THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 274, No. 39, Issue of September 24, pp. 27590 –27596, 1999
Printed in U.S.A.
Oxidative Stress and Iron Are Implicated in Fragmenting Vacuoles
of Saccharomyces cerevisiae Lacking Cu,Zn-Superoxide Dismutase*
(Received for publication, April 19, 1999, and in revised form, July 15, 1999)
Laura B. Corson‡§, Janet Folmer¶, Jeffrey J. Strain¶, Valeria C. Culotta¶,
and Don W. Cleveland§i**
From the ‡Predoctoral Program in Human Genetics, Johns Hopkins University, Baltimore, Maryland 21205,
the §Ludwig Institute for Cancer Research, La Jolla, California 92093, the ¶Department of Environmental Health
Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, Maryland 21205, and the
iDepartments of Medicine and Neuroscience, University of California at San Diego, La Jolla, California 92093
The absence of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) is shown here to cause vacuolar
fragmentation in Saccharomyces cerevisiae. Wild-type
yeast have 1–3 large vacuoles whereas the sod1D yeast
have as many as 50 smaller vacuoles. Evidence that this
fragmentation is oxygen-mediated includes the findings
that aerobically (but not anaerobically) grown sod1D
yeast exhibit aberrant vacuoles and genetic suppressors
of other oxygen-dependent sod1 null phenotypes rescue
the vacuole defect. Surprisingly, iron also is implicated
in the fragmentation process as iron addition exacerbates the sod1D vacuole defect while iron starvation
ameliorates it. Because the vacuole is reported to be a
site of iron storage and iron reacts avidly with reactive
oxygen species to generate toxic side products, we propose that vacuole damage in sod1D cells arises from an
elevation of iron-mediated oxidation within the vacuole
or from elevated pools of “free” iron that may bind nonproductively to vacuolar ligands. Furthermore, additional pleiotropic phenotypes of sod1D cells (including
increased sensitivity to pH, nutrient deprivation, and
metals) may be secondary to vacuolar compromise. Our
findings support the hypothesis that oxidative stress
alters cellular iron homeostasis which in turn increases
oxidative damage. Thus, our findings may have medical
relevance as both oxidative stress and alterations in
iron homeostasis have been implicated in diverse human disease processes. Our findings suggest that strategies to decrease intracellular iron may significantly
reduce oxidatively induced cellular damage.
Aerobic organisms are chronically exposed to potentially
harmful reactive oxygen species generated as by-products of
cellular metabolism. One antioxidant enzyme Cu,Zn-superoxide dismutase 1 (Sod1p) plays an important role in detoxifying
superoxide radicals (O2. ). Superoxide radicals can oxidize ironsulfur cluster proteins liberating iron (1– 4). Thus, Sod1p-deficient yeast (sod1D yeast) may have increased pools of reactive
iron (5) as has been shown for SOD1-deficient Escherichia coli
* This work was supported in part by National Institute of Health
Grants NS 27036 (to D. W. C.) and GM 50016 (to V. C. C.) and the Johns
Hopkins University National Institute on Environmental Health Sciences Center. The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
** Supported by the Ludwig Institute for Cancer Research. To whom
correspondence should be addressed: Ludwig Institute for Cancer Research, 9500 Gilman Dr., La Jolla, CA 92032-0660. Tel.: 619-534-7811;
Fax: 619-534-7659; E-mail: [email protected].
1
The abbreviation used is: SOD, superoxide dismutase.
(4). “Free” iron can react with hydrogen peroxide (H2O2), and
possibly other reactive oxygen species, to generate toxic hydroxyl radicals (OH2) by Fenton chemistry (6, 7). Hydroxyl
radicals have the potential to damage proteins, nucleic acids,
and membranes (4, 8 –12).
In recent years, insight into oxidative defense, and SOD1 in
particular, has come from studies using the unicellular eukaryote, Saccharomyces cerevisiae. Yeast lacking Sod1p grow
slowly in air (13), are sensitive to superoxide generating agents
(e.g. paraquat) (13, 14), and exhibit as yet poorly understood
metabolic and biosynthetic defects (13–17). To further our understanding of oxidative damage to the cell, we examined the
effects of superoxide dismutase deficiency on overall cell structure and organelle morphology and asked whether any such
abnormalities may be tied into iron-mediated toxicity. We report here that oxidative stress to sod1D yeast results in fragmentation of the vacuole, an organelle analogous to the lysosome and thought to play a role in iron homeostasis (Refs. 18
and 19). Furthermore, we find lowering iron availability significantly reduces damage to the vacuole, consistent with disruption of iron handling as part of the pathway through which
Sod1p deficiency causes vacuole fragmentation.
EXPERIMENTAL PROCEDURES
Yeast Strains, Media, and Growth Conditions.—The S. cerevisiae
strains used in these studies are listed in Table I. sod1D strains were
generated by substituting the SOD1 coding region with SOD1 replacement plasmids pKS1 (sod1D::LEU2) or pKS3 (sod1D::TRP1) as described previously (20). Strains for morphological analysis were propagated in YPD medium (1% yeast extract, 2% peptone, 2% glucose) or in
YPG medium (1% yeast extract, 2% peptone, 2% glycerol) in air or in
CO2-enriched anaerobic culture jars shaking at 100 –120 rpm at 30 °C.
For iron starvation studies, 50 mM of the iron chelator bathophenanthrolinedisulfonic acid was added to YPD, and for iron overload studies 2
mM FeCl3 was added to YPD.
Electron Microscopy—For ultrastructural analysis of yeast by electron microscopy, strains were maintained in log phase for 24 h prior to
harvesting. Cells were pelleted, washed in 0.9 M NaCl, washed twice in
0.1 M sodium cacodylate, pH 7.2, fixed in 3% gluteraldehyde, 0.1 M
cacodylate at room temperature for 3 h, washed three times in 0.1 M
cacodylate, post-fixed in 4% KMnO4, 0.1 M cacodylate for 1 h at 4 °C,
washed several times in water, and incubated in 2% uranyl acetate for
1 h at room temperature. Cells were dehydrated with 10-min serial
ethanol washes (35, 50, 70, 95, and four times in 100%) and infiltrated
with 50% spurrs, 50% ethanol on a rotator overnight at 4 °C. Cells were
pelleted, resuspended in 100% spurrs for 2 h on rotator at 25 °C,
repelleted, and resuspended in 100% spurrs an additional 2 h at room
temperature. The cell pellet was transferred to capsules, overlaid with
fresh spurrs, and polymerized at 65 °C for 24 – 48 h. Thin sections were
cut, stained with lead citrate, and observed on an Hitachi HU12A
transmission electron microscope.
Vital Staining of Vacuoles—For analysis of yeast vacuoles by light
microscopy, strains were maintained in log phase for at least 10 h prior
to harvesting. Procedures for vital vacuole membrane staining using
27590
This paper is available on line at http://www.jbc.org
Vacuole Fragmentation in sod1 Null Yeast
27591
TABLE I
Yeast strains used in the morphological analysis of sod1D yeast
Yeast strain
Genotype
Source
1783
KS104
KS105
JS001
JS002
KS109
MATa
MATa
MATa
MATa
MATa
MATa
leu2
leu2
leu2
leu2
leu2
leu2
his4
his4
his4
his4
his4
his4
trp1
trp1
trp1
trp1
trp1
trp1
ura3
ura3
ura3
ura3
ura3
ura3
sod1D::LEU2
sod1D::TRP1
sod1D::LEU2 sod2D::URA3
sod1D::URA3
sod1D::TRP1 pmr1D::LEU2
Ref. 34
K. Slekar and V. Culotta, unpublished
Ref. 34
J. Strain and V. Culotta, unpublished
Ref. 61
Ref. 34
SEY6210
BHY152
TVY1
SEY6210-sod1
TVY1-sod1
MATa
MATa
MATa
MATa
MATa
leu2
leu2
leu2
leu2
leu2
his3
his3
his3
his3
his3
trp1
trp1
trp1
trp1
trp1
ura3
ura3
ura3
ura3
ura3
lys2
lys2
lys2
lys2
lys2
Ref. 62
Ref. 63
T. Vida, unpublished
This study
This study
YPH250
YPH250-fet3
SL202
SL203
MATa
MATa
MATa
MATa
leu2
leu2
leu2
leu2
his3
his3
his3
his3
trp1
trp1
trp1
trp1
lys2
lys2
lys2
lys2
ade2
ade2
ade2
ade2
suc2
suc2
suc2
suc2
suc2
vps5D::HIS3
pep4D::LEU2
sod1D::TRP1
pep4D::LEU2 sod1D::TRP1
ura3
ura3 fet3D::TRP1
ura3 sod1D::LEU2
ura3 fet3D::TRP1 sod1D::LEU2
FM4-64 (Molecular Probes) were as described previously (21). Briefly,
cells were harvested and resuspended in YPD medium 1 30 mM FM4-64
for 15 min at 30 °C. Cells were pelleted, resuspended in fresh YPD
medium, and incubated at 30 °C for an additional 2 h, placed on slides
and viewed with standard fluorescence microscopy. Procedures for
quinacrine staining were as described previously (22). Logarithmically
growing cells were pelleted, resuspended in YPD, 50 mM Na2HPO4, pH
7.5, with 200 mM quinacrine and incubated at 30 °C for 5 min. Cells
were pelleted, washed, and viewed on slides using standard fluorescence microscopy. All fluorescent images were photographed using a
MetaMorph Imaging System (Universal Imaging Corp.) in conjunction
with a CCD camera (Princeton Instruments).
Vacuolar Protein Trafficking—Trafficking of carboxypeptidase Y was
assayed as described previously (23). Briefly, isogenic strains 1783
(SOD1) and KS105 (sod1D) and isogenic strains SEY6210 (VPS5) and
BHY152 (vps5) were pulse labeled with Trans35S-label (ICN) for 10
min. Carboxypeptidase Y was immunoprecipitated from cell lysates,
electrophoresed on an SDS-polyacrylamide electrophoresis gel, and visualized by fluorography.
pH, Metal, and Starvation Sensitivity Assays—To assay the ability of
yeast to grow over a broad pH range, 1783 and KS105 yeast strains
were diluted to an OD600 of 0.05 in minimal medium (synthetic dextrose
supplemented with lysine, methionine, cysteine, leucine, tryptophan,
histidine, and uracil) (24) which was adjusted to the indicated pH
(covering a range from 1.0 to 8.5 in 0.5 increments). Cultures were
incubated overnight, and culture growth was measured by optical
density.
To assay zinc sensitivity, SOD1 and sod1D yeast were grown overnight in minimal medium, diluted back to an OD600 of 0.1 in minimal
medium, inoculated with the appropriate concentration of zinc chloride,
permitted to grow for 20 h in air or in an anaerobic chamber, and
culture growth was measured by optical density.
Starvation sensitivity was assayed as described previously (25). 1783
and KS105 yeast were maintained in log phase for 8 h in synthetic
complete medium (24). 108 cells were pelleted by centrifugation, washed
in water, resuspended in minimal sporulation media or water, and
incubated at 30 °C for 7 days. Cells were counted with a hemocytometer. 500 cells from each culture were plated out onto YPD medium and
permitted to grow for 3 days at 30 °C. The number of colonies formed
per 500 cells was taken as the measurement of cell survival.
RESULTS
Aberrant Vacuole Morphology in Cu,Zn-Superoxide Dismutase-deficient (sod1D) Yeast—To determine effects of oxidative stress on cell morphology, aerated wild-type (1783) and
sod1D (KS105) yeast were examined using electron microscopy
(Fig. 1). Because mitochondria are thought to be a major source
of superoxide radicals (e.g. Refs. 17 and 26), with leakage of
electrons from the respiratory electron transport chain converting up to 5% of consumed oxygen into oxygen radicals (8), we
anticipated mitochondrial abnormalities in sod1D yeast. However, the mitochondria of sod1D mutant yeast (arrowheads,
Fig. 1, C and D) were indistinguishable from those in wild-type
cells (arrowheads, Fig. 1, A and B). What was striking about
Ref. 64
D. Kosman, SUNY, Buffalo
Ref. 64
S. Lin and V. Culotta, unpublished
FIG. 1. Electron microscopic analysis reveals aberrant vacuole
morphology in sod1D yeast. SOD1 (1783) and sod1D (KS105) yeast
were grown in rich media containing glycerol (YPG) to induce mitochondrial proliferation and respiration. Labels are N, nucleus; and V, vacuole. Arrowheads point to mitochondria. Arrows point to aberrant membrane-bound vesicles frequently found in sod1D yeast.
the ultrastructure of the sod1D yeast was the apparent fragmentation of the yeast vacuole. While the wild-type strain
(1783) typically exhibited 1–2 large vacuoles (Fig. 1, A and B),
the sod1D mutant yeast (KS105) exhibited 5–15 smaller vacuoles in cross-section (Fig. 1,C and D). Staining such yeast with
a vacuole-specific fluorescent dye quinacrine revealed a similar
fragmented appearance (see Fig. 2). Imaging through multiple
focal planes with a fluorescence microscope revealed that the
sod1D yeast contained 25–50 small, individual vacuoles per cell
compared with 1–3 vacuoles in wild-type yeast. Electron micrographs demonstrated that the electron densities of many
smaller sod1D vacuoles were comparable to full-size wild-type
vacuoles indicating that the vacuolar concentration of glycosylated molecules (which contribute significantly to the electron
density (27)) was similar. However, some membrane-bound,
non-uniform, electron transparent vesicles (arrows, Fig. 1D)
were also evident in the vicinity of the vacuoles and may
represent vacuole remnants or other aberrant organelles in the
sod1D yeast.
Vacuole Fragmentation in sod1-deficient Yeast Is Oxygen
Dependent—Because both the vacuole and Sod1p have been
implicated in copper homeostasis, an initial hypothesis was
that the aberrant vacuole morphology may reflect altered copper handling in sod1D yeast. Sod1p was identified in a genetic
screen for copper-binding metallothioneins and was demon-
27592
Vacuole Fragmentation in sod1 Null Yeast
FIG. 2. Vacuole fragmentation of sod1D yeast is oxygen dependent. Quinacrine vacuole lumenal staining of SOD1 (1783) and
sod1D (KS105) grown for 10 h in rich media (YPD) in air (A and C) or in
an anaerobic chamber (B and D).
strated to buffer intracellular copper under anaerobic as well
as aerobic conditions, suggesting that the role in copper homeostasis is independent of the superoxide scavenging activity
(20). Similarly, the vacuole plays a role in copper detoxification
(28, 29). To distinguish whether the sod1D vacuolar fragmentation is due to the loss of copper buffering capability or to the
absence of superoxide scavenging activity, the morphologies of
aerobically and anaerobically grown sod1D and wild-type yeast
were compared using electron microscopy (not shown) or quinacrine vital staining to identify the vacuolar lumens (Fig. 2).
Vacuoles were fragmented when sod1D yeast were grown under aerobic conditions (Fig. 2C) but not under anaerobic conditions (Fig. 2D). The degree of vacuolar fragmentation in the
sod1D strain was exacerbated with increased aeration (data not
shown). Similarly, non-aerated sod1D vacuoles were found to
fragment within 3 h of transfer to aerated conditions, and the
fragmented vacuoles of aerated sod1D yeast recovered within a
4-h period following removal from aeration (data not shown).
Since the cell cycle is 2–2.5 h in these yeast, it would appear
that vacuolar repair is occurring over this 4-h period rather
than synthesis of new vacuoles concomitant with cell division
gradually replacing fragmented vacuoles. Altogether, these results indicate that the vacuole fragmentation is an oxygen-dependent, reversible defect presumably mediated by oxygen-free
radicals and suggest that accumulation of oxidative damage
over time is required to induce such fragmentation.
Vacuolar Fragmentation Can Be Rescued by the Addition of
Superoxide Radical Scavenging Mechanisms—To further elucidate the nature of the vacuole fragmentation, genetic suppressors of various sod1D phenotypes were employed. The first
class of suppressors function in redox active metal homeostasis
and apparently provide the cell with cytosolic superoxide scavenging activity. Mutations in PMR1, a Golgi P-type ATPase,
lead to accumulations of manganese in the cytosol (30) which
can scavenge free radicals (31–33). pmr1 mutations have been
shown to rescue all known sod1D oxygen-dependent defects
including amino acid biosynthetic deficits and paraquat sensitivity (30). As seen by fluorescent FM4-64 vacuole membrane
staining in Fig. 3C and quantified in Fig. 4, the pmr1D mutation restores 8 wild-type vacuolar morphology to the sod1D
mutant, suggesting that removal of oxygen-free radicals is sufficient to prevent vacuole fragmentation. Treatment of sod1D
yeast with 1 mM Mn21 was also sufficient to rescue fragmentation (data not shown) providing additional confirmation that
cytosolic superoxide scavenging activity is sufficient to prevent
vacuolar abnormalities.
FIG. 3. Vacuole fragmentation of sod1D yeast can be rescued
by suppressors that reduce intracellular oxygen radical levels.
FM4-64 vacuolar membrane staining of aerated yeast strains (A) wildtype (1783), (B) sod1D (KS104), (C) sod1D pmr1D (KS109), and (D)
KS104 transformed with a 2-mM TKL1 plasmid.
FIG. 4. Quantitative effect of sod1D, sod2D, pmr1D, and TKL1
on vacuole fragmentation. Strains (all isogenic to 1783) were maintained in logarithmic phase of growth in rich media (YPD) shaking at
120 rpm for 8 h, then stained with FM4-64, and the number of vacuoles
per cell counted. Values represent 300 cells for each genotype scored in
three independent experiments (;100 cells scored per genotype per
experiment). Standard deviations between experiments were less than
10%.
A second class of suppressors alleviates only a subset of sod1
null deficiencies that apparently arise from a decreased level of
NADPH in sod1D yeast (34). sod1D yeast exist in a more oxidized state than wild-type yeast presumably due to utilization
of cellular reserves of reductants (such as GSH and NADPH)
for the spontaneous reduction of reactive oxygen species (5).
Overexpression of transketolase (TKL1) stimulates generation
of NADPH by the pentose phosphate pathways and thus rescues the methionine auxotrophy and slow growth defect of the
sod1D yeast (34). Despite the ability to rescue these aspects of
the sod1D phenotype, overexpression of TKL1 does not rescue
the vacuole fragmentation (Fig. 3D). Quantification (Fig. 4)
confirms that, for the most part, vacuoles remain fragmented
with increased Tkl1p, suggesting that the fragmentation is not
related to lowered NADPH levels.
Vacuole Fragmentation Is Specific to the Cytosolic Cu,ZnSuperoxide Dismutase Deficiency—In addition to Cu,Zn SOD1
which is predominantly cytosolic, eukaryotes also have a nuclear-encoded manganese SOD2 localized within the mitochondrial matrix. To determine whether vacuole fragmentation is a
general defect of any superoxide dismutase deficiency, vacuole
morphologies of aerated wild-type (1783), sod1D (KS105),
sod2D (JS002), and sod1D sod2D (JS001) strains were com-
Vacuole Fragmentation in sod1 Null Yeast
27593
FIG. 5. Vacuolar fragmentation is specific to a deficiency in
the cytosolic Cu,Zn-SOD1. The indicated isogenic yeast strains
SOD1 SOD2 (1783), sod1D SOD2 (KS105), SOD1 sod2D (JS002), and
sod1D sod2D (JS001) were grown aerobically for 10 h in rich media prior
to staining with vacuolar membrane specific vital dye FM4-64.
pared using FM4-64 (Fig. 5), as well as by electron microscopy
(data not shown). Yeast deficient for the mitochondrial manganese SOD2 (JS002) had vacuole morphologies indistinguishable from those of wild-type yeast (1783), while sod1D (KS105)
and the double mutant sod1Dsod2D (JS001) yeast exhibited
vacuolar fragmentation (Fig. 5; quantified in Fig. 4). This provides evidence that vacuole aberrations are specific to Sod1p
deficiency and suggests that the free radicals responsible for
vacuole fragmentation are cytosolic or vacuolar in location.
Vacuole Function Is Modestly Compromised in sod1D
Yeast—To determine the potential metabolic consequences of
the aberrant vacuoles in sod1D yeast, we examined various
vacuole-related functions. The yeast vacuole, like the mammalian lysosome, is an acidic compartment containing abundant
hydrolases responsible for macromolecular degradation. In addition, the yeast vacuole plays a role in metabolite storage, pH
homeostasis, and sequestration of potentially toxic substances
such as metals (for review, see Ref. 35).
Staining with the pH-sensitive vital dye, quinacrine, indicated that the sod1D vacuoles were appropriately acidified (Fig.
2C). However, yeast lacking sod1 were unable to grow at a pH
below 3.0 or above 7.0, whereas the wild-type yeast maintained
viability over a broader pH range of 2.5 to 7.5 (Fig. 6A). This
would suggest that sod1D yeast may not be able to regulate the
cytosolic [H1] when extracellular proton levels are particularly
high or low. Indeed, other known yeast mutants with a sensitivity to pH are vacuolar mutants (27), suggesting that this
sod1D pH sensitivity is vacuole related.
The vacuole is responsible for the storage of a subset of
amino acids and serves as a nitrogen and phosphate reserve
(35). When yeast are exposed to adverse starvation conditions,
such as that which occur when yeast sporulate or enter stationary phase, they normally up-regulate vacuolar hydrolases
to turnover macromolecules to recycle necessary nutrients (36 –
38). Consistent with previous reports (16, 17), we found that
when starved for nitrogen, phosphate, or amino acids, the
sod1D yeast exhibited decreased viability (Fig. 6B). This suggests that the fragmented vacuoles may not provide adequate
stores of nutrients for starvation conditions or that vacuole-dependent macromolecular turnover processes may be compromised. This is similar to pep vacuolar mutants which also
exhibit a sporulation defect and inability to survive starvation
conditions (39).
FIG. 6. Additional sod1D phenotypes, potentially vacuole related, include increased sensitivity to pH, starvation, and zinc.
A, assay of ability of SOD1 (1783) and sod1D (KS105) yeast to grow over
a broad pH range. B, assay of ability to survive 1 week in water (amino
acid, sugar, nitrogen, and phosphate starvation) or in minimal sporulation media (sugar, nitrogen, phosphate starvation). C, assay of ability
to grow in the presence of increasing levels of zinc.
The vacuole (like the mammalian lysosome) additionally
plays a role in the sequestration of metals. Mutations in vacuolar components Pep3p and Pep5p result in aberrant vacuole
morphology and increased sensitivity to iron, cadmium, and
zinc (28). Similarly, sod1D yeast not only have aberrant vacuoles but also an increased sensitivity to iron (40), cadmium (41),
and zinc (Fig. 6C), and like the vacuole abnormalities, the
sensitivity to these metals is oxygen-dependent. Thus, metal
homeostasis and/or sequestration may be altered in these
sod1D cells. Altogether, the evidence presented here suggests
that oxidatively damaged sod1D vacuoles may be compromised
in several aspects of function.
Novel Mechanism of Vacuolar Fragmentation in sod1D
Yeast—Because aberrant vacuole morphology has been described in a number of known yeast mutants, we examined
whether the sod1D fragmentation may be occurring through a
previously identified pathway. Vacuole fragmentation has been
described in yeast mutants, known as Class B vps mutants,
which exhibit aberrant trafficking of vacuole proteins (27). To
investigate whether loss of Sod1p affects vacuolar protein trafficking, the maturation of the vacuolar protease carboxypepti-
27594
Vacuole Fragmentation in sod1 Null Yeast
FIG. 7. Aberrant sod1D vacuole morphology does not arise
from vacuole protein mistrafficking or increased autophagy. A,
carboxypeptidase Y (Cpy) forms (69-kDa Golgi precursor and 61-kDa
mature vacuolar product) immunoprecipitated from 35S-pulse labeled
yeast lysate of strains SOD1 (1783), sod1D (KS105), VPS5 (SEY6210),
and vps5 (BHY152) visualized by autoradiography after separation on
an SDS-polyacrylamide electrophoresis gel. B, FM4-64 vacuolar membrane staining of SOD1 and sod1D yeast with (PEP4) or without
(pep4D) the Pep4p protease.
dase Y was examined. Like many vacuolar proteins, carboxypeptidase Y is synthesized in the endoplasmic reticulum
(yielding a polypeptide that migrates with a mobility corresponding to 67 kDa), glycosylated in the Golgi (mobility of 69
kDa), and cleaved upon arrival into the vacuole (mobility of 61
kDa). As seen in Fig. 7A, carboxypeptidase Y maturation was
similar in sod1D mutants and in wild-type yeast indicating that
the fragmentation is not likely due to a global aberration in
vacuolar protein trafficking (as is typical of vps mutants (lane
4, Fig. 7A)) but is occurring via a different mechanism.
Autophagic yeast mutants also exhibit aberrant vacuole
morphology (42, 43). Autophagy is a process in which vesicles of
cytosolic material fuse with vesicles containing lysosomal/vacuolar hydrolases in order to degrade cellular components. We
hypothesized that aberrant sod1D vacuoles may be the manifestation of increased autophagy, stimulated to turnover oxidatively damaged macromolecules. Pep4p is a vacuolar protease required for the maturation and activation of other
hydrolases (44) and without which, autophagy is severely crippled (42). Analysis of sod1D pep4D yeast demonstrated that
elimination of Pep4p-dependent autophagy did not affect vacuole fragmentation (Fig. 7B), indicating that elevated autophagy (or at least Pep4p-dependent autophagy) is not a likely
cause of or contributor to sod1D fragmentation.
Since drug-induced microtubule disruption has been demonstrated to yield a fragmented appearance of vacuoles (45), we
tested whether sod1D vacuole fragmentation arose, at least in
part, from microtubule disruption. By immunofluorescence microscopy, we observed a normal staining pattern of microtubules (data not shown). Thus, free radical-induced microtubule
disarray is not likely responsible for sod1D vacuole fragmentation either.
Iron Is Implicated in Aberrant Vacuolar Morphology—Because iron homeostasis is altered in sod1-deficient cells and
subcellular fractionation of iron-loaded cells suggest the vacu-
FIG. 8. Vacuole fragmentation is iron-dependent. FM4-64 vacuolar membrane staining of isogenic SOD1 fet3D (YPH250-fet3) and
sod1D fet3D (SL203) yeast grown in low-iron media (A and B) (YPD 1 50
mM bathophenanthrolinedisulfonic acid), SOD1 FET3 (YPH250), and
sod1D FET3 (SL202) yeast grown in standard media (C and D) or
iron-rich media (E and F) (YPD 1 2 mM iron).
ole is a major site for iron sequestration (18, 19), one additional
explanation for vacuole fragmentation in sod1D yeast may be
that the vacuole is particularly prone to damage due to higher
levels of adverse iron-mediated reactions. Superoxide has been
demonstrated to react with labile iron-sulfur clusters of dehydratase enzymes such as aconitase and dehydrogenases (3, 46,
47) to liberate iron, which may then be trafficked to the vacuole/lysosome. Free iron can react readily with the reactive
oxygen species to generate toxic hydroxyl radicals which have
an estimated lifetime of ;2 ns and radius of diffusion of ;20 Å
in aqueous solution (reviewed in Ref. 48). Thus, if hydroxyl
radicals were generated by vacuolar iron pools, they would
induce damage primarily within the vacuole.
To determine the degree to which iron contributes to vacuole
damage, sod1D yeast were either starved for iron or loaded
with excess iron and vacuolar morphologies were examined. By
deleting Fet3p oxidase (which is thought to convert Fe21 to
Fe31 for import by the Ftr1p iron transporter (49 –51)), highaffinity iron uptake was impaired in both SOD1 fet3D and
sod1D fet3D yeast. As seen in Fig. 8B, absence of the Fet3p
transport component ameliorated fragmentation (Fig. 8B).
fet3D did not rescue other oxygen-dependent sod1D phenotypes
(such as the lysine biosynthetic defect) suggesting that fet3D
acts by decreasing intracellular (and vacuolar) iron, thereby
diminishing the iron catalyst necessary for adverse chemistry
rather than simply acting globally to reduce oxygen radicals in
sod1D yeast. Furthermore, growth in iron-rich media increased
sod1D fragmentation (Fig. 8F), again consistent with reactive
oxygen species reacting with vacuolar iron to provoke local
damage.
DISCUSSION
Because mitochondria are the primary intracellular sources
of superoxide radicals, we anticipated mitochondrial damage in
sod1D yeast. However, the most obvious sod1D defects were not
in the mitochondria, but rather in the vacuole. Oxidative stress
to Sod1p-deficient yeast leads to vacuolar fragmentation
through an iron-dependent process. Evidence that the vacuole
fragmentation is oxygen (presumably superoxide radical) mediated includes the findings that only aerobically grown sod1D
Vacuole Fragmentation in sod1 Null Yeast
yeast exhibit aberrant vacuoles and genetic supressors (such as
pmr1D) capable of rescuing all other known aerobic sod1D
deficits can also rescue this fragmentation phenotype. Iron
deprivation (as in fet3D sod1D strains) also alleviates the fragmentation without providing resistance to O2. generating reagents or ameliorating other oxygen-dependent phenotypes of
sod1D yeast. Thus, reactive oxygen species arising in the absence of Sod1p, in combination with iron, provoke damage to
vacuoles.
The notion of iron exacerbating damage under conditions of
oxidative stress has been raised previously in other contexts.
Fe21 can react with physiological levels of H2O2 (and possibly
other reactive oxygen species) to produce toxic hydroxyl radicals (reviewed in Ref. 48). In vivo, iron is not found typically in
a free state but is sequestered as iron complexes or is bound
protectively to enzymes, proteins, or iron carriers (for review,
see Refs. 48 and 52). However, superoxide can alter this cellular iron homeostasis. Several groups have found in vivo evidence that superoxide radicals oxidize labile iron-sulfur clusters to liberate iron (1, 2, 3, 46, 47) although the fate and redox
state of the “liberated” iron is not known. Additionally, genetic
screens have found that mutations in iron-sulfur cluster assembly proteins lessen oxidative damage in sod1D yeast, suggesting that reducing the number of iron-sulfur targets for
oxygen radical damage lessens damage in these oxidatively
stressed cells (53). Similarly, superoxide radicals have been
shown to oxidize iron storage components to liberate iron which
is released in a form capable of catalyzing hydroxyl radical
production (54). More generally, Wisnicka et al. (40) have suggested that superoxide radicals (in sod1D yeast) may influence
the redox status of cellular iron pools by reducing Fe31 to the
more reactive Fe21 state. Although total iron is not elevated in
sod1D yeast,2 the cellular distribution of iron, the ratio of
ferrous to ferric iron, and the level of free iron may be altered.
In any case, much evidence does suggest that oxygen radicals
liberate iron which in turn may exacerbate oxidative damage.
In yeast, the vacuole plays a role in iron homeostasis. Subcellular fractionation of iron-loaded cells suggests that the vacuole is the major site of iron sequestration (18) and yeast
lacking a recognizable vacuole structure are unable to accumulate high levels of iron (19). Thus, an iron-enriched yeast vacuole may be particularly susceptible to iron-mediated damage
in superoxide-rich sod1D yeast. One possibility is that the
vacuole damage may result from a disruption of the iron storage state in the vacuole. Under the influence of increased
superoxide levels (which perhaps may be found throughout the
Sod1p-deficient cell), vacuolar iron pools may be converted to a
more accessible and active Fe21 state that can catalyze hydroxyl radical production and damage macromolecules in the
immediate vicinity of the vacuole. A second, not mutually exclusive, possibility is that alterations in iron handling and
homeostasis within the cell may result in an increased trafficking of free iron to the vacuole. Increased vacuolar iron may
result in increased iron-mediated oxidative reactions within
the vacuole or alternatively, the increased iron may simply
bind nonproductively to various sites in the vacuole (i.e. sulfur,
nitrogen, or oxygen containing ligands). We cannot rule out the
possibility that the primary damage actually occurs to cytosolic
components that then secondarily afflict vacuolar structure
and function. Indeed, we should stress, however, that there is
not a global defect in trafficking to the vacuole or vacuole
assembly as judged by normal carboxypeptidase Y trafficking
results. In any case, both reactive oxygen species and iron
contribute to vacuole changes.
2
V. C. Culotta, unpublished results.
27595
Iron-mediated damage occurs not just in the yeast vacuole
but also in the mammalian analogue, the lysosome. Similar to
the yeast vacuole, the lysosome plays a role in intracellular iron
storage, homeostasis, and detoxification (55, 56). Iron overload
in the rat liver has been shown to increase lysosomal fragility
(56). Although lysosomal fragility has been partially attributed
to lipid peroxidation, we should stress that similar lipid membrane damage is not the cause of sod1D vacuole abnormalities
because S. cerevisiae do not synthesize the polyunsaturated
fatty acids that are susceptible to lipid peroxidation (57). Damage to vacuolar membrane proteins (as well as lysosomal membrane proteins) is a more likely possibility.
By examining structural changes in cellular architecture
caused by Sod1p deficiency, we have uncovered a plausible
explanation for various puzzling defects in the sod1D yeast:
some of the pleiotropic sod1D phenotypes may arise as secondary consequences of a compromised vacuole. For example,
sod1D yeast have a sporulation defect (16) and an increased
death rate upon entering stationary phase (17). Previously, this
sensitivity to adverse conditions was attributed to mitochondrial insufficiency (16, 17). However, although sod1D yeast do
not grow as robustly on non-fermentable carbon sources as
wild-type yeast, they nevertheless are respiration proficient
(e.g. Ref. 15). Instead, we suggest these sod1D abnormalities
may be, in part, vacuole related. When yeast are exposed to
such adverse conditions, they normally up-regulate vacuolar
hydrolases to turnover intravacuolar reserves and cytosolic
macromolecules in order to recycle necessary nutrients (36 –
38). The fact that the sod1D yeast cannot survive through such
starvation conditions may be an indication that either vacuolar
nutrient storage or the vacuolar-dependent macromolecular
turnover processes are compromised. Indeed, many known vacuolar mutants also cannot survive adverse conditions (39).
Furthermore, we demonstrate that sod1D yeast have a sensitivity to pH which cannot easily be explained without invoking
the role of the vacuole in pH homeostasis. Likewise, an increased sensitivity to transition metals may be directly or indirectly related to vacuole abnormalities. Thus, some of the
curious metabolic and biochemical abnormalities in sod1D
yeast may in fact be attributable to vacuolar aberrations.
In summary, we demonstrate that oxidative stress to sod1D
yeast results in vacuolar fragmentation and either removal of
oxygen or iron can ameliorate the damage. This may have
medical relevance in that oxidative damage and alterations in
iron homeostasis have been implicated in a number of disease
states including atherosclerosis, neurodegeneration, arthritis,
and aging (for review, see Ref. 48). In some cases oxidative
insult is thought to be the primary cause of damage. For example, brain and spinal cord deterioration after ischemic or
traumatic injury often appears excessive for the level of trauma
(58). One explanation put forth is that iron-binding capacity in
the central nervous system and in the cerebral spinal fluid
bathing the central nervous system is particularly low. Thus,
release of iron by oxidatively or mechanically damaged cells,
organelles, and proteins may catalyze toxic oxidative side reactions causing further cell injury. Consistent with this concept, preliminary trials of treatment with iron-chelating agents
have had success in diminishing post-traumatic degeneration
of brain and spinal cord (59, 60). Thus, iron-mediated oxidative
damage is likely a common event in aerobic organisms, and
dissection and understanding of the process (and ways of prevention) in yeast may give insight into human disease.
Acknowledgments—We thank Steve Gould for advice on EM analysis
of yeast and Scott Emr for advice and reagents to analyze vacuoles. We
also thank Dan Kosman (SUNY, Buffalo) for providing yeast strain
YPH250-fet3D.
27596
Vacuole Fragmentation in sod1 Null Yeast
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