared with whole cells. Ascorbate stabilization showed a similar
increase but reached a plateau at the stationary phase. CoQ exerts its
antioxidant function when it is reduced and requires an appropriate
equilibrium with its reductase, such as cytochrome
b5 reductase, at the plasma membrane (35). This
behavior during growth is similar to that observed for other plasma
membrane redox activities in yeast (36). The coq3 mutant
strain also showed a slight increase in ascorbate stabilization during
the first hours of log phase growth, but instead of reaching a plateau,
the ascorbate stabilization quickly returned to basal levels of
activity. Thus, this activity may be due to another component that was
increased during the growth.
Exogenous CoQ6 was incorporated in the plasma membrane of
both wild type and coq3 strains, although the latter showed
a very high capacity to incorporate CoQ6. As a consequence
of incubation with exogenous CoQ6, ascorbate stabilization
was stimulated in wild type and was restored to wild type levels in the
coq3 mutant. Exogenous CoQ stimulates the trans-plasma
membrane electron transport (9) and significantly increases the
ascorbate stabilization in animal cells (35). CoQ also acts through the
plasma membrane redox system to replace pyruvate as an essential medium
component required for the growth of mitochondria-deficient
°
Namalwa cells (32).
The data presented here show that ascorbate stabilization was not
absent in the CoQ6 mutant yeast strains and thus cannot depend exclusively on CoQ6. This
CoQ6-independent activity is not due to changes in pH or
copper uptake, because FTRUNB1 strain lacking the copper transporter
(25) shows the same ascorbate stabilization activity as the wild type
strain. The contribution of CoQ6 biosynthetic intermediates
to the CoQ6-independent ascorbate stabilization can be
excluded by the examination of the coq2 yeast mutant strain.
Such mutants are defective in transferring the polyprenyl group to
p-hydroxybenzoic acid (the aromatic ring precursor of CoQ)
and hence are incapable of generating any prenylated CoQ biosynthetic
intermediates (12). Other phenolic compounds display antioxidant
properties and may be able to reduce peroxidation damage (37, 38). For
example, 1,4-benzoquinone is a plasma membrane redox system component
thought to provide a defense against free radicals produced by the
mycelial fungus Phanerochaete chrysosporium during lignin
peroxidase-mediated mineralization (39). But the absence of an effect
produced by either 1,4-benzoquinone or the antioxidant
-tocopherol
tend to argue against the role of other antioxidants in the ascorbate
stabilization.
It seems more likely that the remaining activity is due to another
plasma membrane electron transport system in which CoQ6 is
not involved. A possible candidate is the system responsible for the
reduction of ferric iron, a necessary step in iron assimilation (40,
41). In agreement with previous studies (4), this system is modulated
by the presence or absence of iron in the culture medium (Fig.
5B). The ascorbate stabilization is also modulated by iron
in all strains examined (Fig. 5A) in a manner that is
independent of the CoQ6 content (Table V). The most
pronounced effect was displayed in the coq3 strain, in which
the ascorbate stabilization was abolished when cells were cultured in
iron-supplemented media. In this situation, the lack of
CoQ6 at the plasma membrane, combined with the repression
of iron reduction, results in the absence of ascorbate stabilization.
The data indicate that ascorbate stabilization depends on two redox
systems at the plasma membrane, one that is
CoQ6-dependent and the ferrireductase complex.
The contribution of the ferrireductase complex accounts for the
increase in ascorbate stabilization observed in the coq3
stain during log phase growth, because iron reduction was also
increased during this phase. Thus, the high level of iron reduction in
the coq3 mutant strain accounts for the stabilization of
ascorbate despite the absence of the
CoQ6-dependent system.
In conclusion, ascorbate stabilization at the plasma membrane of
S. cerevisiae is in part dependent on CoQ6,
whereas the remaining activity is due to the action of the plasma
membrane iron reductase system. Both of these systems cooperate to
maintain a reduced environment, based on ascorbate, at the
apoplast.