(Received for publication, December 29, 1995; and in revised form, February 28, 1996)
From the
An Arabidopsis thaliana ATP sulfurylase cDNA (ASA1), encoding a putative chloroplastic isoform, has been
cloned by functional complementation of a Saccharomyces cerevisiae (met3) ATP sulfurylase mutant which also has a poor
sulfate transport capacity. Homologous complementation of the yeast
mutant with the ATP sulfurylase gene restores both ATP sulfurylase
function and sulfate transport. Heterologous complementation restores
only ATP sulfurylase function as demonstrated by low
[S]sulfate influx measurements and selenate
resistance. A structural relationship between ATP sulfurylase and
sulfate membrane transporters in yeast is proposed. The sequence of ASA1 is homologous to deduced plant and animal ATP sulfurylase
sequences. Analyses indicate a potential tyrosine phosphorylation site
which is unique to higher eukaryote sequences. ASA1 is
specified by a single copy gene that is part of a multigene family in A. thaliana. At least two ASA1 copies are found in Brassica napus plants. ASA1 transcripts were found in
all organs examined, with the highest transcript abundance and ATP
sulfurylase activity in leaves or cotyledons. Absence of sulfate from
culture media transiently increased B. napus transcript
abundance, indicating that initially, the response to sulfate
deprivation is transcriptionally regulated.
Sulfur is an essential mineral nutrient for plant and animal
growth which, in its reduced form, is incorporated into sulfur amino
acids, other sulfur-containing metabolites, and coenzymes. In its
oxidized form, it is incorporated into sulfolipids which are the major
components of the chloroplast membrane(1) . In both plants and
microorganisms, active uptake of sulfate through specific transporters
is followed by reduction to sulfide. As sulfate has a very low
oxidation/reduction potential relative to available cellular
reductants, the primary step in assimilation requires its activation
via an ATP-dependent reaction(2) . This reaction is catalyzed
by ATP sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4) and
leads to the formation of adenosine 5`-phosphosulfate
(APS)()(1) . The equilibrium for the formation of
this product is thermodynamically unfavorable and, as the efficiency of
APS removal by subsequent reactions seems insufficient for energetic
compensation, shift of the thermodynamic balance through subcellular
compartmentalization or substrate channeling may occur (2) .
ATP sulfurylase has been purified from a wide range of sources and characterized extensively at the biochemical level from plants(3, 4, 5) , animals(6, 7) , and fungi(8, 9) . In plants, leaves are considered to be the main site of sulfur assimilation with ATP sulfurylase activity found predominantly in chloroplasts and at low levels in the cytosol (4, 5) . All the enzyme activities involved in assimilatory sulfate reduction have also been detected in plant root proplastids(10) . Although ATP sulfurylase isoforms with different biochemical properties have been purified from higher plants, no specific cellular function has been attributed to any of them.
An ATP sulfurylase gene was first cloned from Saccharomyces cerevisiae(11) . Genes have subsequently been cloned from prokaryotes (12, 13, 14) and another lower eukaryote(15) , and several cDNAs have been cloned from plants (16, 17, 18, 19) and animals(20, 21) . The prokaryotic enzymes are heterodimers with a catalytic subunit and a subunit that acts as a stimulatory GTPase(2) . The yeast and plant enzymes are homooligomers, dimeric and tetrameric(5, 11, 19) , and do not respond to GTP(15) . In animals, ATP sulfurylase and APS kinase reside on a single bifunctional protein(7, 20, 21) .
Using an Arabidopsis thaliana cDNA library, we have functionally complemented an ATP sulfurylase-defective yeast mutant. We report the isolation of the corresponding ATP sulfurylase cDNA clone that restores yeast methionine heterotrophy and ATP sulfurylase activity. The clone was sequenced and found to be identical with one of the three previously cloned A. thaliana ATP sulfurylases (18, 19) , except for differences in the 3` sequence. Comparison of yeast mutants complemented by the heterologous cDNA and homologous gene led us to propose the existence of a structural relationship between ATP sulfurylase and membrane sulfate transporters in yeast. ATP sulfurylase activity has been shown to increase under sulfur limiting conditions in plants(22, 23) , but the mechanism by which this response is induced has not been determined. We have carried out ATP sulfurylase expression studies in relation to sulfur availability in A. thaliana and Brassica napus.
For Northern blot hybridization, total RNA was isolated from frozen organs using the guanidinium thiocyanate/phenol/chloroform extraction method(36) . Approximately 10 µg of total RNA were denatured and separated in a 0.66 M formaldehyde, agarose (1.2% w/v) gel in MOPS buffer(25) . The gel was then washed twice for 20 min in 3 M NaCl, 0.3 M trisodium citrate, pH 7.0, before transfer of RNA to nitrocellulose and hybridizations as described above. For slot-blot analysis, approximately 1 µg of total RNA per slot was denatured and applied to nitrocellulose (Sartorius) using the Minifold II slot-blot manifold (Schleicher & Schuell) as described (37) ; duplicates of each sample and nonspecific controls of 50 µg of E. coli tRNA were loaded. After hybridization, as described above, blots were exposed to preflashed film (38) for 50-70 h at -80 °C with intensifying screens. Quantification of Northern blots was carried out using the Imager system (Appligene) in conjunction with the National Institutes of Health Image program(39) .
Figure 1:
Alignment of A. thaliana ASA1 with other eukaryotic ATP sulfurylase amino acid sequences. The
amino acids of the open reading frame encoded by the A. thaliana
ASA1 cDNA are compared to the ATP sulfurylase sequences APS1(17) and APS3 (A.
thaliana)(19) , StMet3-1 and StMet3-2 (S. tuberosum)(16) , MET3 (S.
cerevisiae)(11) , and APS (P.
chrysogenum) (15) , and U. caupo(21) represents the sequence of bifunctional ATP
sulfurylase/APS kinase sequence. The alignments were obtained using the
PILEUP GCG program. The full sequence for ASA1 is shown, but
for the other clones only the sequence containing the homology blocks,
discussed in the text, are shown. Roman numerals refer to
blocks of homology. Boxed areas represent regions where all
but one of the sequences are identical or all the sequences show
similarity. Positions where all residues are identical are shaded.
Numbers at the ends of lines indicate the position of the most 3`
amino acid relative to the start of the protein. * indicates the
position of putative tyrosine phosphorylation and indicates the ASA1 putative transit peptide cleavage site. Residues underlined correspond to those identical with the N-terminal
sequence from purified spinach ATP sulfurylase(5) . Dashes indicate gaps in the sequence to yield the best
alignment.
Figure 2:
Southern blot analysis of the genomic DNA
from A. thaliana and B. napus. The genomic DNA (10
µg) was digested by restriction enzymes (E, EcoRI; H, HindIII; B, BamHI; RV, EcoRV), separated by agarose gel
(0.8%) electrophoresis, transferred onto nitrocellulose, and then
hybridized with P-labeled ASA1 DNA
fragments.
Figure 3:
Growth curve of yeast strains transformed
with ASA1 or MET3. Yeast were grown from 50
milliunits initial absorbance, measured at 650 nm, in synthetic media
supplemented with 0.4 mM sodium sulfate as the sole sulfur
source and the required auxotrophy factors. Symbols correspond
as follows: , Met3
strain W303-1A;
, met3 mutant strain CC371-4C;
, met3 mutant
strain CC371-4C transformed with pFL61-ASA1;
, met3 mutant strain CC371-4C transformed with
pM3-32.
In addition to the lack of ATP sulfurylase
activity, yeast strains which have the met3 mutation have been
shown to be defective in sulfate transport(44) . In order to
ascertain the effect of complementation on sulfate transport, we used
selenate, as a toxic structural analogue of sulfate, in drop assays.
When the strains were grown on sulfateless media (containing
homocysteine as the sole sulfur source) in the presence of 4 mM sodium selenate, only the wild-type W303-1A and the mutant
complemented by the yeast gene (CC371-4C + pM3-32) were
unable to grow, whereas the mutant complemented by the A. thaliana ATP sulfurylase cDNA (CC371-4C + pFL61-ASA1) grew as
well as the noncomplemented Met3 mutant (Fig. 4). To investigate whether the selenate growth effect was
related to sulfate transport, [
S]sulfate
influxes were measured (Table 1). The heterologous expression of
pFL61-ASA1 partly restored the sulfate uptake function to the
mutant, but to less than 20% of the sulfate uptake capacity of the
yeast control. When the mutant was complemented by the homologous MET3 ATP sulfurylase gene, the
[
S]sulfate influx was almost 4-fold higher than
in the yeast control.
Figure 4:
Test for selenate resistance. Yeast were
grown in sulfur-free synthetic liquid media supplemented with 0.1
mM homocysteine as the sole sulfur source and the required
auxotrophy factors. Yeast were washed and 30 µl (at 0.8
milliunit absorbance at 650 nm) were dropped onto agarose sulfate-free
media supplemented with 0.1 mM homocysteine which contained
either no selenate (-Se) or 4 mM sodium
selenate (+Se). WT corresponds to W303-1A strain
which has a wild type yeast MET3 gene. CC371-4C is a met3 mutant and is either not complemented (Mut), transformed
with a plasmid containing the yeast MET3 gene (Met3-2), or transformed with a plasmid containing
the A. thaliana cDNA ASA1 (ASA1).
Figure 5:
Northern blot analysis of total RNA from A. thaliana and B. napus grown with and without
sulfate. Total RNA (10 µg) from plants grown in the presence
(+) or absence(-) of sulfate (S) was isolated from
leaves (L), cotyledons (C), or roots (R) and
electrophoresed on an agarose (1.2%) gel, transferred onto a
nitrocellulose filter, and then hybridized with P-labeled
DNA fragments either ASA1 or
actin.
Figure 6:
Northern blot analysis and ATP sulfurylase
activity of B. napus organs grown with and without sulfate.
Total RNA or soluble protein was isolated from cotyledons (Co), hypocotyls (H), and roots (R) after
growth in the presence (+4) or absence of sulfate for 2 (-2) or 4 (-4) days. RNA (10 µg) was
electrophoresed on an agarose (1.2%) gel, transferred onto a
nitrocellulose filter, and then hybridized with P-labeled
DNA fragments either ASA1 or actin. A, the relative
abundances of ASA1 hybridizing transcripts in these organs,
determined from RNA slot-blot analysis, standardized to actin
hybridization, and compared for each organ to the sulfate (+)
value. Duplicate slots were carried out for each sample. B,
the relative ATP sulfurylase specific activities in these organs
compared for each organ to the sulfate + value. C, the
ATP sulfurylase specific activities measured in these organs, expressed
as nmol
min
mg
of
protein, are the mean of three replicates.
In this paper we report the cloning, by functional complementation of a yeast mutant, of a cDNA which encodes an A. thaliana ATP sulfurylase (ASA1). The sequence of this clone is identical with the previously reported AtMet3-1(18) and APS2 cDNAs(19) , except that the latter part of its 3`-untranslated sequence is different. Heterogeneity at the 3` ends of mRNA encoded by a single plant gene has been shown to result from polyadenylation of the transcripts at multiple sites(46) . The poly(A) of the two ASA1 homologous sequences and an homologous EST (accession number Z26572) are located downstream of the ASA1 poly(A) which seems to agree with the observed preference for polyadenylation in response to the second site after the open reading frame(46) .
Yeast met3 mutants complemented with ASA1 had a lower growth rate
than wild type or the mutant complemented by the homologous gene. This
result may reflect differences between constitutive expression of ASA1 by the phosphoglycerate kinase promoter and expression of
the yeast ATP sulfurylase gene by its own promoter. Alternatively, the
presence of a chloroplast transit peptide or differences in primary
sequence between the yeast and ASA1 could significantly alter
the activity of the plant enzyme. Putative cytosolic and chloroplastic
forms of ATP sulfurylase have been cloned from potato by functional
complementation(16) ; however, only the activity of the
cytosolic form expressed in yeast was presented. Activity measurements
for all complemented met3 mutants were lower than for the
noncomplemented Met3 strain, but as the growth curve
of the mutant complemented with the homologous gene is similar to that
of the wild type (Fig. 3) there does not seem to be any growth
limitation due to insufficient ATP sulfurylase activity, suggesting
that more activity than required for growth is expressed in the wild
type strain. Expression of ASA1 in the Met3
strain reduced the ATP sulfurylase activity by 30% compared to
when it was expressed in the met3 mutants and reduced the
activity of the noncomplemented Met3
strain by 50% (Table 1). This result implies that the plant ATP sulfurylase
protein or cDNA has an effect on either the yeast protein activity or
the yeast gene expression. For example, ATP sulfurylase is a
homomultimeric protein in plants(5, 19) and
yeast(9) , and it is possible that in the non-mutant
Met3
strain the co-expressed yeast and plant proteins
form chimeric structures with reduced or no ATP sulfurylase activity.
In contrast, the activity measured for pM3-32 in the
Met3
strain was 20-fold higher than that when the
plasmid complemented the mutant strains and 4-fold greater than the
noncomplemented Met3
strain which supports the
suggestion that, in the wild type strain, ATP sulfurylase activity
levels are not growth-limiting.
Yeast strains which have the met3 mutation have been shown to be defective in sulfate
transport as well as in ATP sulfurylase activity(44) . These
mutants, unlike wild type strains, can grow on media containing
selenate, a toxic analogue of sulfate (Fig. 4). Comparison of
strains, expressing either the homologous or heterologous ATP
sulfurylases, by growth on selenate-containing media showed that only
the strain expressing the heterologous protein was able to grow (Fig. 4). This was confirmed to result from reduced
[S]sulfate transport capacity (Table 1)
and demonstrates that ASA1 does not fully complement the met3 mutant phenotype. This suggests that the observed
differences in growth kinetics (Fig. 3) probably result from the
low sulfate uptake rate in the pFL61-ASA1 complemented mutant
rather than differences in expression level or structure. The loss of
sulfate transport when the ATP sulfurylase gene is mutated indicates
that structural interactions, which are essential for efficient sulfate
uptake, may exist in yeast between some sulfate transporters and ATP
sulfurylase. In plants, such an interaction between the sulfate
transporter and ATP sulfurylase may not exist or may depend on specific
recognition domains which are absent or different from those of yeast.
Alternatively, the presence of the transit peptide may affect protein
conformation and this interaction. Interestingly, a yeast mutant
defective in sulfate transport exhibits normal ATP sulfurylase activity (49) . Interaction between the sulfate transporter and the
first enzyme in the sulfate metabolic pathway may be advantageous as it
could result in a degree of sulfate channeling from the transporter
into the sulfate binding and activation site of the ATP sulfurylase.
This could push the unfavorable thermodynamic equilibrium of the ATP
sulfurylase reaction toward forming the APS product. Such a channeling
mechanism has been suggested in plants for the cysteine synthase
complex(50, 51) and for the animal bifunctional ATP
sulfurylase/adenosine 5`-phosphosulfate
kinase(7, 20, 21) .
Sequence analysis indicates that ASA1 has a 62-amino acid putative transit peptide. Homology of the proposed mature ASA1 protein to N-terminal sequence of a spinach chloroplast isozyme (5) suggests its chloroplastic location. In addition to blocks of homology common to all eukaryotic ATP sulfurylase sequences, a putative tyrosine phosphorylation site was identified (Fig. 1, block II) which is conserved only in the higher eukaryote sequences. This site is situated between the conserved block proposed to form part of the catalytic site (Fig. 1, block I) (15) and a region of homology including the putative phosphate binding loop sequence (Fig. 1, block III)(14) . Translational control of enzyme activity through tyrosine phosphorylation has been demonstrated extensively in animals. In plants, a tyrosine kinase has only recently been cloned (52) , and the regulation of a plant enzyme by tyrosine phosphorylation has just been demonstrated in vivo(53) . Prokaryotic ATP sulfurylases have been found to contain a GTP binding motif and to be stimulated by GTP(2) . GTP has little effect on the activity of plant, yeast, and fungal enzymes (15) and, in common with other eukaryotic ATP sulfurylase sequences, no GTP binding motif is found in the ASA1 sequence. Labeling of cysteine residues in P. chrysogenum has identified two buried cysteine residues which may have important structural functions, one of which is in a region whose sequence is conserved in A. nidulans and S. cerevisiae(15) . Although this cysteine is not conserved in the plant sequences, there is a conserved cysteine in the plant ATP sulfurylases which belongs to a highly conserved block at the 3` end (Fig. 1, block V).
Genomic Southern analysis (Fig. 2) demonstrated that ASA1 is encoded by a unique gene in agreement with the results of Murillo and Leustek (19) for APS2. Under low stringency conditions, an additional 2-3 fragments were detected in each A. thaliana genomic DNA digest (not shown) indicating at least 2 related ATP sulfurylase genes. This agrees with the cloning of two different full-length A. thaliana ATP sulfurylase cDNAs, APS1 and APS3(17, 19) . Southern blots probed with these cDNAs indicate that they are also single copy genes, suggesting that the ATP sulfurylase gene family in A. thaliana has just these three members(19) . Our data base searches have, however, identified six additional ASA1 homologous A. thaliana ESTs (accession numbers T21966, T42953, R29819, T88260, T45338, and T21042) which appear to encode one or several related ATP sulfurylases. Analyses using the deduced protein sequence from the 6 combined sequences showed 62, 68, and 72% identity, respectively, to ASA1, APS1, and APS3. These values are sufficiently different to suggest a fourth ATP sulfurylase gene in A. thaliana which may not cross-hybridize, even under low stringency conditions. As the six ESTs were all identified from the same systematic sequencing program(54) , they may represent an organ or treatment-specific ATP sulfurylase form. In A. thaliana, the existence of a fifth ATP sulfurylase gene expressed in the cytosol might be expected since cell fractionation and enzyme activity studies on spinach revealed a cytosolic form(4) . In addition, a cDNA without a transit peptide has been cloned from S. tuberosum(16) . Therefore, the A. thaliana ATP sulfurylase family probably consists of at least four chloroplastic and perhaps one cytosolic isoform. The identification of at least 2 ASA1-like gene copies in B. napus is consistent with the allotetraploid state of this species(55) .
Northern blot
analyses of A. thaliana and B. napus identified a
transcript of 1.9 kb in all organs examined, with highest
expression in leaves (Fig. 5). An APS1 probe also
identified a transcript (1.85 kb) in leaf and root total RNA (17) which was most abundant in leaves. An increase in
transcript abundance in roots of both A. thaliana and B.
napus was observed on sulfur deprivation, 1.8-fold and 1.5-fold,
respectively, indicating that the expression of the ASA1 gene
responds to the availability of sulfate. The apparent absence of
response in A. thaliana leaves to sulfate starvation seems in
contrast to the 1.3-fold increase observed in B. napus. This
can be attributed to the difference in the age of the organs used,
9-day-old cotyledons compared to 3-week-old leaves, and is more likely
to reflect the higher sulfur requirement of the young organs. ATP
sulfurylase specific activity has been shown to be high in young
leaves, decreasing as the leaves mature (23, 56) indicating that the sulfur requirement of
mature leaves is low.
As effects of sulfate starvation on the relative abundance of ASA1 hybridizing transcripts in B. napus were similar to those observed in A. thaliana, although the cDNA probe was heterologous, the use of B. napus in these studies was considered appropriate. ATP sulfurylase activity and Northern analysis carried out on the same B. napus organs showed similar patterns up to two days of sulfate starvation with the largest relative increase in roots and highest in cotyledons (Fig. 6, A and C). Specific activities increased in all organs with sulfur starvation, although in hypocotyls after 2 days they decreased then increased. This indicates that in these organs initial changes in enzyme activity on sulfur starvation are probably transcriptionally regulated. Increased ATP sulfurylase activity in response to the absence of sulfate in the external media has previously been observed in higher plants(22, 23) . In M. atropurpureum, this difference was also found to be largest in roots, with only a slight initial activity increase in leaves. Large increases in relative transcript abundance for the recently cloned plant sulfate transporters have also been observed for roots(49) . Sulfate translocation studies have demonstrated that roots are the predominant sulfur sink during sulfate deprivation(23) . These results, together might indicate that roots have priority for sulfate utilization. Such a priority could be envisaged to improve plant survival under sulfate limiting conditions by augmenting the sulfate foraging ability of roots.
After 4 days of sulfate deprivation, although the enzyme activity had increased, the RNA abundance decreased in both cotyledons and roots. A difference between relative ATP sulfurylase activity and RNA abundance profiles was also observed for hypocotyls (Fig. 6, A and B). B. napus probably expresses multiple forms of ATP sulfurylases whose combined enzyme activities might differ, on sulfur starvation, from the profile of one transcript type. The observed differences could, however, result from translational control, but as the observed increases in activity, observed on deprivation, are greater than increases in ASA1 RNA abundance, simultaneous increases in expression of several transcript types seems likely.
The increase in transcript abundance
observed in B. napus leaves suggests that the absence of
sulfate in the root external media is perceived by leaves. Sulfate
uptake and transport from roots to shoots have been shown to be
inhibited by glutathione (57) which has been proposed to act as
a quantitative signal informing the plant of its sulfur
status(58) . Glutathione levels have been found to decrease on
sulfate starvation, ()and this could induce the changes in
transcript abundance on sulfate starvation.
We have cloned a cDNA which encodes an A. thaliana ATP sulfurylase (ASA1) with a putative chloroplast transit peptide. ASA1 is encoded by a single copy gene that is part of a multigene family in A. thaliana, probably consisting of at least four members. Yeast mutants deficient in ATP sulfurylase also lack sulfate uptake capacity. Comparison of these mutants transformed with the heterologous ASA1 plant cDNA to those transformed with the homologous yeast MET3 ATP sulfurylase gene showed that, although ATP sulfurylase activity is completely restored, the sulfate uptake ability is not fully complemented. We propose a model involving structural interaction between the yeast plasma membrane sulfate carrier and the cytosolic ATP sulfurylase. We plan to verify this model in yeast and examine the possibility of such an interaction in plants.
A putative tyrosine phosphorylation site was found in the ASA1 sequence which is conserved, but only in higher eukaryote ATP sulfurylases. This tyrosine is situated between two homology blocks proposed to correspond to part of the catalytic site and bind ATP(14, 15) . Investigation of this region with regard to possible regulation and catalytic functions should be informative. Northern blot analysis showed that ASA1 is expressed in all A. thaliana and B. napus organs examined with highest expression in leaves or cotyledons, respectively. The relative RNA abundance and ATP sulfurylase activity in B. napus were found to increase in cotyledons, and to a greater degree in roots, after 2 days of sulfur starvation. This indicates that the initial response to sulfate starvation is probably at the transcriptional level. Subsequently relative ASA1 RNA abundance decreases whereas ATP sulfurylase activity continues to increase, this could be as a result of translational control or other members of the ATP sulfurylase gene family having different response profiles to sulfate starvation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U40715[GenBank].