From the Plant Gene Expression Center, U. S. Department of Agriculture-Agricultural Research Service, Albany,
California 94710 and the § Department of Plant and Microbial
Biology, University of California, Berkeley, California 94720
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ABSTRACT |
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A cadmium-hypersensitive mutant of the fission
yeast Schizosaccharomyces pombe was found to accumulate
abnormally high levels of sulfide. The gene required for normal
regulation of sulfide levels, hmt2+, was cloned
by complementation of the cadmium-hypersensitive phenotype of the
mutant. Cell fractionation and immunocytochemistry indicated that HMT2
protein is localized to mitochondria. Sequence analysis revealed
homology between HMT2 and sulfide dehydrogenases from photosynthetic
bacteria. HMT2 protein, produced in and purified from Escherichia
coli, was soluble, bound FAD, and catalyzed the reduction of
quinone (coenzyme Q2) by sulfide. HMT2 activity was also
detected in isolated fission yeast mitochondria. We propose that HMT2
functions as a sulfide:quinone oxidoreductase. Homologous enzymes may
be widespread in higher organisms, as sulfide-oxidizing activities have
been described previously in animal mitochondria, and genes of unknown
function, but with similarity to hmt2+, are
present in the genomes of flies, worms, rats, mice, and humans.
The oxidation of sulfide can provide energy for chemolithotrophic
or photosynthetic growth of bacteria. This capacity allows some
bacteria to thrive in such unlikely environments as hot sulfur springs
and deep-sea thermal vents. Sulfide-based anoxygenic photosynthesis appeared quite early in evolution. Today, it is widespread in the green
and purple phototrophic bacteria and has been reported in cyanobacteria
(1). Enzymes mediating sulfide oxidation have been described in the
photosynthetic bacteria Chlorobium limicola (2, 3),
Oscillatoria limnetica (4), Rhodobacter
capsulatus (5), and Chromatium vinosum (6); the
complete sequence of the latter two proteins has been described.
In recent years, it has become known that sulfide oxidation is not the
province solely of bacteria. Some animals from sulfide-rich aquatic
sediments, such as the gutless clam (Solemya reidi) and the
lugworm (Arenicola marina) are able to oxidize sulfide
within their own tissues without the aid of bacterial symbionts
(7-12). This capacity allows them to detoxify sulfide that enters
their bodies from the surrounding environment that would otherwise
poison aerobic metabolism. The enzymes responsible for sulfide
oxidation in these eukaryotes have not been isolated, and their nature
and evolutionary origin remain unknown. In particular, it is not clear if sulfide oxidation in this exotic group of organisms bears any relationship with the better-understood pathways of bacteria.
The lower eukaryote Schizosaccharomyces pombe (fission
yeast) has not been described to live in sulfide-rich habitats,
although cells are exposed to sulfide generated internally during
assimilation of inorganic sulfur. After reduction of sulfate to sulfite
over several steps, the enzyme sulfite reductase catalyzes the reaction [H2SO3 + 3 NADPH + 3H+ In the course of studying the response of fission yeast to heavy
metals, we uncovered a mutant with an unusual defect in sulfur metabolism. This led to the cloning and characterization of a new gene
encoding a mitochondrial enzyme that can oxidize sulfide. The protein
has sequence homology to sulfide-oxidizing enzymes of bacterial
photosynthesis, suggesting a common evolutionary origin of sulfide
metabolism between prokaryotes and eukaryotes. Interestingly, potential
homologues of this enzyme appear in the genomes of nematodes, fruit
flies, mice, rats, and humans. This raises the possibility that sulfide
oxidation is more widespread among organisms, and might occur in a
wider diversity of habitats, than has previously been imagined.
Genetic Materials--
S. pombe strains Sp223
(h Growth Conditions--
Cells were grown at 30 °C on complete
medium YG (2% glucose, 0.5% yeast extract) or minimal medium SG (2%
glucose, 0.67% yeast nitrogen base without amino acids; supplemented
with 20 µg/ml uracil and/or 100 µg/ml leucine as needed). JS21
cells were mutagenized by a 45-min exposure to 175 µg/ml
MNNG.1 Media for comparison
of growth on glucose or glycerol consisted of either 1% yeast extract,
2% glucose or 1% yeast extract, 4% glycerol, 0.1% glucose, respectively.
Sulfide Analysis--
S2
For 35S-labeling of sulfide in vivo, cells
incubated for 20 min in 4 ml of low sulfur medium (MM medium (15) minus
sodium sulfate) were spiked with 80 µCi
Na235SO4. After 15 min, the cells
were harvested, washed with phosphate-buffered saline, and resuspended
in 200 ml of YG containing 200 µM cadmium. Cells at
various time points were collected and frozen in liquid nitrogen for
high performance liquid chromatography analysis.
For analysis of S2 Molecular Cloning--
S. pombe genomic and cDNA
libraries were described previously (16). Transformation of S. pombe was performed essentially as described (17). For gene
disruption, the EcoRV to SphI fragment of pJV1,
containing part of the hmt2+ coding sequence,
was replaced by the S. cerevisiae URA3+ gene. A
linear 4.1-kb XbaI fragment from this pJV17 plasmid was transformed into JS21, and colonies prototrophic for uracil were selected. For Northern analysis of hmt2+
expression, RNA was prepared as described (16). Blots were hybridized
to random primer-labeled hmt2+ cDNA and,
following stripping of the blot, were reprobed with an end-labeled
oligonucleotide representing a short antisense sequence of the S. pombe 18 S rRNA. The mutant hmt2
pJV37 contains the hmt2+ coding sequence in the
pQE12 Escherichia coli expression vector (Qiagen). The
hmt2+ genomic clone was cleaved with
PacI. Ends were made flush with T4 DNA polymerase and
ligated to BglII linkers. The DNA was cleaved with
BglII, followed by HincII that released a
fragment containing all but the first two and the last codons of
hmt2+. This fragment was ligated to
pQE12 that had been cleaved with BamHI, made flush with the
Klenow fragment of DNA polymerase I, and subsequently cleaved with
BglII. The resulting construct encodes an
hmt2+ fusion protein having four new
vector-derived N-terminal amino acids and nine new vector-derived
C-terminal amino acids, the last 6 of which are histidines. The DNA
encoding the entire fusion protein from pJV37 was cloned into pART1
under the control of the alcohol dehydrogenase promoter to form pJV40.
This construct was transformed into JS563, and cadmium sensitivity was
compared with JS563 and JS23 containing the empty vector pART1.
Cell Fractionation--
Cells grown in SG were vortexed with
glass beads in an equal volume of 10 mM Tris-Cl, pH 7, 0.15 M NaCl, 0.25 M sucrose, 1 mM each
phenylmethylsulfonyl fluoride, EDTA, EGTA, dithiothreitol, and
benzamidine HCl. Unbroken cells and debris were removed by centrifugation at 1,000 × g for 10 min. The total
protein preparation was further fractionated by centrifugation at
100,000 × g for 1 h. Protein was quantified using
an assay kit (Bio-Rad) in the presence of 0.05% CHAPS for
membrane-containing samples.
Mitochondria were isolated using published procedures (19, 20). Marker
enzymes corresponding to various cell compartments were assayed:
cytochrome c oxidase (mitochondria), cytochrome c
reductase (endoplasmic reticulum), Antibody Production and Immunodetection--
HMT2 protein was
purified under denaturing conditions from XL1-Blue E. coli
(Stratagene) carrying pJV37, according to the QIAexpress kit protocol
(Qiagen). Purified protein was electroeluted from a preparative
SDS-polyacrylamide electrophoresis gel, mixed with MPL+ TDM + CWS
Emulsion (RIBI ImmunoChem Research), and used to immunize two rabbits.
For Western blots, anti-HMT2 antisera were typically used at a dilution
of 1/30,000. Chemiluminescent detection of Western blots has been
described (23). The polyclonal antisera specifically recognize the
recombinant protein in Western blots of extracts from E. coli expressing tagged HMT2, but not from E. coli
bearing the empty expression vector.
Nondenaturing Protein Purification--
E. coli
XL1-Blue carrying pJV37 was grown to A600 = 0.6 at 30 °C in 4 liters of LB, 1 M sorbitol, 2.5 mM betaine, 50 µg/ml carbenicillin, and induced for
2.5 h with 1 mM IPTG. Cells were harvested by
centrifugation and resuspended in 100 ml of 50 mM NaPO4, pH 7.8, 50 mM NaCl, 10% glycerol, 0.1%
Triton X-100. After incubation with 0.1 mg/ml lysozyme on ice for 15 min and three rapid freeze-thaw cycles, extracts were sonicated briefly
and centrifuged at 10,000 × g for 20 min. The
supernatant was applied to a DEAE-Sephadex column (Amersham Pharmacia
Biotech) equilibrated in the same buffer, and the first, highly
UV-absorbing flow-through peak was collected. This fraction was stirred
with 1 ml of Pro-Bond nickel chelate resin at 4 °C for 2 h. The
resin was then loaded onto a column, washed with 10 ml of the same
buffer, and then eluted with the same buffer containing 500 mM imidazole. Fractions containing HMT2 were pooled,
subjected to several cycles of dilution with imidazole-free buffer, and
concentrated by ultrafiltration (Centricon 30). At each step in the
procedure, sulfide:quinone reductase activity copurified with anti-HMT2
immunoreactive protein peaks. The final preparation, which showed an
~180-fold enrichment of enzyme specific activity, was adjusted to
50% glycerol and stored at HMT2 Activity Assay--
Sulfide:quinone oxidoreductase activity
was measured under air at room temperature. A typical 250-µl reaction
contained 20 mM Tris-Cl, pH 7.8, 40 µM
coenzyme Q2 (Sigma), and 0.5 µg of purified HMT2, to
which 400 µM Na2S was added to begin the
reaction. Reduction of Q2 was measured by loss of 285 nm
absorption. The decrease in A285 was followed
for 30 s and was linear during this period for all experiments.
The mM extinction coefficient " Mutant Phenotype--
S. pombe mutant strain JS563
bears a cadmium-hypersensitivity trait that segregates as a single
locus. As this locus affects heavy metal
tolerance, it was given the genetic designation
hmt2. When compared with the wild-type parent strain JS21,
which can grow at up to 800 µM cadmium, the
hmt Sulfide Hyperaccumulation and Hypersensitivity--
A noticeable
trait of the mutant is that the colonies turn bright yellow in the
presence of cadmium, suggesting the formation of CdS. Over an 18-h
period, JS563 accumulated >6-fold as much acid-labile sulfide as the
wild-type JS23 (Table I). S. pombe is known to increase sulfide production during cadmium
stress, and a higher level of sulfide production was observed in both strains grown in 200 µM cadmium, but the
hmt Defect Not in Sulfate Assimilation--
One described route of
sulfide production in S. pombe is the sulfur assimilatory
pathway, where inorganic sulfur is routed from sulfate to sulfite to
sulfide and then to cysteine. A defect in this pathway might increase
the sulfide pool through either attenuating sulfide incorporation to
cysteine or by overproducing sulfide directly. Both possibilities were
examined by the following experiments. First, when cultured in minimal
medium, JS563 grew as well as the wild type, suggesting that cysteine
production is sufficient for cell growth. Further supplementation with
cysteine (100 µM) did not increase its growth rate.
Therefore, a defect in the incorporation of S2
Second, if sulfide overaccumulation were because of hyperactivity in
sulfate assimilation, a genetic block in this pathway should abolish
sulfide hyperaccumulation. The double mutant JV3 contains mutations in
both hmt2 and sulfite reductase. Like the parent
sulfite-reductase mutant strain DS31, JV3 is unable to convert sulfate
to cysteine and therefore requires cysteine supplementation. For this
reason, sulfide assays were carried out on cells grown in the complete
YG medium. DS31 accumulated far less sulfide than either wild-type or
JS563 (Table I), as expected from its lack of sulfite reductase
activity. However, DS31 also accumulated ~3-fold less sulfide than
JV3, suggesting that the hmt Kinetics of Sulfide Consumption--
To address if the
hmt2 Isolation of hmt2+ Gene--
JS563 was transformed
with a S. pombe genomic library. A single plasmid clone,
pJV1, with an 8.5-kb insert, restores cadmium tolerance to the mutant
(Fig. 2). A deletion analysis of the
8.5-kb insert showed that a 1.9-kb SalI/ScaI
subclone, in pJV26, complements the poor growth of the mutant on
cadmium, hydrogen peroxide, diamide, or glycerol as well as restores
wild-type sulfide levels (Table I). The 1.9-kb genomic fragment was
used to isolate a 1834-base pair cDNA. In Northern blots, wild-type
and mutant cells showed a single band of ~1.9 kb that hybridizes to
the hmt2+ cDNA (Fig.
3A). Accumulation of this
transcript increased by ~2-fold when cells were exposed to 200 µM cadmium (Fig. 3B).
An Engineered Gene Disruption--
To test whether the cloned DNA
represented a wild-type allele of the genetic lesion or an extragenic
suppressor, the wild-type allele was disrupted through integration of a
S. cerevisiae URA3+ marker (Fig. 2). Polymerase
chain reaction and Southern analysis indicated that, in the disruption
strain JV5, the coding region of the gene is intact, but two copies of
the disruption construct have integrated in tandem upstream of the
hmt2 coding region, disrupting the promoter and
possibly part of the untranslated leader sequence. As a result, the
1.9-kb mRNA is deficient in JV5 (Fig. 3B). A shorter
transcript in JV5 can be attributed to the truncated hmt2
gene on the disruption construct itself because this band is also
present in JV11, a strain bearing a random integration of the
disruption construct. An additional transcript in JV5 of ~4 kb
hybridizes faintly to both URA3+ (not shown) and
hmt2+ probes. This band might represent
transcriptional read-through from the upstream
URA3+ gene through hmt2. Neither
transcript detected in JV5 is expected to yield a functional
hmt2+ product.
The disruption phenotype in JV5 is very similar, though not identical,
to that of the original mutant. As with JS563, JV5 is hypersensitive to
cadmium, hyperaccumulates sulfide (Table I), and exhibits poorer growth
after heat shock, or in the presence of hydrogen peroxide or
Na2S. Surprisingly, no significant difference is observed
in the presence of diamide or with glycerol as the major carbon source.
This anomaly could be attributed to a leaky allele. For example, the
transcription of a functional mRNA in JV5, though severely reduced,
might not be entirely abolished. When JV5 was crossed to wild-type, the
disruption phenotype segregated as a single locus and was linked with
the URA3+ marker (53 cadmium-hypersensitive,
yellow, URA3+ progeny: 46 cadmium-resistant,
white, URA3 Sequence Change from hmt2+ to
hmt2
The mutant allele was cloned and sequenced across the entire coding
region. Comparison with the wild-type sequence revealed a G to A
transition that changed amino acid 396 from glutamate to lysine. When
the mutant allele, in the vector pART1, is reintroduced into JS563, it
is unable to complement (Fig. 2, pJV30). When a 300-base pair region
surrounding amino acid 396 is replaced by wild-type sequence, the
hybrid allele is fully functional (Fig. 2, pJV34). This result
demonstrates that this single point mutation is sufficient to account
for the mutant phenotype.
Similar Genes--
BLAST searches (26) of protein and nucleotide
sequence data bases found sequence similarity between the encoded
protein, HMT2, and a variety of oxidoreductases, some of which are
listed in Table II. The two best matches,
C. vinosum flavocytochrome c (6, 27) and R. capsulatus sulfide quinone reductase (28), are bacterial enzymes
that oxidize sulfide. Overall sequence identity between HMT2 and these
two enzymes is low (~20%). However, potentially functional features
are conserved among these sequences (Fig. 4; and see the Discussion).
The sequences with greater similarity to HMT2 have unknown function.
Apparent full-length genes from human, mouse, Caenorhabditis elegans, and the cyanobacterium Synechocystis sp.
PCC6803 all appear to have > 30% overall sequence identity with
HMT2. A fragmentary expressed sequence tag (EST) from rat appears
similarly well conserved. ESTs from Drosophila,
Chloroflexus, and Schistosoma all have >20% identity
with HMT2. These proteins are predicted to have unusually high pI:
human, pI ~ 9.5; mouse, pI ~ 9.1; C. elegans,
pI ~ 9.8; Synechocystis, pI ~ 8.9. This
feature is shared with HMT2 (pI ~ 9.9), sulfide quinone
reductase (pI ~ 9.5), and NADH dehydrogenase (pI ~ 9.4).
Mitochondrial Localization--
The N-terminal 24 amino acids of
HMT2 display features characteristic of mitochondrial targeting
sequences (29). The PSORT protein-targeting analysis program predicted
with up to a 59% probability that HMT2 might be directed to
mitochondria (30). In fission yeast extracts, a single anti-HMT2
immunoreactive band shows an apparent molecular mass of ~48 kDa.
Abundance of this protein band correlates to hmt2 mRNA
accumulation. It is found in both hmt2+ and in
hmt2
The bulk of HMT2 immunoreactive protein is pelleted by 100,000 × g centrifugation (Fig.
5A), suggesting that HMT2 is
membrane-associated or enclosed within an organelle. In purified
mitochondrial fractions (6-fold enriched in cytochrome oxidase specific
activity), HMT2 is >5-fold enriched (Fig. 5B). A
mitochondrial localization for HMT2 was supported by immunofluorescence
microscopy experiments (not shown). The mitochondrial fractions were
further subfractionated at pH 7.4. The mitochondrial membrane-enriched,
fumarase-depleted (0.5×) pellet showed increased immunoreactivity for
HMT2, suggesting that HMT2 may be membrane-associated (Fig.
5C). HMT2 is not predicted to have any hydrophobic regions
capable of forming transmembrane helices. However, subfractionation at
pH 11 increases the yield of soluble HMT2 (Fig. 5D). This
property is characteristic of some peripheral membrane proteins.
HMT2 Purification--
To facilitate purification of HMT2, a gene
fusion construct, pJV40, was made to encode a modified HMT2 protein
with additional histidine residues. This construct was able to
complement fully the hmt2 Flavin Binding--
Sequence analysis predicted that HMT2 might
bind FAD. The purified protein from E. coli was visibly
yellow, and light spectroscopy revealed absorption maxima at ~375 and
~455 nm in addition to the main protein peak at 280 nm (Fig.
6A). This profile is
characteristic of a flavoprotein. Similar flavin absorption peaks are
visible in free FAD (Fig. 6B). Free flavins exhibit
fluorescence, with excitation maxima at ~375 and ~450 nm and an
emission maximum at 520 nm. Although the purified protein was
nonfluorescent, boiling for 3 min denatured the protein and released a
soluble component having the fluorescence profile expected of a flavin.
This indicates that the protein binds flavin noncovalently and that the
fluorescence is quenched in situ.
Analysis of the fluorescence properties of the dissociated flavin (31)
indicated that it consists predominantly of flavin adenine dinucleotide
(93%); traces of FMN may represent breakdown products. Although
sequence analysis predicts a 1:1 molar ratio of FAD to polypeptide, we
experimentally obtained a ratio of ~1:3. This discrepancy may be
because of incomplete binding of flavin in the heterologous expression
system, losses of flavin during purification, or truncation of the protein.
Sulfide:Quinone Oxidoreductase Activity--
The addition of
sulfide caused rapid bleaching of the 450-nm absorption peak in HMT2
(Fig. 6A), but not in free flavin (Fig. 6B) or in
another flavoprotein, glutathione reductase (not shown). The bleaching
of HMT2 could be reversed when the sulfide was removed by
ultrafiltration. This reversible bleaching is consistent with the
reduction of the flavin by sulfide. The sequence similarity of HMT2 to
sulfide-oxidizing enzymes, and the sensitivity of its absorption
profile to sulfide, suggested that it might be capable of using sulfide
as a substrate in a redox reaction. By analogy with the activity of the
R. capsulatus sulfide quinone reductase, we tested the
ability of HMT2 to catalyze the reaction S2 Substrate Specificity--
We tested the ability of other electron
acceptors to replace coenzyme Q2 in the HMT2-catalyzed
reaction. Sulfide reacts rapidly and spontaneously with cytochrome
c, 2,6-dichloroindophenol, and ferricytochrome, and the
addition of HMT2 did not increase reaction rates. Sulfide fails to
reduce menadione, NAD+, or NADP+ spontaneously,
and HMT2 was unable to stimulate these reactions. Likewise, sulfite,
thiosulfate, cysteine, glutathione, Reaction Stoichiometry--
Sulfide and oxidized quinone
concentrations were determined independently at the end of a 10-min
reaction of 50 nmol each of sulfide and coenzyme Q2 with 5 µg of purified HMT2. Sulfide and oxidized quinone were consumed in an
approximately 1:1 molar ratio (7.85 ± 1.45 nmol
S2 Kinetic Parameters--
The sulfide-quinone reaction catalyzed by
HMT2 is saturable with increasing concentrations of substrate; the
apparent Km for coenzyme Q2 is 2 mM. It is important to note that coenzyme Q2 is
a water-soluble analogue of the more likely physiological electron
acceptor, ubiquinone, and therefore the Km obtained may not reflect the affinity of HMT2 for its true substrate.
Additionally, because of the insolubility of high quinone
concentrations in the reaction buffer, it was not possible to assay
Km for sulfide under saturating concentrations of
quinone. At lower quinone concentrations (40 µM),
Km for sulfide was calculated to be 2 mM. Using saturating S2 Sulfide:Quinone Oxidoreductase Activity in Mitochondria--
We tested the native protein in its physiological context for
the ability to carry out the same reaction. Mitochondria were isolated
from wild-type (JS23) and mutant (JS563) cells. The specific activity
of fumarase, an enzyme of the mitochondrial matrix, was equivalent in
the two preparations. Mitochondria from both strains were able to
reduce exogenous coenzyme Q2 at low rates in the absence of
added electron donor (JS23: 75 ± 51 nmol/min·mg; JS563: 68 ± 23 nmol/min·mg). The addition of sulfide significantly increased the rate of quinone reduction in JS23 mitochondria, and the spectrum of
the reaction mixture showed a rapid decrease in the 285-nm absorption
peak (Fig. 7C, b). Sulfide:quinone reductase
activity, corrected for the background rate of quinone reduction, is
significantly higher in JS23 (144 ± 45 nmol/min·mg) than in
JS563 mitochondria (21 ± 12 nmol/min·mg, p < 0.05). Therefore, fission yeast mitochondria possess a sulfide:quinone
oxidoreductase activity that correlates to the presence of functional
HMT2 protein.
Purified recombinant HMT2 protein is ~25-fold enriched over
isolated mitochondria in both protein abundance and in sulfide:quinone oxidoreductase-specific activity. This suggests that the activity of
HMT2 itself could account for all of the sulfide:quinone oxidoreductase activity measured in fission yeast mitochondria. The apparent Km for sulfide is relatively high (2 mM), when determined with a water-soluble analogue of the
more likely electron acceptor, ubiquinone. Assuming this value is
physiologically meaningful, HMT2 could nonetheless be adequately
supplied with substrate under such conditions as heavy metal exposure,
when whole-cell sulfide levels can exceed 1 mM. Quinone
should also be available, as ubiquinone is abundant in the
mitochondrial inner membrane.
Key features are shared among HMT2 and two sulfide dehydrogenases,
flavocytochrome c from C. vinosum and sulfide quinone
reductase from R. capsulatus. Flavocytochrome c
binds a FAD cofactor via a two-part sequence motif (amino acids 34-64
and 314-324; Fig. 4, black bar) (6, 27) that is also
present in sulfide quinone reductase and in HMT2. A pair of cysteines
(amino acids 191 and 367; Fig. 4, *) forming a disulfide bridge
adjacent to the flavin in flavocytochrome c has been implicated to be
catalytically involved in the redox reaction (6, 32). Aligning
cysteines are also present in sulfide quinone reductase and in HMT2.
Finally, the predicted secondary structure of HMT2 bears striking
similarities with the known structure of flavocytochrome c.
The similarity is greater than to any other protein in the Protein Data
Bank as judged by the program PHDthreader (33). Previously, it has been
suggested that all flavoenzymes known to be involved in sulfur chemistry may be related (27). HMT2 extends this family of related proteins to the eukaryotes.
It is possible that HMT2 functions in the detoxification of endogenous
sulfide. Not all sulfide produced during the assimilation of inorganic
sulfur is immediately incorporated into amino acids, and we can detect
a low level of acid-labile sulfide even in wild-type cells. Sulfide is
a potent inhibitor of cytochrome c oxidase (34), and
accumulation of sulfide in mitochondria would be expected to poison
respiration. Therefore, a mitochondrial sulfide dehydrogenase might
play a role in ensuring that local sulfide concentration near
cytochrome c oxidase is kept low. Consistent with this
hypothesis, hmt2 Mitochondrial sulfide oxidation is not unprecedented. Mitochondria of
several species of marine animals have been shown to couple sulfide
oxidation to the production of ATP (7, 9-12), but the proteins or
genes responsible for the initial oxidation step have not been purified
or cloned. In the lugworm Arenicola marina, electrons from
sulfide appear to enter the electron transport chain at the level of
ubiquinone (12), and the authors postulated that the enzyme involved
might be similar to the sulfide quinone reductase of R. capsulatus. HMT2, a mitochondrial protein with homology to the
R. capsulatus sulfide quinone reductase, strengthens this
hypothesis by providing a genetic link between mitochondrial and
bacterial sulfide oxidation. Intriguingly, genes similar to hmt2+ appear in worms, flies, mice, rats, and
humans. A heat-labile sulfide oxidizing activity has been reported in
rat liver mitochondria (35, 36). It is possible that the machinery for
capturing electrons from sulfide has been conserved in evolution,
although it has been adapted to new physiological roles.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S2
+ 3 NADP+ + 3 H2O ]. Much of
this S2
is incorporated into cysteine, but the cell still
accumulates measurable amounts of acid-labile S2
under
normal laboratory conditions. S2
increases during
exposure to heavy metals and is involved in resistance to cadmium and
cisplatin (13).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, ade6.216, ura4.294, leu1.32)
and B1048 (h+, ade7.50, ura4.294)
have been described (14). JS21 (h
,
ura4.294, leu1.32) and JS23 (h+,
ura4.294, leu1.32) were derived from the mating of Sp223 with B1048. A cadmium-hypersensitive mutant of JS21 was crossed to JS23 to
yield JS563 (h+, ura4.294, leu1.32,
hmt2
) and JV7 (h
, ura4.294,
leu1.32, hmt2
). JV5 (h
,
ura4.294, leu1.32, hmt2::URA3+) harbors a
homologous insertion, whereas JV11 (h
,
ura4.294, leu1.32, URA3+) harbors a random insertion
of an hmt2
disruption construct bearing the
Saccharomyces cerevisiae URA3+ gene.
DS31 (h+, ura4.294, leu1.32,
sir1::LEU2) was generated by disruption of the sulfite
reductase gene with a S. cerevisiae LEU2+
fragment (D. Speiser, USDA-ARS, Albany, CA). JV3 (ura4.294,
leu1.32, sir1::LEU2, hmt2
) was obtained
from the mating of DS31 with JV7.
was collected and assayed
as described (14). S2
content was first normalized to the
dry weight of the cell culture and, to minimize day-to-day assay
variability, was subsequently normalized to each day's value obtained
from wild-type cells grown without cadmium (set to 1.0).
turnover, labeled cell pellets were
homogenized and centrifuged for 2 min at 15,000 × g,
4 °C. Proteins were precipitated with 5% 5-sulfosalicylic acid.
After filtration of supernatants, samples were injected onto a Betasil
Basic-18 high performance liquid chromatography column (Keystone
Scientific) equilibrated in 5% acetonitrile, 95% 0.05%
trifluoroacetic acid in water, and eluted by a linear gradient to
12.5% acetonitrile, 87.5% (0.05% trifluoroacetic acid in water) over
20 min, at a rate of 1 ml/min. 0.7 mg/ml DTNB in 0.3 M
KPO4, pH 7.8, 7.5 mM EDTA was mixed with
post-column effluent at a rate of 0.1 ml/min, and absorption of the
derivatized effluent was monitored at 405 nm. Effluent was then mixed
with an equal amount of Ultima-Flo M scintillation mixture (Packard
Instrument), and radioactive peaks were monitored by flow scintillation
counting. Na2S was used as a standard for peak
identification and quantification.
allele
was cloned into pART1 (18) to form pJV30. A 300-base pair
HindIII to ScaI fragment containing the mutation
was replaced with wild-type DNA to form pJV34.
-mannosidase (vacuole), glucose-6-phosphate dehydrogenase (cytoplasm), catalase (peroxisome), and guanosine diphosphatase (Golgi). The results indicated that mitochondria were ~6-fold enriched over their initial abundance in
the cell homogenate, whereas the abundance of other organelles was
decreased or relatively unchanged. Mitochondrial subfractionation was
carried out with minor modifications from a published protocol (21).
Activity of the soluble matrix enzyme fumarase (22) was used to monitor
mitochondrial breakage. For investigation of the effects of pH on HMT2
solubility, mitochondria were suspended in ice-cold 0.6 M
sucrose, 3 mM MgCl2, 20 mM Tris-Cl,
pH 7.4, or 0.6 M sucrose, 3 mM
MgCl2, 20 mM Na2CO3, pH
11. Mitochondria were sonicated for a total of 4 min, with rest periods
on ice, then centrifuged for 1 h at 100,000 × g.
20 °C.
oxidized-reduced" of Q2, determined empirically by comparing the absorption
of oxidized and NaBH4-reduced Q2 samples in
reaction buffer at 285 nm, was 8.85. S2
from aliquots of
reaction mixtures was trapped in 1 M zinc acetate and
quantified by the methylene blue assay as described (14). Mitochondria,
adjusted to 8 µg of protein in 250 µl, were assayed as above,
except reactions contained 3.2 mM Na2S and 2 mM KCN.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant ceases to grow at 100 µM cadmium and shows reduced yield with as low as 5 µM cadmium. A difference in growth rate was not found in
the presence of high NaCl or sucrose concentrations, but a reduction
relative to the wild type was observed after heat shock at 47 °C or
in the presence of hydrogen peroxide, the thiol-oxidizing agent
diamide, or the nonfermentable substrate glycerol. Because the
catabolism of glycerol requires a functional respiratory pathway, the
defect was suspected to be associated with mitochondrial function.
JS563 continued to exceed wild-type
levels. Exogenous sulfide is toxic to S. pombe at
concentrations in the 100 µM range. Because the
hmt2
mutant already exhibits an elevated level
of endogenous sulfide, it could be more sensitive to an exogenous
supply of this substance. JS563 showed impaired growth relative to the
wild-type strain in the presence of Na2S, with the greatest
differential observed at 200 µM Na2S.
Genetic control of sulfide accumulation
accumulation over 18 h of growth in minimal (SG) or
complete (YG) medium, in the presence or absence of cadmium. Strains
used are wild-type (JS23), hmt2 disruption (JV5) or missense
(JS563) mutant, sulfite reductase mutant (DS31), and
hmt2
/sulfite reductase double mutant (JV3). Where
indicated, the strains harbor the empty vector (pART1) or a
complementing clone (pJV26). Normalized to the value obtained from the
wild type grown in the absence of cadmium.
into
cysteine seems unlikely.
locus in JV3
enhances sulfide accumulation through a pathway separate from inorganic
sulfur assimilation.
mutation affects the consumption of
S2
, cells were pulse-labeled with
35SO42
. During the brief 15-min
labeling period, both wild-type and mutant cells converted
35SO42
into
35S2
(Fig. 1,
time = 0 h). Most of this 35S2
then
disappeared, as it was incorporated into organic sulfur compounds,
leaving approximately equal amounts in mutant and wild-type cells after
2 h. Over the next 20 h, wild-type cells gradually turned
over this pool of 35S2
, but in the
hmt2
mutant this pool was not depleted. During
that time, total (radioactive plus nonradioactive) sulfide levels
accumulated more rapidly in the mutant than in wild type (Fig. 1,
inset). The pattern indicates that the
hmt2
defect lowers the consumption of the
sulfide pool. This alone may account for the higher accumulation of
sulfide, although the data do not rule out the possibility that the
mutation also enhanced de novo S2
synthesis.
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Fig. 1.
S2 turnover and
accumulation. HPLC measurements of extracts from cadmium-induced
wild-type (JS23, filled circles) and mutant (JS563,
open circles). At each time point, 4 ml of radiolabeled
culture was analyzed simultaneously for radioactive (main
panel) and total (inset) sulfide. Normalized
35S2
is the amount of radiolabeled sulfide at
each time point divided by total 35S present in the cell at
time 0.
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Fig. 2.
Genetic analysis of
hmt2+. Deletion derivatives of
the complementing genomic clone pJV1 were transformed into JS563 and
scored for their ability to complement (+) the mutant phenotype. The
alcohol dehydrogenase promoter (Padh) of the pART1 expression vector is
indicated, and lies upstream of the hmt2 coding region in
pJV26, pJV30, and pJV34. The gene disruption construct shown is the
XbaI fragment of pJV17, described under "Experimental
Procedures." Thin line, wild-type genomic DNA; thick
line, JS563 genomic DNA; dashed arrow,
hmt2+ cDNA; open box,
URA3+ insert; asterisk, site of
mutation in JS563.
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Fig. 3.
Northern analysis of
hmt2+ expression. A,
wild-type (JS21) and mutant (JS563) strains (harboring pART1) were
grown in SG and exposed to 200 µM cadmium for 24 h.
30 µg total RNA from these cultures was hybridized with
hmt2+ cDNA, and rehybridized with a probe
specific to the 18S rRNA. B, 30 µg total RNA from a
wild-type strain (JS21), a strain bearing a random insertion of the
hmt2 disruption construct (JV11), and the hmt2
disruption strain (JV5) was hybridized to the
hmt2+ cDNA and to an 18S rRNA probe. Cells
were or were not exposed to cadmium (200 µM cadmium for
24 h) as indicated. The arrow marks the position of the
truncated hmt2 transcript originating from the disruption
construct itself.
progeny,
2 = 0.64).
When crossed to JS563, wild-type recombinants were not recovered (49 cadmium hypersensitive, yellow, URA3+ progeny:
51 cadmium hypersensitive, yellow, URA3
progeny,
2 = 0.04). This genetic linkage between the
original mutant locus and the disruption locus is consistent with the
cloned gene corresponding to the genetic lesion in JS563.
--
The hmt2+ genomic
and cDNA clones were sequenced on both DNA strands.
hmt2+ contains a putative 5' untranslated region
of 357 nucleotides, longer than average for S. pombe (24).
This region contains multiple poly-T and poly(A) stretches. The
putative start methionine occurs at position 358, embedded in the
sequence taaaaatgt, which matches the most commonly observed S. pombe translational start motif at 8 of 9 positions (25).
Comparison of genomic and cDNA sequences reveals that
hmt2+ lacks introns. The deduced
hmt2+ open reading frame is predicted to encode
a protein of 459 amino acids, with a molecular mass of 51,574 Da and an
isoelectric point of 9.5-10.
Sequence similarity to HMT2
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Fig. 4.
Sequence alignment of HMT2 with putative
homologues. Multiple sequence alignment of HMT2 from S. pombe (underlined) with putative distant homologues
from C. vinosum and R. capsulatus and putative
near homologues from Homo sapiens, Synechocystis
sp. PCC6803, and C. elegans. Residues that are
identical (black shading) or similar (gray
shading) among at least three of the sequences are highlighted.
The bipartite FAD-binding motif is indicated by black bars;
the putative redox-active cysteines, by asterisks; and the
site of the missense mutation in JS563, by a cross.
(JS563) cells, is more abundant in cells
containing hmt2+ on a multicopy plasmid, and is barely
detectable in cells containing a disruption of the hmt2
promoter (JV5). The data are consistent with the 48-kDa band being the
translation product of the hmt2+ gene. The
~3-kDa reduction from the predicted size of HMT2 is consistent with
the presumed cleavage of the putative mitochondrial targeting sequence
during translocation from cytoplasm to mitochondria.
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Fig. 5.
Western blots of cell fractions.
A, 30 µg total protein (T), and the supernatant
(S) and pellet (P) resulting from a 1-h,
100,000 × g centrifugation of an equivalent 30-µg
sample. B, 1, 5, or 25 µg total protein from an initial
S. pombe homogenate and from mitochondria purified from the
same homogenate were blotted and probed with antibodies to HMT2.
C, whole mitochondria (M) were osmotically
shocked and divided into a supernatant fraction (S1) and a
pellet. The pellet was sonicated and centrifuged to separate a second
supernatant (S2) and pellet (P). Each lane
contains 2.5 µg of protein. D, whole mitochondria were
sonicated at pH 7.4 or pH 11, then separated into supernatant
(S) and pellet (P) fractions by centrifugation at
100,000 × g for 1 h. 2 µg of protein from each
fraction was blotted and probed with antibodies to HMT2.
mutant phenotype,
suggesting that the additional amino acid residues do not interfere
with the in vivo activity of the protein. We therefore
assumed that the His6-tagged protein is suitable material for initial biochemical characterization. The final protein preparation consisted of two major bands of ~52 and ~50 kDa, both of which were
immunoreactive with anti-HMT2 antibodies. The smaller band may result
from an alternative translation start site or may be a degradation
product of the full-length protein.
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Fig. 6.
Absorbance spectra of HMT2 and FAD.
Absorbance spectra were recorded before and immediately after
(+S2 ) addition of 1.25 mM sulfide to purified
HMT2 (A) or free FAD (B). Indicated are
absorbance maxima (375 and 450 nm) of free FAD. Each sample was
adjusted to 2.5 µM flavin, assuming a mM
extinction coefficient of 11.3 at 450 nm. After addition of sulfide,
the increase of absorbance at ~230 nm is because of sulfide
itself.
+ coenzyme
Q2(oxidized)
[S(oxidized)] + coenzyme
Q2(reduced). Coenzyme Q2 is a water-soluble
ubiquinone analogue whose oxidized (Fig.
7A, a) and reduced
(Fig. 7A, b) forms can be distinguished by their
absorption profiles in the UV range. Addition of sulfide to a cuvette
containing coenzyme Q2 and purified recombinant HMT2 causes
a rapid decrease in the 285-nm peak (Fig. 7B, b).
The reaction between coenzyme Q2 and sulfide in the absence
of HMT2, or in the presence of an equivalent quantity of free FAD
(0.43 ± 0.08 nmol/min), is nearly 8-fold slower than the
enzyme-catalyzed rate (3.35 ± 0.54 nmol/min, significant at
p < 0.05).
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Fig. 7.
Absorbance spectra of oxidized
versus reduced coenzyme
Q2. A, absorbance spectra of 40 µM coenzyme Q2 before (a) and
after (b) chemical reduction with NaBH4.
B, absorbance spectra of reaction mixture containing 40 µM coenzyme Q2 and 0.5 µg of HMT2 before
(a) and after (b) addition of 400 µM sulfide. The additional absorbance at ~230 nm is
because of sulfide itself. C, absorbance spectra of a
sulfide:quinone reaction catalyzed by mitochondria. Spectra were taken
immediately after the addition of sulfide (a) and 4 min
later (b).
-mercaptoethanol, succinate, and
pyridine nucleotides were all unable to replace sulfide in the
HMT2-catalyzed reduction of Q2. Therefore, HMT2 appears to
possess a specific sulfide:quinone oxidoreductase activity.
/7.6 ± 0.99 nmol quinone, n = 2.). Because flavoproteins and quinones both commonly carry two
electrons, the stoichiometry of the reaction suggests that sulfide is
oxidized to elemental sulfur and donates two electrons to coenzyme
Q2.
(3 mM)
and the highest concentration of quinone (1.5 mM) that can
be assayed, we obtained an apparent Vmax of 1.36 µM coenzyme Q2 reduced per s·µg of
enzyme, and kcat of 52/sec (assuming an active
enzyme concentration of 22.3 nM, based on incomplete FAD binding).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells grow poorly on a
nonfermentable carbon source, suggesting that unchecked accumulation of
sulfide may interfere with respiration. The exact role of HMT2 in
cadmium tolerance is not yet clear, but a likely possibility is to
detoxify excess sulfide generated during cadmium stress.
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ACKNOWLEDGEMENT |
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We thank H. A. Koshinsky for assistance with the preparation of figures.
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FOOTNOTES |
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* This work was supported by U. S. Department of Energy Grant EM96-55278 (to D. W. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF042283.
¶ Present address: MicroGenomics Inc., 11211 Sorrento Valley Rd., San Diego, CA 92121.
To whom correspondence should be addressed: Plant Gene
Expression Center, USDA-ARS, 800 Buchanan St., Albany, CA 94710. Tel.: 510-559-5909; Fax: 510-559-5678; Email: ow{at}pgec.ars.usda.gov.
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ABBREVIATIONS |
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The abbreviations used are:
MNNG, N-methyl-N'-nitro-N-nitrosoguanidine;
DTNB, 5,5'-dithiobis(2-nitrobenzoic acid);
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
kb, kilobase(s);
IPTG, isopropyl-1-thio--D-galactopyranoside.
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REFERENCES |
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