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?????
???. 14: 527?533 (1998)
Identification of YHR019 in Saccharomyces cerevisiae
Chromosome VIII as the Gene for the Cytosolic
Asparaginyl-tRNA Synthetase
ISABELLE LANDRIEU1,2*, MICHELINE VANDENBOL1, REUBEN LEBERMAN2,
DANIEL PORTETELLE1 AND MICHAEL HARTLEIN2
1
2
Unite? de Microbiologie, Faculte? Universitaire des Sciences Agronomiques de Gembloux, Gembloux, Belgium
EMBL Grenoble Outstation, Grenoble, France
Received 10 September 1997; accepted 9 November 1997
Exploiting the asparagine auxotrophy of the Saccharomyces cerevisiae mutant strain 8556a, we have isolated the
gene for the cytosolic asparaginyl-tRNA synthetase (AsnRS) of S. cerevisiae, by functional complementation of the
mutation affecting this strain. The isolated gene could be identified to the open reading frame YHR019, called
DED81, located on chromosome VIII. The mutant gene from the 8556a strain, asnrs -1, was amplified from
genomic DNA by PCR. This gene contains a point mutation, leading to the replacement of a glycine residue by a
serine in a region of the protein probably important for the asparaginyl-adenylate recognition. The protein encoded
by YHR019 is very similar to cytosolic AsnRS from other eukaryotic sources. In a phylogenetic analysis based on
AsnRS sequences from various organisms, the eukaryotic sequences were clustered. Expression of YHR019 in
Escherichia coli demonstrated that a yeast AsnRS activity was produced. The recombinant enzyme was purified to
homogeneity in three chromatography steps. We showed that the recombinant S. cerevisiae AsnRS was able to
charge unfractionated yeast tRNA, but not E. coli tRNA, with asparagine. 1998 John Wiley & Sons, Ltd.
Yeast 14: 527?533, 1998.
??? ????? ? Saccharomyces cerevisiae; YHR019; chromosome VIII; asparaginyl-tRNA synthetase
INTRODUCTION
Aminoacyl-tRNA synthetases (aaRSs) are crucial
enzymes in protein biosynthesis since they
catalyse the specific attachment of amino acids to
their cognate tRNAs. The catalysis is a two-step
reaction, in which the amino acid is first activated by formation of an aminoacyl-adenylate in
the presence of ATP and magnesium, before
being transferred to the 3 end of a cognate
tRNA. Two classes of synthetases of ten members each have been defined, based on their
primary and tertiary structures (Eriani et al.,
1990; Cusack et al., 1990). Asparaginyl-tRNA
*Correspondence to: I. Landrieu, Unite? de Microbiologie,
Faculte? Universitaire des Sciences Agronomiques de
Gembloux, 6 Avenue Mare?chal Juin, 5030 Gembloux, Belgium.
Tel: (+32) 81622355; fax: (+32) 81611555; e-mail:
microbio@fsagx.ac.be
Contract/grant sponsor: Fonds National de la Recherche
Scientifique
CCC 0749?503X/98/060527?07 $17.50
1998 John Wiley & Sons, Ltd.
synthetase (AsnRS) is a class II synthetase. This
class is defined by three sequence motifs: motif 1
is part of the dimer interface, motifs 2 and 3 are
constituents of the catalytic site. AsparaginyltRNA synthetase is a class IIb aaRS and is
grouped with the LysRS and AspRS on the basis
of similarities in the N-terminal extensions of
their catalytic domains (Cusack et al., 1991).
Aminoacyl-tRNA synthetases are described as
ubiquitous enzymes but restrictions have to be
made concerning the AsnRS. It has been proposed recently that an AsnRS function could be
absent at least from certain archaebacteria, if not
from the whole archaea domain (Curnow et al.,
1996; Ibba et al., 1997). In the absence of AsnRS,
Asn-tRNAAsn is obtained in a tRNA-dependent
way, involving a misacylation step by the AspRS,
followed by a transamidation in the presence of
Asn or Gln as amide donor (Curnow et al.,
1996).
?. ???????? ?? ??.
528
The AsnRS from the nematode Brugia malayi is
the only eukaryotic AsnRS that has been described
so far (Perrine et al., 1988; Kron et al., 1995).
Although the amino acid sequences of 15 Saccharomyces cerevisiae cytosolic aaRSs have been
determined previously, the information on the
structure and function of eukaryotic aaRSs is far
less abundant than that concerning prokaryotic
aaRSs.
We have recently reported the characterization of the mitochondrial S. cerevisiae AsnRS
(Landrieu et al., 1997). We describe below the
isolation and properties of the yeast cytosolic
AsnRS. Since a conditional defective mutant yeast
strain, lacking a functional cytosolic AsnRS
(Ramos and Wiame, 1979) was available, it was
complemented with a plasmid pool containing
random fragments of yeast genomic DNA. It was
possible to isolate a 6�kb DNA fragment containing the cytosolic AsnRS gene. The mutation
affecting this asparagine auxotroph, called 8556a
strain, was determined. The isolated AsnRS gene
was expressed in Escherichia coli and the purified
recombinant protein was characterized.
MATERIALS AND METHODS
Materials used were obtained from the following
suppliers: Expand Long Template PCR system,
unfractionated E. coli tRNA and unfractionated
brewer?s yeast tRNA from Boehringer; low protein molecular mass standards from BioRad;
and
[35S]dATP[S]
from
L-[14C]asparagine
Amersham; LigATor kit, pET17 plasmid and
BL21(DE3) strain from Invitrogen; oligonucleotides from Genosys; Sequenase version 2�DNA
sequencing kit from US Biochemicals; DEAE
Sepharose CL-6B resin and Sepharose CL-4B resin
from Pharmacia, AcA44 resin from BioSepra.
Yeast, bacteria and growth media
The wild-type S. cerevisiae strain used in this
study was �78b (Bechet et al., 1970). The
asnrs -1 mutant strain 8556a was received from
Dr F. Ramos and is a derivative of �78b previously described by Ramos and Wiame (1979).
The ura3 , asnrs -1 mutant strain S450 was
obtained by mating strains 8556a (MATa,
asnrs -1) and 382?7a (MAT�, ura3 , isogenic to
�78b) and selecting a ura3 , asnrs -1 double
mutant in the diploid progeny, on a minimal
ammonium medium. The AB972 strain is isogenic
to S288C and non-isogenic to �78b.
1998 John Wiley & Sons, Ltd.
YPD medium, minimal ammonium medium and
sporulation medium were prepared as described
elsewhere (Ausubel et al., 1990). Composition of
medium with asparagine as nitrogen source was
0� asparagine, 0�5% yeast nitrogen base without ammonium sulfate and amino acids and 2%
glucose.
E. coli strain BL21(DE3), carrying the isopropyl
thio-�-D-galactoside (IPTG) inducible T7 RNA
polymerase gene (Studier et al., 1990) under the
control of the lacUV5 promoter, was used as host
for production of yeast cytosolic asparaginyltRNA synthetase. Bacterial cultures were grown
on Luria-Bertani medium supplemented with
50 靏/ml of ampicillin.
Gene bank, plasmid and yeast DNA manipulations
The gene bank of �78b chromosomal DNA
was a gift from Dr S. Vissers, ULB-Belgium. It was
constructed by inserting 6?10 kb Sau3A genomic
DNA fragments into the BamHI site of the
centromeric shuttle plasmid pFL38 (Bonneaud
et al., 1991).
Plasmid pG5 contained a 6�kb insert at the
BamHI restriction site of pFL38, including the
1665 bp of an open reading frame corresponding to YHR019 (cosmid YSCH8082, gb/10399/,
position: 3081?1417), 630 bp upstream of the ATG
start codon of YHR019 and 4400 bp downstream
of YHR019.
Yeast strain S450 was transformed by the
lithium acetate procedure of Ito et al. (1983).
Plasmid DNA from yeast transformants were
isolated as described in Ausubel et al. (1990) and
amplified in E. coli. Yeast genomic DNA extraction was adapted from Hoffman and Winston
(1987).
Amplification of the asnrs -1 gene by PCR
Two oligonucleotides of 26 bp (sequence from
144 to 119) and 24 bp (complementary to
sequence from 1666 to 1643), corresponding to
YHR019 flanking sequences, were used respectively as direct and reverse primers, to amplify by
PCR the asnrs -1 mutant gene, directly from
8556a genomic DNA (100 ng). The amplified
fragment contained asnrs -1 flanked by 144 bp
upstream and 1 bp downstream. The unique
fragment obtained under those conditions was
directly cloned in the T-tailed pTAg vector, following the instructions of the ?LigAtor kit?. Clones
from two independent DNA amplifications were
?????
???. 14: 527?533 (1998)
????????? ???????????-t??? ?????????? ????
used to determine the asnrs -1 sequence, in order
to ensure the absence of additional mutations introduced during the PCR by the DNA
polymerase.
Construction of the recombinant plasmid for
expression of YHR019 in E. coli
An NdeI restriction site was created at the ATG
start codon of YHR019 by site-directed mutagenesis using PCR with the pG5 plasmid as template. Synthetic primers allowed the amplification
of a 308 bp fragment. The PCR product was
directly cloned into a pTAg vector and sequenced
to verify the presence of an NdeI site. An 85 bp
NdeI-NdeI fragment and a 2032 bp NdeI-SacI
fragment were subcloned in a pET17 plasmid to
allow the reconstitution of the full-length YHR019
sequence. The correct orientation was controlled
by restriction analysis. The resulting construct was
transferred into E. coli BL21(DE3) strain.
Production and purification of the recombinant
S. cerevisiae cytosolic AsnRS
E. coli BL21(DE3) strain carrying the recombinant plasmid was used to express YHR019.
Expression was induced by addition of IPTG to a
final concentration of 0�m? to the bacterial culture grown at 30C to an A600 of 0�1. The
incubation of the culture was continued for an
additional 3 h. For enzyme isolation, the bacterial
culture was scaled up to an 18 l fermentor and
expression obtained under the same conditions.
The cellular extract was prepared by lysozyme
treatment according to Leberman et al. (1980). The
lysate was centrifuged at 10,000 g and the supernatant subsequently fractionated on a DEAE
Sepharose CL-6B column, equilibrated in Tris?
HCl buffer, pH 7� The elution was performed
with a linear gradient from 10 m?-NaCl to
200 m?-NaCl in Tris?HCl buffer, pH 7� Fractions were collected and assayed using unfractionated yeast and E. coli tRNA. The yeast AsnRS
activity was eluted between 25 and 50 m?-NaCl
and the endogenous E. coli AsnRS between 75 and
100 m?-NaCl. Fractions containing the yeast
AsnRS activity were applied on a Tris?HCl,
pH 7�buffer pre-equilibrated AcA44 gel filtration
column. Active fractions were pooled and ammonium sulfate was added to a concentration of
1�?. This pool was then applied on a CL-4B
Sepharose column equilibrated in Tris?HCl pH 7�buffer containing 1�?-ammonium sulfate. The
1998 John Wiley & Sons, Ltd.
529
recombinant yeast AsnRS was not retained on this
column under those conditions, unlike most of the
remaining minor protein contaminants.
Enzyme activity
The aminoacylation reaction mixture contained 50 m?-Tris, pH 7� 10 m?-MgCl2,
5 m?-ATP
(Boehringer),
0�m?-spermidine,
0�m?-L-[14C]asparagine diluted to 100 cpm/pmol
(Amersham), 8 mg/ml of unfractionated E. coli
tRNA or 8 mg/ml of unfractionated yeast tRNA
and a suitable amount of enzyme. After incubation
at 30C for various times, the reaction was
quenched on Whatman GF/A filters pre-wetted
with 5% TCA. Filters were washed three times
with 5% TCA, once with ethanol, with ethanol/
ether 50:50 (v/v) and finally ether, then dried and
counted.
RESULTS
Isolation of a genomic DNA fragment able to
suppress the mutation of S. cerevisiae strain 8556a
The cytosolic AsnRS gene was isolated by functional complementation of the asparagine auxotrophic 8556a strain previously described as having
a deficient cytosolic AsnRS (Ramos and Wiame,
1979). The corresponding mutant gene was called
asnrs -1 (Ramos and Wiame, 1979). This strain is
only viable on a medium containing asparagine as
the only nitrogen source. A ura3 derivative of
8556a was obtained as described in Materials and
Methods, and called S450. The S450 strain was
transformed with the �78b genomic DNA
library, based on the centromeric plasmid
pFL38, containing the URA3 gene. Restoration of
asparagine prototrophy for transformants was
screened on minimal medium, without uracil and
containing ammonium as nitrogen source. A
recombinant plasmid, called pG5, was isolated
from all uracil prototroph transformants able to
grow on the ammonium medium. We showed that
restoration of the wild-type character was linked to
this recombinant plasmid, as in non-selective cultures, clones loosing the uracil prototrophy also
lost the asparagine prototrophy (data not shown).
The 6�kb Sau3A insertion of the pG5 plasmid
was analysed by restriction site mapping and
partial sequencing. Comparison (BESTFIT from
the University of Wisconsin GCG package) of this
partial sequence with sequences released by the
systematic sequencing programme of the yeast
?????
???. 14: 527?533 (1998)
?. ???????? ?? ??.
530
genome showed that we had isolated a fragment of
chromosome VIII, containing the 1665 bp open
reading frame YHR019 called DED81, the ARG4
gene (YHR018c) and a 1157 bp open reading
frame called YHR017w (Johnston et al., 1994).
Protein encoded by YHR019 is highly similar to
eukaryotic AsnRS
The YHR017w open reading frame was predicted to encode a protein that is not significantly
similar to any sequence in the public databases.
The deduced 555 amino acid sequence encoded
by YHR019 showed 45% amino acid identities
with the AsnRS from B. malayi (Perrine et al.,
1988; Kron et al., 1995). Identities with bacterial
AsnRS were lower (31% with E. coli). The protein
encoded by YHR019 shared 28% identical residues
and 52% similarities with the S. cerevisiae mitochondrial AsnRS (BESTFIT from the University
of Wisconsin GCG package). Alignment of AsnRS
conserved sequences of the enzyme from E. coli,
S. cerevisiae and B. malayi can be found in Kron
et al. (1995).
A multiple sequence alignment of the available
AsnRS protein sequences and the protein encoded
by YHR019 was performed using CLUSTALV
(Higgins and Sharp, 1988). The N-terminal
extensions of the eukaryotic AsnRS, of about 100
amino acids, were not taken into account in the
alignment.
A protein distance matrix was calculated with
the PROTDIST program (PHYLIP package;
Felsenstein, 1993), from the AsnRS multiple
alignment. The phylogenetic tree based on this
matrix was built using the UPGMA option of the
NEIGHBOR-JOINING
program
(PHYLIP
package).
Positions of the organisms in the unrooted
phylogenetic tree, based on the multiple alignment, clearly showed the close relationship of the
protein encoded by YHR019 and other eukaryotic
AsnRS (Figure 1), as they formed a separate
group. The mitochondrial AsnRS emerged alone,
diverging from the eubacterial Gram-positive and
Gram-negative groups, as well as from the
eukaryote group. The Thermus thermophilus
AsnRS, a protein from a Gram-negative bacteria,
was grouped with the Gram-positive Bacillus subtilis. This deviation from the classical phylogenetic
classification of the organisms may be due to the
particular amino acid composition of the T.
thermophilus AsnRS, caused by adaptation to high
growth temperature of this organism.
1998 John Wiley & Sons, Ltd.
Figure 1. Unrooted phylogenetic tree of AsnRS sequences in
radial representation. Sequences used are from the following
organisms: NRSLB from Lactobacillus bulgaricus (P54262),
NRSBS from Bacillus subtilis (P39772), NRSTT from Thermus
thermophilus (P54263), NRSCS from Synechocystis sp.
(P52276), NRSEC from E. coli (P17242), NRSHI from Haemophilus influenzae (P43829), NRSMG from Mycoplasma genitalium (P47359), NRSYC from S. cerevisiae, cytosolic (P38707),
NRSBM from B. malayi (P10723), NRSHS from Homo sapiens
(unpublished sequence, Dr M. Ha?rtlein, personal communication), NRSYM from S. cerevisiae, mitochondrial (P25345).
Isolation of the single mutation leading to the
asparagine auxotrophy of the 8556a strain
Three nucleotides located at positions 107, 1226
and 1256 of the YHR019 open reading frame are
different between the sequence cloned by complementation and the sequence from the GenBank
database. These differences are silent mutations,
probably due to strain polymorphism, as YHR019
was sequenced from strain AB972, which is
non-isogenic to our working strain �78b.
The single mutation responsible for the
asparagine auxotrophy of strain 8556a was shown
to be located at position 1437 of asnrs -1, where a
guanosine is replaced by an adenosine. This substitution was compatible with the chemical mutagen, ethyl methyl sulfonate, used to obtain this
asparagine auxotroph (Ramos and Wiame, 1979).
The replacement of a GGT codon by a AGT
codon causes the substitution of a glycine by a
serine, at position 479, in the encoded protein. A
glycine is strictly conserved at this position in all
known AsnRS sequences.
Expression and purification of a recombinant yeast
cytosolic AsnRS
A T7 RNA polymerase expression system was
used for the expression and purification of the
YHR019 gene product. The YHR019 was subcloned such that the translation initiated at the
?????
???. 14: 527?533 (1998)
????????? ???????????-t??? ?????????? ????
Figure 2. Purification profile of recombinant yeast cytosolic
AsnRS from E. coli. Proteins separated by SDS?PAGE
(Laemmli, 1970) were vizualized by Coomassie Blue staining.
Lane 1: soluble cell extract from induced cells; lane 2: pool of
fractions containing the yeast AsnRS activity after DEAE
Sepharose CL-6B chromatography; lane 3: pool of fractions
containing the AsnRS activity after gel filtration on AcA44
resin; lane 4: pool of the unbound fractions on a Sepharose
CL-4B resin equilibrated in 1�?-ammonium sulfate. M: molecular mass marker proteins. The arrow marks the position of
the recombinant S. cerevisiae cytosolic AsnRS.
original ATG codon. After 3 h of induction, the
yeast cytosolic AsnRS was accumulated to a level
of about 2% total E. coli proteins, as judged by
SDS/PAGE.
The purification protocol is described in
Materials and Methods. The yeast recombinant
AsnRS and the endogenous E. coli AsnRS were
separated after anion exchange chromatography
(data not shown). An SDS/PAGE analysis of the
yeast AsnRS showed that the enzyme has the
expected molecular mass of 62 kDa (Figure 2). The
final product was about 95% pure as judged by
SDS/PAGE. 14C-labelled asparagine incorporation into unfractionated yeast tRNA confirmed
the AsnRS activity of the recombinant protein
(Figure 3).
The tRNA charging specificity of the S.
cerevisiae cytosolic AsnRS enzyme was tested with
unfractionated E. coli tRNA. Cytosolic AsnRS
was unable to aminoacylate efficiently E. coli
tRNA (Figure 3).
DISCUSSION
We propose that the gene DED81, corresponding
to the open reading frame YHR019, encodes the
1998 John Wiley & Sons, Ltd.
531
Figure 3. Aminoacylation of unfractionated E. coli or yeast
tRNA with 14C-labelled asparagine by the recombinant S.
cerevisiae cytosolic AsnRS. Asn-accepting tRNA activity
measured in the unfractionated yeast tRNA with the cognate
enzyme was 3 �. tRNAAsn concentration in the unfractionated
E. coli was 3�� as calculated from the supplier indication.
1 � of enzyme was used for the assay. The activity is expressed
in pmoles of charged tRNA.
S. cerevisiae cytosolic AsnRS, based on the following observations. The DED81 gene was isolated by
screening for functional complementation of a
mutant cytosolic AsnRS in yeast. Enzymatic assay
after expression of YHR019 in E. coli and
purification of the recombinant gene product
showed that this protein has a yeast AsnRS
activity. The existence of high sequence similarities
between the protein encoded by DED81 and
AsnRS from other organisms reinforces the conclusion that the DED81 gene encodes a yeast
AsnRS. The possibility that DED81 could encode
the mitochondrial AsnRS was excluded. Indeed,
with the exception of ValRS and HisRS (Chatton
et al., 1988; Natsoulis et al., 1986), the cytosolic
and mitochondrial aaRSs are encoded by separate
genes in S. cerevisiae. Since the product of the
YCR24 open reading frame, located on chromosome III, was shown to be encoding the mitochondrial AsnRS (Landrieu et al., 1997), we can
reasonably postulate that DED81 is encoding the
cytosolic counterpart. Furthermore, phylogenetic
analysis based on AsnRS sequences showed that
the DED81 gene product clustered with the
eukaryotic enzymes, which would not be expected
for a mitochondrial enzyme, due to the endosymbiotic origin of mitochondria.
The aaRS substrate recognition may have been
modified during evolution as both tRNAs and
aaRS have undergone mutations. We found differences in tRNA specificities between yeast cytosolic
?????
???. 14: 527?533 (1998)
?. ???????? ?? ??.
532
AsnRS and E. coli or yeast mitochondrial AsnRS.
The S. cerevisiae cytosolic AsnRS is not able to use
the heterologous E. coli tRNA as substrate. In
contrast the mitochondrial AsnRS is able to
aminoacylate the E. coli tRNA while yeast unfractionated tRNA is a poor substrate for the
mitochondrial enzyme (Landrieu et al., 1997).
As there are only a few reported yeast mutants
with defective cytosolic aaRS (Hartwell and
McLaughlin, 1968; McLaughlin and Hartwell,
1969; Mitchell and Ludmerer, 1984), only a small
number of the aaRS genes have been cloned by
complementation (Ludmerer and Schimmel, 1985;
Hohmann and Thevelein, 1992; Walter et al., 1983;
Meussdoerffer and Fink, 1983). The 8856a mutant
strain is the first one to be characterized at the
molecular level. It has been shown previously that
the mutant protein encoded by asnrs -1 has a
10-fold increase in Km for asparagine as compared
to that of a wild-type strain (Ramos and Wiame,
1979). We found that the replacement of Gly479
by Ser479 in the cytosolic AsnRS is responsible
for the decreased affinity of the mutant protein
for asparagine. The equivalent position of the
AspRSs, which are closely related to the AsnRSs,
identified with a multiple alignment (data not
shown), is less conserved. Interestingly, a serine
is found at this position in the eukaryotic and
archaebacterial AspRS and a serine or a glycine in
the prokaryotic AspRS. This equivalent position
in the S. cerevisiae AspRS, whose threedimensional structure is known (Ruff et al., 1991;
Cavarelli et al., 1993), is located in the sevenstranded �-sheet of the catalytic core. This position
is flanked by residues important in the binding
of the Asp-adenylate, like the arginine 485 (S.
cerevisiae AspRS numbering, arginine 483 in the
S. cerevisiae AsnRS numbering). The importance of the numerous glycines located in the
�-sheet characteristic of the catalytic site of the
class II synthetases has previously been reported
(Cavarelli et al., 1994). They allow the formation
of a cavity with only a few side chains pointing
towards the substrate (Cavarelli et al., 1994). We
suggest that the introduction of a lateral chain at
position 479 of the cytosolic AsnRS could disturb
the asparaginyl-adenylate formation.
ACKNOWLEDGEMENTS
We thank Dr F. Ramos for providing the 8556a
strain and for encouragement, Dr S. Vissers for the
�78b gene bank and Dr G. Bec for the T7 RNA
1998 John Wiley & Sons, Ltd.
polymerase. This work was supported by a grant
from the Fonds National de la Recherche
Scientifique. I. L. is the recipient of a fellowship
from the Fonds National de la Recherche
Scientifique.
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