Sulfur is a major component of cells. Proteins, sulfated
polysaccharides, sulfolipids, coenzymes, and other sulfur-containing
secondary compounds are actively involved in cellular metabolism.
Sulfate must be activated in order to be used in cellular metabolism.
This activation is achieved by the enzymes ATP sulfurylase-forming
adenosine 5`-phosphosulfate (APS) (
)and the APS
kinase-forming adenosine 3`-phosphate 5`-phosphosulfate
(PAPS)(1, 2) . The PAPS is further reduced to sulfite
and sulfide, which contribute sulfur to cysteine and methionine. Many
intermediates and their derivatives in the sulfur assimilation pathway,
like sulfite(3) , PAPS(4, 5) , cysteine
conjugate(6, 7) , and sulfide (8) are toxic to
the cell when accumulated beyond certain levels. Cell's demand
for sulfur varies according to its growth status. To avoid accumulation
of these compounds to toxic levels, a sensitive flux (rate of flow) of
the intermediates in the sulfur assimilation pathway is necessary. A
3`(2`),5`-diphosphonucleoside 3`(2`)-phosphohydrolase (DPNPase) was
reported from Chlorella to catalyze the conversion of PAPS to
APS in
vitro(2, 9, 10, 11) . This
enzyme is widely distributed and suggests the presence of a futile
cycle (substrate cycle) in plants to control the sulfur activation
pathway. Futile cycle is one of the most sensitive flux control systems
in metabolic pathways (12, 13, 14) .
In
yeast, a halotolerance gene (HAL2) was identified by
functional assay of supporting the growth of cells under high salinity
stress(15) . The yeast HAL2 gene is identical to MET22(15) . The yeast met22 mutant is
methionine auxotroph and can not use sulfate, sulfite, or sulfide as
sulfur sources(16) . However, the mutant exhibits wild-type
activities of the enzymes necessary to assimilate sulfate and has
normal sulfur uptake system. HAL2 also has high homology with
the Escherichia coli cysQ gene(4) . The cysQ mutant is cysteine auxotroph, but mutations that resulted in
sulfate transport defects compensated for cysQ mutation.
Although it was proposed that cysQ may control the pool of
PAPS(4) , the function of cysQ is not confirmed. Since
over expression of HAL2 in yeast improved salt
tolerance(15) , it is of interest to determine the role(s) this
gene plays in sulfur assimilatory pathway and whether such a gene
exists in plants. Overexpression of this gene may improve plants'
salt tolerance.
Very recently, the protein encoded by yeast HAL2 gene was shown to have the activity of 3`(2`),5`-bisphosphate
nucleotidase with both PAPS and PAP as substrates(17) . It was
proposed that the enzyme helps sulfite synthesis by removing PAP, a
byproduct of the sulfite synthesis reaction. In this paper, we report
the cloning and characterizing of a rice HAL2-like (RHL) cDNA
clone. The RHL cDNA complemented both cysQ and met22 mutations. Thus, we demonstrated that the proteins encoded by cysQ, HAL2, and RHL genes have the same function in
sulfur assimilation pathways in E. coli, yeast, and plants.
Our results also demonstrated that the enzyme encoded by the RHL gene
prefers PAPS over PAP as native substrate. This enzyme, together with
APS kinase, appears to catalyze a futile cycle in sulfur assimilation
pathway.
MATERIALS AND METHODS
Plant Material and Microbes
Rice (Oryza
sativa L., cv. Tallahamsa) seeds were germinated on 3-mm
filter paper at 28 °C in a growth chamber. 5-day-old seedlings were
harvested and frozen in liquid nitrogen for RNA preparation. Yeast
mutant met22 (CC364-18C, MAT
, his 3, ura 3 met22) was kindly provided by Dr. Dominique
Thomas(16) . E. coli cysQ mutant (DBan 41;
594 with cysQ::Tn5tac 1) was kindly provided by Dr.
Douglas E. Berg(4) . E. coli strains Top10 F` and
lys-s were from Invitrogen Inc.
Construction of cDNA Expression Library
Total RNA
was prepared from 5-day-old rice seedlings by the method of Verwoerd et al.(18) . Poly(A)
RNA was isolated
by oligo(dT)-cellulose fractionation. A Not T primer was used
to prime the first-strand cDNA synthesis. The Not T primer is
39 base pairs with 18 T residues and a NotI restriction site.
After addition of BstXI adapters, the cDNA was digested with NotI and size fractionated on an agarose gel. Molecules of
larger than 500 base pairs were electroeluted from the gel and cloned
into BstXI and NotI-linearized pcDNAII plasmid
(Invitrogen, Inc.). The ligation mixture was transformed into E.
coli TOP10 F` by electroporation. The primary recombinants were
about 5.33
10
.
Cloning of Rice HAL2-like Gene
By comparison of
HAL2 and CysQ amino acid sequences, two degenerate primers were
designed: 1) GA(TC)CCNAT(T/C/A)GA(T/C)GGNACNAA and
2)CCNGCNGC(A/G)TG(A/G)TCCCA(A/T/G)AT. Rice total RNA was used as
template for reverse transcription and PCR. An amplified fragment
(about 460 base pairs) was cloned into pUC19 SmaI site by
blunt-end ligation and sequenced. One clone with highest homology to
the yeast HAL2 gene was used for screening the rice cDNA
library following the procedure of Invitrogen Inc.
Expression of RHL cDNA in E. coli and Protein
Purification
A BamHI site was created by PCR right
before the ATG start codon. The entire reading frame including the
3`-end of the RHL cDNA was translationally inserted into the expression
vector pRSET C (Invitrogen Inc.) to generate a construct pRSET-RHL1 at
the BamHI and XhoI sites. This vector contains a
coding sequence for a polyhistidine that binds metal and facilitates
the purification of the expressed proteins. The construct was then
transformed into E. coli Lys-s cells. The transformed cells
were grown in LB medium, and the cells were induced for 3.5 h by the
addition of 1 mM IPTG at A
of 0.8. The
protein was purified by nickel-affinity chromatography in nondenaturing
condition following the manufacture's instructions (Invitrogen
Inc.). The purified protein was used for enzyme assay.
Enzyme Assay
The enzyme activity was determined by
quantifying the inorganic phosphate released from substrate using a
colorimetric method(19) . A standard assay was conducted in a
200-µl reaction mixture including 50 mM Tris, pH 8.5, 0.5
mM PAP (Sigma), or 0.5 mM PAPS, 5 mM
Mg
, and 0.5 µg of the purified protein. The
mixture was incubated at 30 °C for 30 min. Under this condition,
the enzyme activity was linear with protein quantity (up to 1 µg)
and reaction time (up to 1 h). The reaction was stopped by adding a
one-fifth volume of 40% trichloroacetic acid. The blank (control)
included substrate and Mg
but no enzyme. For
determination of K
, K
, pH
optimum, and Mg
-dependent enzyme profile, the
corresponding factors were changed. The Li
was removed
from PAPS (Sigma) by passing over three P11 cation exchange columns at
4 °C just before use. This procedure removed twice the amount of
Ag
ion monitored by Cl
. The PAPS
concentration was determined by UV absorption.For radioactive
substrate, the reaction condition was the same as standard assay, but
the volume was reduced to 20 µl. The [
S]PAPS
(DuPont NEN), [
S]APS, and protein quantities
were as indicated. Standard [
S]APS was converted
from [
S]PAPS by nuclease P1. The reaction
products were analyzed on a PEI cellulose thin-layer chromatography
sheet, which was developed by 1 M lithium chloride (20) and detected by PhosphorImager.
Functional Complementation
To complement yeast
mutant met22, the RHL cDNA was cloned into a yeast expression
vector pYEUra3 (Clontech Inc.) at BamHI and XhoI
sites. The yeast mutant cells were transformed by electroporation with
corresponding vectors and spread on uracil selection medium. The
colonies were replicated to methionine selection medium. The cysQ mutant cells grown with Kan (50 mg/liter) were transformed by
electroporation. The transformed cells were spread on LB plate with
both Amp (100 mg/liter) and Kan (50 mg/liter). The resulting colonies
were streaked on modified M9 medium with Amp (100 mg/liter), Kan (50
mg/liter), and IPTG (1 mM)(4) . For the medium with
sulfite and cysteine, sulfite concentration was from 0.1 to 50
mM, as indicated, and cysteine was 0.2 mM. The
sulfite, cysteine, calcium chloride, magnesium sulfate, ammonium
acetate, and antibiotics were added after autoclaving the media.
Mutagenesis
The mutagenesis was carried out by
PCR. At the two domains that we intended to mutate, each had a unique
DNA restriction enzyme site, Pf1m I located at nucleotide
position 579 and BglII located at nucleotide position 1024.
For D141N, D141A, and D144N, the mutation primers included the Pflm I site, and the other end primer included a PstI site, a
unique site located at nucleotide position 261 of the RHL cDNA. For
W291L, D292A, and D292N, the mutation primers included the BglII site, and the other end primer was (M13) -40
primer, which covers a XhoI site of the vector. The mutated
fragments generated by PCR were sequenced and then replaced the
wild-type fragments using the restriction enzyme sites included in the
primers.
RESULTS
Isolation of a RHL Gene and Its Level of Transcription
in Different Tissues
The yeast HAL2 gene belongs to a
phosphatase gene family identified by Neuwald et
al.(21) . The genes in this family have two conservative
domains, DPIDGT and WDXAAG. We designed two degenerate primers
based on the sequences of yeast HAL2 and cysQ at
these domains. Rice total RNA was used as template for reverse
transcriptase and PCR, which yielded a fragment of about 460 base
pairs. Sequence analysis of this fragment showed highest homology with
the yeast HAL2 and E. coli cysQ genes. This fragment
was used as a probe to screen a rice cDNA library. The restriction
mapping of 10 clones, out of the 300 positives, showed that they were
identical, except one of them had one restriction enzyme site
difference at the 3`-noncoding region. The longest cDNA clone was 1506
base pairs in length with an open reading frame encoding a polypetide
of 358 amino acids with a predicted molecular mass of about 40 kDa. The
159-base pair 5`-noncoding region contains 74% GC with a hairpin
structure just three base pairs before the ATG start codon. The context
around the start codon contains a typical start codon consensus
sequence CCACCATG(22) . The deduced amino acid sequence from
RHL cDNA showed significant homology (Fig. 1) with the yeast
Hal2 protein (38% identity and 57% similarity) and E. coli CysQ protein (30% identity and 49% similarity). It also had two
characteristic motifs of the inositol monophosphatase gene family (21) . The northern hybridization results showed that this gene
was constitutively transcribed in both roots and shoots, but the
transcript level in shoots was about twice of that in roots (data not
shown). Light had no affect on the transcription of this gene.
Figure 1:
Amino acid sequence alignment of rice
Hal2-like protein, yeast HAL2 protein (15) and E. coli cysQ protein(4) .
RHL Gene Complemented Yeast HAL2 Mutant, met22, and E.
coli cysQ Mutant
To determine whether the products of RHL and
yeast HAL2 genes have similar function, a complementation
experiment was conducted. The yeast met22 mutant was
transformed by electroporation with pYEUra3 vector carrying RHL cDNA.
The transformed cells were selected on uracil
minimum
medium and then replicated on to the methionine medium. The RHL cDNA
complemented met22 mutant on the medium with sulfate as sole
sulfur source as well as on the medium with sulfite (Fig. 2, A and B). The mutant cells did not grow on medium with sulfite (Fig. 2B). Thus, it is clear from these data that RHL
or yeast HAL2 gene is not directly involved in sulfite
synthesis.
Figure 2:
A and B, complementation of yeast
mutant, met22 (16). 1, the met22 mutant
harboring pYEUra3 vector with RHL gene; 2, the met22 mutant harboring vector pYEUra3 only; 3, the met22 mutant. A, growth on minimum medium with ammonium sulfate
5 mg/liter, histidine 10 mg/liter, and galactose 20 g/liter (3.5 days
at 30 °C). B, the same as in A except 0.5 mM sulfite was added (3 days at 30°C); C and D,
complementation of E. coli cysQ mutant(4) . 1, the cysQ mutant harboring pcDNAII inserted with
RHL gene; 2, the cysQ mutant harboring vector pcDNAII
only; 3, the cysQ mutant; 4, wild-type E. coli. C, growth on modified M9 medium (Amp, 100
mg/liter, Kan, 50 mg/liter, and IPTG, 1 mM; 3.5 days at 37
°C). D, the same as in C, but 0.2 mM sulfite was added (3 days at 37
°C).
The cysQ gene in E. coli is involved in
sulfur metabolism(4) . This gene shares high homology with both
yeast HAL2 as well as the RHL gene. The cysQ mutant
is a cysteine auxotroph under aerobic condition and is a conditional
mutant (depends on IPTG) that was reported to be leaky(4) . To
determine whether RHL gene could complement cysQ, the
transformed cysQ cells were plated on LB medium with
antibiotics, and then streaked to a triple selection M9 medium (Amp 100
mg/l, Kan 50 mg/liter, IPTG 1 mM, and no cysteine). The
results showed (Fig. 2C) that all cells harboring RHL
cDNA grew while cysQ mutant or cysQ mutant harboring
empty plasmid did not grow.
The complementation of both met22 and cysQ with RHL gene demonstrated that these three
genes have the same function. However, according to previous reports, cysQ mutant grew on sulfite medium but yeast met22 did not grow on sulfite medium(4, 16) . We
carefully observed cysQ mutant cells' behavior on M9
medium with sulfite (Fig. 2D). The growth of cysQ mutant was poor compared with the complemented or wild-type cells.
With time, the difference between the cysQ mutant cells and
the complemented or wild-type cells became more significant. This
growth phenotype was the same from 0.1 mM to 30 mM sulfite. In the case of met22, the cells did not grow on
minimum medium with sulfite (Fig. 2B).
The Enzyme Encoded by RHL Gene Converts PAPS to APS and
PAP to AMP
In order to study the function of RHL gene, we
expressed RHL cDNA in E. coli. Following induction with IPTG,
the soluble proteins were passed through a nickel-agarose affinity
column, and the enzyme was purified to homogeneity (Fig. 3A). The calculated size of the fused protein is about
44 kDa (4 kDa from the polyhistidine leader sequence). The protein band
on SDS-polyacrylamide gel electrophoresis appeared to be about 47 kDa.
The E. coli inositol monophosphatase, a protein of the same
family, also appears larger than calculated size on SDS-polyacrylamide
gel electrophoresis(23) . The purified enzyme converted PAPS to
APS (Fig. 3B). Since the enzymes of the inositol
monophosphatase family are sensitive to Li
and the
commercially available PAPS (Sigma) is supplied with 4 mol of
lithium/mol of PAPS, we removed the Li
by
cation-exchange columns right before use. Although PAPS degrades up to
17%/day at 37 °C (Sigma instruction), no PAPS decomposition was
detected in the Li
removing process conducted at 4
°C. Several compounds were tested for substrates (Table 1).
The RHL enzyme had high activity with PAPS (100%) and 3`-PAP (92%),
while low or no activity with other tested compounds. The K
for PAPS was 100 µM, while the K
for PAP was 240 µM (Table 1).
The results suggest that PAPS is the favored substrate.
Figure 3:
A,
overexpression and purification of RHL protein. Proteins were separated
on a 10% SDS-polyacrylamide gel. Lane 1, molecular weight
markers; lane 2, crude extract from E. coli harboring
empty vector pRSET C; lane 3, crude extract from E. coli with RHL cDNA inserted in pRSET C and induced for 3.5 h with 1
mM IPTG; lane 4, the purified RHL protein. B, separation of [
S]PAPS and
[
S]APS by polyethyleneimine cellulose thin-layer
chromatography. The thin-layer chromatography sheet was developed with
1 M LiCl. 2 µl of end product of the reaction mixture was
loaded on each lane. The reactions were conducted under conditions
described under ``Materials and Methods.'' Lane 1,
PAPS standard; lane 2, reaction mixture containing 0.25 µg
of RHL protein, 0.5 mM PAPS, and 4 µCi of
[
S]PAPS; lane 3, reaction mixture
containing 1 µg of RHL protein, 0.5 mM PAPS, and 4 µCi
of [
S]PAPS; lane 4, reaction mixture
containing 0.25 µg of RHL protein, 0.5 mM APS, and 4
µCi of [
S]APS; lane 5,
[
S]APS standard.
The RHL
enzyme activity was found to be Mg
-dependent. The
optimal Mg
concentration was 5 mM (Fig. 4A), but the enzyme had high activity from 0.5 to
30 mM Mg
. The optimal pH was around 8.5
(data not shown). The enzyme activity was inhibited by
Ca
, Li
, and Na
(Fig. 4B). The enzyme inhibition by
Ca
depended on the ratio of
Ca
/Mg
, and the inhibition by
Ca
was eliminated by high Mg
concentration (Fig. 4B). The relationship of
Ca
and Mg
was competitive. With
Mg
concentrations of 5 mM, 0.5 mM,
and 0.1 mM, the K
for Ca
was 0.8, 0.05, and 0.01 mM, respectively. The K
for Li
was 0.85 mM and
for Na
was 55 mM. The RHL enzyme was
significantly activated by K
, and the inhibition by
Na
was eliminated by elevated K
(Fig. 4C).
Figure 4:
A, Mg
-dependent profile
of RHL enzyme activity. The reaction was performed under standard
conditions as described under ``Materials and Methods,''
except that Mg
concentrations were as indicated. The
activities are expressed as percentage of that with 5 mM Mg
(12.35 µmol of P
/h/mg of
protein). The results are the mean of three independent experiments. B, effect of cations on the RHL enzyme activity. The reactions
were conducted under standard conditions as described under
``Materials and Methods'' except that cations were as
indicated. The activity is expressed as percentage of that with
standard condition (12.17 µmol of P
/h/mg of protein).
Results are the mean of three independent experiments.
,
inhibition by Ca
with Mg
at 0.1
mM;
, inhibition by Ca
with
Mg
at 0.5 mM;
, inhibition by
Ca
with Mg
at 5 mM;
, inhibition by Li
with Mg
at
5 mM;
, inhibition by Na
with
Mg
at 5 mM; and
, the restoration of
RHL enzyme activity by Mg
with Ca
at 1 mM. C, the activation of the RHL enzyme
activity by K
. The reactions were conducted under
standard conditions, except K
and Na
were included as indicated. The activities were present as
percentage of that with standard condition (12.21 µmol of
P
/h/mg of protein). Results were the mean of three
independent experiments.
, without Na
;
,
with 55 mM Na
.
Site-directed Mutagenesis Indicated That RHL Enzyme Has
Similar Mg
Binding Sites as Inositol
Monophosphatase
The RHL gene belongs to inositol monophosphatase
family. Many residues are conserved in the whole gene family at two
domains(21) . We mutated several residues in these regions to
determine whether they are essential for the RHL enzyme activity. We
mutagenized D141N, D141A, D144N, W291L, D292A, and D292N. All of these
mutations did not complement cysQ mutant. Direct enzyme assay
showed that all of the mutants had less than 5% enzyme activity
compared with the wild-type. Crystal structure studies and mutagenesis
of the human inositol monophosphatase proved that Asp-90, Asp-93, and
Asp-220 bind Mg
(24) . The Trp-219 binds to a
water molecule that stabilizes phosphate group of inositol
monophosphate by binding with it(25) . These four residues in
inositol monophosphatase correspond to the four residues (Asp-141,
Asp-144, Asp-292, and Trp-291) of RHL enzyme that we mutated. Our
mutagenesis results indicated that the functions of these four residues
are conserved between RHL enzyme and inositol monophosphatase, although
the substrates are different. Studies with inositol monophosphatase
indicated that it requires two Mg
for activity. The
RHL enzyme kinetics data showed that Ca
and
Mg
have a competitive relationship. It is highly
possible that replacing one of the two Mg
ions by
Ca
renders the RHL enzyme inactive.
DISCUSSION
The RHL Gene Product Has Properties in Common with
Chlorella DPNPase
It has been reported that DPNPase from Chlorella can use both PAPS and PAP as
substrates(2, 9, 10, 11) , and its
activity is Mg
-dependent. The enzyme activity is
inhibited by Ca
, and the optimal pH is around 9. The
optimum Mg
concentration of Chlorella DPNPase is about 25 mM. The RHL enzyme has 70% activity
(data not shown) at this Mg
concentration. Since the
RHL enzyme and the Chlorella DPNPase have the same substrate
specificity and similar kinetics, and both depend on Mg
and are inhibited by Ca
, we believe that the
two are the same enzyme. DPNPase together with APS sulfotransferase
transfers sulfur from PAPS to a thio carrier, which is further reduced.
An intermediate (APS) was detected in this reaction. The DPNPase was
partially purified, but the molecular weight was not
known(11) . Since all of the work on DPNPase was conducted in vitro and many nonspecific phosphatases, 3`-nucleotidases,
and nuclease P1 can convert PAPS to APS in
vitro(26, 27) . Thus, the role of DPNPase in
sulfur reduction pathway was questioned(28) . The isolation of
RHL gene and the complementation of yeast HAL2 and E. coli
cysQ mutants provided direct evidence that DPNPase is involved in
sulfur reduction in plants.The assimilation of sulfur starts with
sulfur activation. The first enzyme ATP sulfurylase catalyzes APS
synthesis. Since the equilibrium for APS formation is far to the left,
the second reaction, catalyzed by APS kinase phosphorylating APS at the
3`-position, plays an important role to pull the first reaction
forward. Consequently, the PAPS accumulates, and an enzyme that
controls the PAPS pool and removes unnecessary PAPS becomes essential.
DPNPase is such an enzyme.
The RHL Enzyme, Together With APS Kinase, May Control a
Futile Cycle in Sulfur Activation Pathway
Since the RHL enzyme
can use both PAPS and PAP as substrates, it is not easy to prove which
one is the native substrate. Murguía et al.(17) proposed that PAP is the substrate for the
yeast HAL2-encoded enzyme, and the function of the HAL2 gene product is to remove PAP, the byproduct of sulfite synthesis
reaction. This hypothesis explains some phenotypes of the HAL2 mutant, met22. However, it does not explain the following
facts. 1) met22 mutant cannot use sulfite as sulfur source ( (16) and Fig. 2B). If the enzyme uses PAP as
substrate, the function of the enzyme should be to help sulfite
synthesis by removing the byproduct of sulfite synthesis reaction. In
this case, addition of sulfite should correct the met22 phenotype. But the fact is that sulfite is lethal to this mutant
(see Fig. 2B). Murguía et al. (17) proposed that accumulated PAP may inhibit
PAPS utilizing enzyme(s). However, the PAPS reductase is normal in met22(16, 29) . PAP was reported to be an
inhibitor of phenol sulfokinase and amine N-sulfotransferase(30, 28) , but these
enzymes are not directly involved in sulfur reduction. Since met22 grows normally on medium with methionine, the mutation is likely
in the sulfur reduction pathway. 2) During the purification of RHL
enzyme, we found another phosphatase in the cysQ mutant cells
(grown with 1 mM IPTG). This phosphotase is about 30 kDa and
is more PAP-specific. (
)The activity was present in the
40-60% ammonium sulfate fraction and eluted from DEAE column at
the fraction between 65 and 100 mM KCl. The enzyme activity
was Mg
-dependent and Na
-sensitive
but not sensitive to Li
. The activity of this enzyme
for PAPS was only 32% of that for PAP. The K
for
PAP was 30 µM and for PAPS was 250 µM.
Brungraber (30) predicted the existence of a PAP-specific
nucleotidase, and our results agree with this prediction. 3) Mutations
that resulted in sulfate transport defects compensated for cysQ mutation(4) . If PAP is the substrate of the cysQ-encoded enzyme and the mutant phenotype of cysQ is due to PAP accumulation, which inhibits sulfite synthesis, then
the mutations of sulfate uptake system will only result in less sulfite
synthesis (due to limited sulfate available and thus the decreased
level of PAPS). The phenotype of cysQ mutant should not be
compensated by the mutation in the sulfur uptake system. 4) Both cysQ and met22 are sulfur reduction pathway mutants,
and the RHL gene complemented both mutations. However, it has been
suggested that plants use APS pathway instead of PAPS
pathway(1, 2) . PAP is not produced in the APS
pathway. The substrate of RHL-, cysQ-, and met22-encoded enzymes should be common to both APS and PAPS
pathways. 5), The RHL enzyme has higher enzyme activity and lower K
for PAPS (Table 1). Considering the facts
presented above, we believe that PAP is not the primary substrate of
these enzymes.The phenotypes of cysQ and met22 can be explained if PAPS is the native substrate. The cysQ gene product converts PAPS to APS, and APS kinase catalyzes APS to
PAPS. The two enzymes run a futile cycle in sulfur activation pathway (Fig. 5). If CysQ is knocked out, the PAPS accumulates
immediately, which is toxic to the
cell(4, 5, 31) . When cysteine is provided to E. coli, the sulfate uptake system is inhibited by feedback
regulation, and the cell stops synthesizing the enzymes involved in
sulfur assimilatory
pathway(32, 33, 1, 2) . Therefore,
the toxic PAPS is not accumulated and the cysQ mutant grows
normally. When sulfite is provided, it can be easily converted to
cysteine without producing PAPS. However, the feedback regulation and
repression due to added sulfite is not as strong as with cysteine, and
the PAPS can still accumulate. Therefore, the cells do not grow well on
sulfite medium (and with time, the differences between wild-type and
mutant cells become more significant). In yeast, the same principles
may apply. Methionine acts as a repressor (34) and inhibits
sulfur assimilatory enzymes and exerts feedback regulation.
Figure 5:
The futile sulfur cycle model in both PAPS
and APS sulfur-reduction pathways. The steps in the PAPS pathway are
shown in boldface with capital
letters.
DPNPase Is a Member of Inositol Monophosphatase
Family
The sequence comparison data identified two motifs in the
rice DPNPase that are conserved and characteristic of the inositol
monophosphatase family. The inositol monophosphatase contains several
characteristic residues that have been shown, by site-directed
mutagenesis and x-ray crystallography, to be involved in Mg
and substrate bindings. Even though the substrate for DPNPase is
different, the residues involved appear to be the same as shown by our
data on site-directed mutagenesis. The rice DPNPase requires
Mg
for activity and is highly sensitive to cations,
suggesting similar structure of this enzyme as that of inositol
monophosphatase.
The Possible Role of RHL Gene Product in Salt
Tolerance
A gene encoding a
Na
/SO
co-transport was
recently isolated from rat(35) . The sulfate uptake by this
transport increases with Na
elevation in the culture
medium. Under Na
stress, this kind of transport system
would increase sulfur uptake, but the metabolism of the cells is slowed
down, and the demand for sulfate may decrease. Therefore, the toxic
sulfur compounds, such as, cysteine conjugates, sulfite, sulfide, and
PAPS may accumulate. The product of RHL gene converts PAPS to APS,
controls the sulfur flux, and thus may reduce the accumulation of the
toxic compounds. Gläser et al.(15) reported that supplying methionine improved salt
tolerance in yeast. This phenotype could be due to the fact that
availability of methionine inhibits sulfate
uptake(32, 34) . Sequestering toxic sulfur compounds
may be a common phenomenon under salt stress. In plants, choline O-sulfate (an osmolyte) is present to sequester extra sulfate
under stress conditions(36, 37) .Our results
indicated that the transcription of RHL gene was not induced by
salt.
But the activity of the RHL enzyme and the yeast HAL2 enzyme were increased by K
. Plants have
high intracellular K
levels and low
Na
levels. Osmotic stress increases K
uptake and keeps the K
level
high(38, 39, 40) . This phenomenon indicates
that the RHL enzyme may response to salt stress at protein level.
Overexpression of the yeast HAL2 under salt stress improves
salt tolerance in yeast, an observation consistent with the above
explanation. Similarly, it is possible that overexpression of RHL in
plants may help increase salinity-induced osmotic stress tolerance.