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J. Biol. Chem., Vol. 276, Issue 45, 41998-42002, November 9, 2001
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From the Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan
Received for publication, August 24, 2001, and in revised form, September 7, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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L-Azetidine-2-carboxylic
acid (AZC), a toxic four-membered ring analogue of
L-proline, is transported into the cells via proline transporters. It causes misfolding of the proteins into which it is
incorporated competitively with L-proline and thereby
inhibits the growth of the cells. We recently have discovered, on the
chromosome of Saccharomyces cerevisiae Some amino acid analogues are known to induce in cells a transient
stress response comparable with that of heat shock stress (1-3). The
addition of certain amino acid analogues or mild heat shock causes a
rapid increase in the synthesis of heat shock proteins in cells. The
elevated levels of synthesis for this set of proteins begins to
decrease shortly after restoration of the normal amino acids or normal
temperature. Therefore, amino acid analogues have proved to be valuable
reagents for studying cellular metabolism and the regulation of
synthesis of macromolecules in both prokaryotic and eukaryotic cells
(1-3). A toxic four-membered ring analogue of L-proline,
L-azetidine-2-carboxylic acid
(AZC)1, is incorporated into
proteins competitively with L-proline and causes the
synthesis of abnormal misfolded proteins, thereby inhibiting cell
growth in both bacterial and animal cells (4-7). In bacteria such as
Escherichia coli and Serratia marcescens,
L-proline biosynthesis from L-glutamate is
regulated mainly through feedback inhibition by L-proline
of To investigate the cryoprotective effect of L-proline on
the freezing stress of yeast, we previously isolated AZC-resistant mutants from an L-proline-nonutilizing strain of
Saccharomyces cerevisiae (12). In the process of this study,
it was noteworthy that S. cerevisiae strain Thus, in the present study, we analyzed the function of the
MPR1 gene required for AZC resistance in S. cerevisiae and E. coli. We show here by Ala-scan
mutagenesis that the MPR1 gene encodes a novel AZC
acetyltransferase and that Mpr1p belongs to a superfamily of NAT. In
addition, a possible mechanism for AZC resistance by Mpr1p in
vivo will be discussed.
Strains and Plasmids--
The S. cerevisiae strains
with a
An E. coli strain JM109 (recA1
Culture Media--
The media used for growth of S. cerevisiae were SD (2% glucose, 0.67% Bacto yeast
nitrogen base without amino acids (Difco Laboratories)) and YPD (2%
glucose, 1% Bacto yeast extract, 1% Bacto peptone). SD medium
contains ammonium sulfate (0.1%) as the nitrogen source. To examine
the MPR1 gene function, 0.1% proline was used instead of
ammonium sulfate as the sole source of nitrogen. When appropriate,
required amino acids were added to the media for auxotrophic strains.
Yeast strains were also cultured on SD agar plates containing
L-proline analogue, AZC, 3,4-dehydro-DL-proline (DHP), or L-thiazolidine-4-carboxylic acid (TAC). All the
analogues were obtained from Sigma. The E. coli recombinant
cells were grown in M9 medium (21) containing 2% casamino acids and 50 µg/ml ampicillin (M9CA). If necessary, 2% agar was added to solidify the medium.
Subcellular Localization of Mpr1p--
Plasmid pRSMPR was first
constructed by subcloning of the 3.7-kb SacI-SacI
fragment containing the MPR1 gene from pMH1 into the
SacI site of pRS416. The 750-bp
XhoI-AatII fragment containing a mutated
Aequorea GFP gene from pGreenscript A (22) was then ligated
into the NcoI site 3 bp upstream of the termination codon in
the MPR1 gene of pRSMPR by blunt-end ligation. The plasmid in which the GFP gene was fused to the carboxyl terminus of Mpr1p in
the sense orientation was designated as pMPRGFP. The S. cerevisiae CKY2 was transformed by pMPRGFP, and the
Ura+ and AZC-resistant transformants were visualized
with a confocal laser scanning microscopy (INSIGHT PLUS; Meridian Instruments).
Expression of the MPR1 Gene in E. coli--
The enzymes used for
DNA manipulations were obtained from Takara Shuzo. To introduce the
NcoI sites on the amino terminus and carboxyl termini,
polymerase chain reaction (PCR) was carried out with pMH1 as a template
and primers 5'-AGCCATGGATGCGGAATC-3' and
5'-GTCCATGGTTATTCCAT TGAGAGGA-3' (the underlined sequence is the position of a NcoI site). The amplified DNA was
digested with NcoI, and the ends were filled with T4 DNA
polymerase. The blunt-ended DNA was ligated into the SmaI
site of pQE30. The plasmid in which the MPR1 gene was placed
under the T5 promoter/lac operator in the sense orientation
was designated as pQE-MPR. The pQE-MPR carries the entire
MPR1 gene with the sequence coding six consecutive histidine
residues and nine additional amino acid residues
(Gly-Ser-Ala-Cys-Glu-Leu-Gly-Thr-Pro) at the amino terminus. The
nucleotide sequence was confirmed with a Model 377 DNA sequencer
(PE Biosystems). The E. coli strain JM109 was
transformed with pQE-MPR, and the recombinant cells were grown at
37 °C in M9CA medium. When absorbance at 600 nm reached 0.5, IPTG
was added to the culture medium to a final concentration of 1 mM to induce gene expression. After cultivation for 4 h at 37 °C, the cells were harvested by centrifugation, and
cell-free extracts were prepared by sonic oscillation under cooling.
The His-tagged fusion proteins in the soluble fraction were then
purified using the nickel-nitrilotriacetic acid-agarose supplied
by Qiagen.
Site-directed Mutagenesis--
The replacements of
Arg145, Gly146, Gln147,
Lys148, Val149, and Gly150 by Ala
and of Lys148 by Gly in Mpr1p were performed by PCR with
oligonucleotide primers R145A+
(5'-GTGCCCATG*C*T*GGTCAGAA-3'), R145A Northern Blot Analysis--
Northern blot analysis was carried
out using Gene Images random-prime labeling and detection system
(Amersham Pharmacia Biotech). Total RNA from S. cerevisiae
was isolated by the method of Köhrer and Domdey (23). RNA was
separated in 1.0% agarose gel and transferred to nylon membrane. As a
DNA probe, the DNA fragments of the GAP1, PUT4,
MPR1, and ACT1 genes were prepared by PCR with
oligonucleotide primers GAP1+ (5'-TTGACGAAACAGGTTCAGGG-3'), GAP1 Western Blot Analysis--
Total cellular proteins from extracts
of S. cerevisiae and E. coli were separated on
SDS-polyacrylamide gel electrophoresis and transferred to
poly(vinylidene difluoride) membrane. To determine whether Mpr1p
contains an asparagine-linked carbohydrate, the S. cerevisiae extracts expressing the MPR1 gene were also
digested with endoglycosidase H (New England Biolabs) under conditions recommended by the supplier. The wild-type and mutant proteins were
detected by Western blot analysis using the Phototope-Star detection
kit (New England Biolabs) and anti-Mpr1p polyclonal antibody prepared
from the recombinant His-tagged Mpr1p overproduced in E. coli.
Assay of Acetyltransferase Activity--
To determine
acetyltransferase activities, the whole-cell extracts of S. cerevisiae and E. coli strains expressing the
MPR1 gene were prepared by vortexing the cells with glass
beads and by sonic oscillation under cooling, respectively, and used as enzyme sources. The acetyltransferase activity was assayed at 30 °C
by monitoring the increase of 5-thio-2-nitrobenzoic acid (TNB) because
of the reaction of acetyl-CoA with 5,5'-dithiobis(2-nitrobenzoic acid)
(DTNB) described previously with some modifications (24). The initial
rate of the increase in absorbance at 412 nm of the reaction mixture
(final volume, 1 ml) containing 50 mM acetate ammonium (pH
7.5), 1 mM EDTA, 1 mM L-proline,
AZC, DHP, or TAC, 1 mM DTNB, 0.1 mM
acetyl-CoA (Wako Pure Chemical) and enzyme solution was measured, and
that obtained for a solution containing all the materials except
L-proline, AZC, DHP, or TAC (blank) was subtracted. The
reaction rate was calculated using an extinction coefficient for TNB of
15,570 M Data Deposition--
The GenBankTM accession
numbers for Mpr1p, Spt10p, Hat1p, Mak3p, spermidine
N-acetyltransferase, and puromycin
N-acetyltransferase are AB031349, L24435, U33335, M95912,
D25276, and M25346, respectively.
Identification of the MPR1 Gene Product--
The MPR1
mRNA was identified by RNA hybridization analysis of total yeast
RNA as a single 1.0-kb transcript in the strains L5685 and CKY2 (pMH1)
in proportion to the copy number of the MPR1 gene (data not
shown). Bands corresponding to the molecular mass of Mpr1p (~26 kDa)
were also detected in the same strains expressing the MPR1
gene by Western blotting using an anti-Mpr1p polyclonal antibody (data
not shown). Mpr1p was shown to be a nonglycoprotein, based on the
finding that the apparent molecular mass after digestion with
endoglycosidase H was similar to that of the untreated protein (data
not shown), consistent with the fact that the Mpr1p sequence lacks
consensus sites for N-linked glycosylation.
MPR1 Does Not Affect the Expression of Genes Involved in Proline
Uptake--
The Mpr1p sequence of 229 amino acid residues was found to
be homologous only to the amino-terminal sequence of the S. cerevisiae SPT10 (SUD1)-encoded protein with 640 amino
acids, a negative transcriptional regulator (25-27). Both
L-proline and AZC are transported into the yeast cells via
two transporters, the general amino acid permease (encoded by
GAP1) and the proline-specific permease (encoded by
PUT4) (28). We therefore examined whether the
MPR1 gene functions as an Spt10p-like transcriptional
regulator, based on the hypothesis that Mpr1p represses GAP1
and PUT4 genes, leading to the AZC-resistant phenotype.
Northern blot analysis showed quantitatively similar amounts of
GAP1 and PUT4 transcripts in the strains L5685,
LD1014, CKY2 (pMH1), and CKY2 (pYES2) in the presence or absence of AZC (data not shown). Also, when the above yeast strains were grown on a
minimal agar medium containing L-proline as the sole
nitrogen source, the growth did not vary from that observed with
ammonium sulfate as the nitrogen source, suggesting that the proline
permeases function normally even in the presence of the MPR1
gene (data not shown).
Further, Mpr1p did not include any amino acid sequences that are
similar to the DNA binding motifs and the amino-terminal basic domains
found in other yeast transcriptional factors (26, 27). A preliminary
experiment showed that Mpr1-GFP fusion protein stayed primarily in the
cytoplasm and did not localize in the nucleus (data not shown). These
findings indicate that Mpr1p involved in AZC resistance is not a
transcriptional regulator, which does not repress the yeast proline
permease genes GAP1 and PUT4.
Effect of the MPR1 Gene on Growth Inhibition by Other
L-Proline Analogues--
To further analyze the function
of the MPR1 gene, various S. cerevisiae strains
were cultured on SD agar plates containing each of three toxic
L-proline analogues (AZC, DHP, and TAC) (Fig. 1). The MPR1 gene was shown to
confer only a four-membered ring analogue-AZC resistance to the yeast
cells, not a five-membered ring DHP and TAC. Therefore, Mpr1p is
supposed to work on AZC directly, and bacterial cells such as E. coli, which are naturally AZC-sensitive, would therefore become
resistant to AZC when the MPR1 gene is successfully
expressed in the cells. In addition, it is worth noting that the strain
with the Acetyltransferase Activity of Mpr1p--
Based on the hypothesis
described above, we constructed an expression plasmid pQE-MPR for the
MPR1 gene under the IPTG-inducible promoter. Surprisingly,
the E. coli JM109 cells expressing the MPR1 gene
in the presence of IPTG were capable of growing on agar plates
containing AZC, thereby acquiring the AZC-resistant phenotype, whereas
the vector-harboring cells failed to grow on AZC-containing plates
(Fig. 2A). Based on the
finding that Mpr1p sequence was a member of the NAT superfamily, we
examined the acetyltransferase activities for L-proline and
various analogues (AZC, DHP, and TAC) in total cell extracts from the
S. cerevisiae L5685, CKY2, and E. coli JM109
cells expressing the MPR1 gene (Fig. 2B). Among the substrates used, only acetyltransferase activities of AZC were
clearly detected in proportion to the level of gene expression. No
measurable activities were observed from the cell extracts harboring
the vector only and with the substrates of L-proline, DHP,
or TAC having a five-membered ring structure (data not shown). These
results showed that the MPR1 gene encodes an AZC
acetyltransferase that does not acetylate the five-membered ring
compound L-proline, DHP, or TAC.
Ala-scan Mutational Analysis of Mpr1p--
The NAT superfamily,
which includes over 50 members, has four conserved regional motifs,
A-D (17). In particular, the most highly conserved motif A would be
involved in the binding of acetyl-CoA (29, 30) and has a short
consensus sequence, (Q/R)XXGX(G/A), in
common with members of the superfamily (Fig.
3A). Further, point mutations
in any of these residues were found to impair the activities of NAT
enzymes (31, 32). In the case of the Mpr1p sequence, the corresponding
region consists of
Arg145-Gly146-Gln147-Lys148-Val149-Gly150,
with high degrees of similarity to other members, excepting position
148. To examine the function of Mpr1p as a member of the NAT
superfamily, the six consecutive residues were individually changed to
Ala by site-directed mutagenesis, and the resulting mutants were
characterized in terms of AZC resistance and acetyltransferase activity. In addition to these mutations, Lys148 was
changed to consensus Gly. All mutant genes were expressed using the
original promoter and terminator of MPR1 to obtain normal levels of gene expression. The S. cerevisiae strain CKY2
with a S288C background was transformed with these plasmids, and the Ura+ transformants were cultivated in SD medium
supplemented with His. The levels of MPR1 gene expression
were almost the same for all mutants, based on densitometric estimates
of the amounts of accumulated Mpr1p in the soluble fraction by Western
blot analysis (Fig. 3B). Fig.
4C shows the growth phenotype
on AZC-containing medium and the AZC acetyltransferase activities of
strains having various mutant Mpr1 proteins. The enzymes in the
whole-cell extracts were assayed without further purification. The Ala
substitution of the highly conserved residues Arg145,
Val149, and Gly150 led to a growth defect in
AZC and simultaneously resulted in the complete loss of AZC
acetyltransferase activity. These residues in Mpr1p that corresponded
to the consensus sequence of the NAT superfamily were found to be
necessary for activity. On the other hand, G146A and Q147A
mutants displaying AZC resistance in the growth assay also retained a
significant level of activity for AZC acetylation. These residues are
unlikely to be crucial for the function of Mpr1p. It was also shown
that Gly or Ala substitution at position 148 had no influence on this
activity. This is probably explained by the fact that Lys or Arg
naturally occupies this position in some NAT members (17). AZC
resistance and acetylation activity in the strains producing the
various Mpr1 proteins correlated with each other. These results
strongly suggest that Mpr1p is a novel member of the NAT
superfamily.
AZC Acetylation by Mpr1p--
The His-tagged fusion Mpr1p was
purified from the recombinant E. coli cells expressing the
MPR1 gene as described under "Experimental Procedures."
A 2-ml reaction mixture containing 100 mM ammonium acetate
(pH 7.5), 2 mM EDTA, 50 mM AZC (1 mg), 50 mM acetyl-CoA, and 15 milliunits of the recombinant
enzyme was incubated at 30 °C. The gradual decrease in AZC in the
reaction mixture was observed with the time of incubation, whereas the
initial content of AZC still remained under this condition without the
enzyme (Fig. 4A). However, no new peak corresponding to
acetylated AZC appeared, possibly because the ninhydrin reagent used
for amino group detection did not bind to acetylated AZC. Based on
these findings and the amino acid sequence similarity with the NAT
superfamily, we now believe that AZC is converted to
N-acetyl AZC by Mpr1p in the cytoplasm (Fig. 4B),
and consequently, N-acetyl AZC no longer replaced
L-proline during the biosynthesis of protein.
In general, unusual imino acid AZC is incorporated into the
nascent polypeptide chain by competition with L-proline in
the formation of AZC-tRNA, given rise to by virtue of its structural similarity to L-proline. Stereochemical considerations
suggest that AZC in the protein will change the normal Mpr1p was found be a novel acetyltransferase with high substrate
specificity for AZC. It is unlikely that this enzyme is responsible for
AZC degradation, because the yeast cells expressing the MPR1 gene were unable to grow in media containing AZC as the sole nitrogen source (data not shown). Our findings suggested that
N-acetylation of AZC is catalyzed by Mpr1p in the cytoplasm,
which presumably transfers acetyl groups from acetyl-CoA to The question arises as to why the S. cerevisiae strain
Neuwald and Landsman (17) reported that proteins belonging to the NAT
superfamily have been grouped by function as histone acetyltransferases, transcriptional regulators, protein
acetyltransferases, metabolic enzymes, participants in detoxification
and drug resistance, or precise function unknown. In particular, the
functional significance of the highly conserved residues in motifs A
and B, involved in the binding of acetyl-CoA, have been confirmed by
site-directed mutagenesis experiments with many enzymes (29, 36, 37). To further analyze the function of Mpr1p as a member of the NAT superfamily, we performed an Ala-scan mutagenesis through a six-amino acid sequence (residues 145-150) possibly involved in the binding of
acetyl-CoA to Mpr1p. These data, shown in Fig. 4, demonstrated a clear
correlation between residues within Mpr1p that are critical for the
acetylation of AZC and residues that are absolutely required for AZC
resistance. V149A and G150A mutations severely affected AZC
acetyltransferase activity, suggesting that Ala substitutions at these
positions may interfere with acetyl-CoA binding to Mpr1p. A more
detailed sequence comparison among members of the superfamily revealed
that branched-chain amino acids such as Ile, Leu, and Val mainly occupy
position X, corresponding to Val149 (17). It is
also possible that the steric hindrance, occurring via the G150A
mutation that introduces a side chain, leads to reduced AZC
acetyltransferase activity in Mpr1p. In contrast, Ala could be replaced
with Lys at position 148 in terms of AZC resistance and
acetyltransferase activity, so the positive charge is not likely to be
indispensable for these functions. Recently, the crystal or solution
structures of several proteins belonging to the NAT superfamily were
determined in a complex with acetyl-CoA (29, 30, 37). In addition to a
short consensus of residues (Q/R)XXGX(G/A)
in motif A, it was also shown that other hydrophobic residues in motifs
A and B also contribute to form an acetyl-CoA binding pocket and to
bind acetyl-CoA, respectively (38). Given that Mpr1p does not contain
any carbohydrates (data not shown) and remains active in the E. coli cells, a crystallization of the recombinant protein
overexpressed in E. coli is now attempted to determine the
tertiary structure.
In terms of medical applications, naturally occurring AZC was
identified as an antimutagen against the spontaneous mutation of
bacteria (39) and was tested for its antitumor activity in tissue
culture (40). Therefore, when the MPR1 gene is expressed in
normal cells, the toxic AZC is expected to be an efficacious agent. The
MPR1 gene may also be promising as a new positive selection marker that confers AZC resistance in a wide variety of organisms, because AZC is incorporated, in place of L-proline, into
the newly synthesized proteins. Construction and availability of
versatile vectors with the MPR1 gene for E. coli,
yeast, and animal cells will be published elsewhere.
1278b, a novel
gene MPR1 required for the resistance of 1278 background
strains to toxic AZC. This gene was missing in the particular yeast
strain used for the genomic sequence determination. Although the
protein sequence was homologous to that of the S. cerevisiae transcriptional regulator, Mpr1p did not affect the
expression of genes involved in proline uptake. However, gene
expression in Escherichia coli and enzymatic analysis showed that the MPR1 gene encodes a novel AZC
acetyltransferase, by which L-proline itself and other
L-proline analogues are not acetylated. Mpr1p was
considered to be a member of the N-acetyltransferase superfamily based on the results of an Ala-scan mutagenesis through the
highly conserved region involved in binding acetyl-CoA in members of
the superfamily. Our findings suggest that Mpr1p detoxifies AZC by
acetylating it in the cytoplasm. This enzyme might be utilized as a
selective marker in a wide variety of organisms, because the cells
expressing the MPR1 gene acquire the AZC-resistant phenotype.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glutamyl kinase, a key enzyme in the pathway (8, 9). Because
this enzyme is also feedback-inhibited by L-proline
analogues such as AZC, L-proline-overproducing strains have
been successfully isolated from
L-proline-analogue-resistant mutants, which have a mutation
in the proB gene coding for -glutamyl kinase that causes
desensitization to feedback inhibition (10, 11).
1278b showed
greater AZC resistance than that of other laboratory strains, including
the genome project strain S288C. The strain 1278b, mainly used in
European laboratories, is known to have unique genetic features for
nitrogen metabolism and morphological characteristics for diploid
pseudohyphal development and haploid invasive growth (13-15). We
recently isolated novel genes involved in AZC resistance from the
genomic library of strain 1278b (16). Intriguingly, the novel genes
MPR1 and MPR2 (sigma 1278b gene for
L-proline-analogue resistance)
required for AZC resistance were present on chromosomes XIV and X of
strain 1278b, respectively, but were absent in other laboratory
strains (S288C etc.). The primary structure of the predicted Mpr
proteins was found to be a member of the N-acetyltransferase
(NAT) superfamily (17), although one amino acid change at position 85 occurred between the MPR1 and MPR2 genes. Both
MPR genes are expressed in other S. cerevisiae
strains, where they play global roles in AZC resistance (16). Previous
reports have described only a point mutation or a deletion of a few
bases, based on comparisons of certain genes between 1278b and S288C
strains (18-20). To our knowledge, ours is the first report of novel
genes present in strain 1278b and absent in other laboratory
strains. However, the detailed function of the MPR genes in
AZC resistance has been unclear.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1278b background used in this study were L5685
(MATa ura3-52 trp1 MPR1 MPR2) (supplied by G. Fink)
and LD1014 (MATa ura3-52 trp1 mpr1::URA3
mpr2::TRP1). For MPR1 and MPR2
genes disruption, the 3.3-kb SacI-SacI fragment containing mpr1::URA3 and the 3.7-kb
SacI-SacI fragment containing mpr1::TRP1 were integrated into the
MPR1 and MPR2 locus in strain L5685 by
transformation (16). Strains CKY2 (MATa ura3-52 his4-619) with a S288C background (supplied by C. Kaiser) was also used in this
study. Plasmids pMH1 (16) and pMPR-BM, both of which contain the
MPR1 gene inserted into a yeast multicopy plasmid pYES2
(Invitrogen), were used to express the MPR1 gene in S. cerevisiae strain S288C. A centromere plasmid pRS416 (Stratagene)
harboring URA3 gene was used for construction of chimeric
gene encoding Mpr1p and green fluorescent protein (GFP).
(lac-proAB) endA1 gyrA96 thi-1 hsdR17 relA1
supE44/(F' traD36 proAB+
lacIq ZM15)) and the
isopropyl-
-D-thiogalactopyranoside (IPTG)-inducible vector pQE30 (Qiagen) with the sequence coding six consecutive histidine residues at the 5' end of cloning sites were used for the
expression of the MPR1 gene in E. coli.
(5'-TTCTGACCA*G*C*ATGGGCAC-3'), G146A+ (5'-CCCATAGAGC*TCAGAAGCTT-3'),
G146A (5'-AACCTTCTGAG*CTCTATGGG-3'), Q147A+ (5'-ATAGAGGTG*C*T*AAG
GTTGGCTAC-3'), Q147A (5'-GTAGCCAACCTTA*G*C*ACCTCTAT-3'), K148A+
(5'-GAGGTCAGG*C*T*GTTGGCTA-3'), K148A (5'-TAGCCAACA*G*C*CTGACCTC-3'), V149A+ (5'-GTCAGAAGGC*TGGCTAC-3'), V149A (5'-GTAGCCAG*CCTTCTGAC-3'), G150A+ (5'-AGAAGGTTGC*T*TACAGGCTTG-3'), G150A (5'-CAAGCCTGTA A*G*CAACCTTCT-3'), K148G+ (5'-AGGTCAGG*G*T*GTTGGCTAC-3'), and K148G
(5'-GTAGCCAACA*C*C*CTGACCT-3'), respectively, using a Gene Amp PCR
system 2400 (PE Biosystems). The asterisks show the locations of
mismatches. A plasmid pMPR-BM was constructed by ligating the 1.6-kb
BglII-MluI fragment containing the
MPR1 gene from pMH1 to the large fragment of pYES2 digested
with BamHI and MluI and used as a template DNA
for site-directed mutagenesis. In addition, primers 5'-CACAG
TTTGACAAAACAGGG-3' and 5'-CGACGCGTCGTTATTCGTTC-3' were
synthesized to complement regions upstream and downstream of the
EcoRI restriction sites in pMPR-BM. The unique
amplified band of 630 bp was digested with EcoRI to recover
the 340-bp fragment and ligated to a 6.9-kb fragment of plasmid pMPR-BM
digested with EcoRI. The mutations were confirmed by DNA sequencing.
(5'-TGCT GGGATGAAAAGCTTCC-3'), PUT4+ (5'-GATTATGGACGTGGACTTGG-3'),
PUT4 (5'-AATGAGAGAGAACCACACGG-3'), MPR1+
(5'-GCTCGAGAAGCTTCGAATGC-3'), MPR1 (5'-TCAAAATTCCGGCATGAGGC-3'), ACT1+ (5'-CGGAATTCCTCTCCCATAA CCTCCTA-3'), and ACT1
(5'-CGGGATCCGGGCTCTGAATCTTTCGT-3'), respectively. Each unique amplified
band was purified from agarose gel, denatured, and labeled according to
the protocol recommended by the supplier.
1 cm1. One unit is
defined as the amount of enzyme catalyzing the formation of 1 µmol
TNB/min at 30 °C. Protein concentrations were determined with a
Bio-Rad protein assay kit. Bovine serum albumin was used as the
standard protein. The amount of AZC in the reaction mixture was
quantified with an amino acid analyzer (L-8500A; Hitachi).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1278b background, regardless of the MPR genes,
was less sensitive to DHP and TAC relative to the strain with the S288C
background.
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Fig. 1.
Mpr1p confers only AZC resistance to the
yeast cells. The structural formula of each L-proline
analogue is shown. Approximately 106 cells of each strain
and serial dilutions of 10 1 to 103 (from
left to right) were spotted onto SD plates in the
absence (analogue) and presence (+AZC,
+DHP, and +TAC) of each analogue. Plates were
incubated at 30 °C for 3 days.
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Fig. 2.
The MPR1 gene encodes a
novel AZC acetyltransferase. A, expression of the
MPR1 gene in E. coli. Approximately
107 cells of E. coli JM109 carrying the
MPR1 gene on the IPTG-inducible expression plasmid and the
serial dilution 10 1 to 103 (from
left to right) were spotted onto M9CA plates with
0.1 mM IPTG in the absence () and presence 100 µg/ml AZC (+). Plates were incubated at 37 °C for 1 day. B, AZC acetyltransferase activities of the E. coli and S. cerevisiae cells expressing the
MPR1 gene. The soluble fractions from the indicated strains
were used as enzyme sources. The data shown are means from three
independent experiments. The variations in the values are less than
5%.
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Fig. 3.
Mpr1p belongs to the NAT superfamily.
A, amino acid sequence alignment of a motif A involved in
acetyl-CoA binding in the NAT superfamily members with yeast Hat1p
(yHAT1), yeast Spt10p (ySPT10), yeast Mak3p
(yMAK3), E. coli spermidine NAT
(eSNAT), and Streptomyces alboniger puromycin NAT
(sPNAT). The numbering above the amino acid
sequences refers to Mpr1p. B, Western blot analysis of the
extracts of S. cerevisiae CKY2 transformants. Total cellular
proteins (~30 µg) were subjected to a 15% polyacrylamide gel and
were detected by using an anti-Mpr1p polyclonal antibody. C,
growth phenotype on AZC-containing medium and AZC acetyltransferase
activities of S. cerevisiae CKY2 transformants.
Approximately 106 cells of each strain and serial dilutions
of 10 1 to 103 (from left to
right) were spotted onto SD plates containing
L-histidine and 300 µg/ml AZC. Plates were incubated at
30 °C for 3 days. AZC acetyltransferase activity was assayed by
using the enzyme solution prepared from each strain grown in SD medium
at 30 °C. The data shown are means from three independent
experiments. The variations in the values are less than 5%.
View larger version (19K):
[in a new window]
Fig. 4.
Mpr1p is supposed to be an AZC
N-acetyltransferase. A, time course of
AZC concentration in the reaction mixture. The enzymatic reaction was
performed as described under "Results." Purified His-tagged fusion
Mpr1p (closed circles) and the soluble proteins from the
E. coli cells carrying vector pQE30 (open
circles) were used as the enzyme sources. AZC content at the
indicated time was quantified with an amino acid analyzer.
B, proposed scheme for the AZC acetyltransferase reaction.
AZC is converted to N-acetyl AZC by Mpr1p.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helical
structure of a polypeptide to one having an angle about 15° smaller
than that by L-proline, leading to an altered tertiary
structure of the protein (5). Therefore, AZC shows growth-inhibitory
and toxic activities in a wide variety of cells (4-7). It is
noteworthy that AZC is widely distributed in many plants belonging to
the Lilaceae family (33, 34), which includes Convallaria
majalis (lily of the valley) and Polygonatum
multiflorum (a Solomon's seal), at high concentrations. Although
the physiological role of AZC in Lilaceae plants is not clear, AZC is
supposed to protect them from attack by vertebrates and/or microbes.
Some plant species that produce AZC were found to have
L-proline-activating enzymes that differentiate between
L-proline and AZC (34). It is speculated that only
L-prolyl-tRNA is then formed so that L-proline
alone is incorporated into proteins. In recent studies on the
biosynthesis of AZC, labeled L-methionine was incorporated
into AZC in lily of the valley plants (35), the result of which was in
agreement with the earlier hypothesis by Leete (33) that AZC is formed by the intramolecular displacement of thiomethyladenosine by the -amino group of S-adenosylmethionine. In contrast, little
is known about its metabolic pathway in plants.
-imino
groups of AZC. It can be assumed that N-acetyl AZC is no
longer recognized by L-prolyl-tRNA synthetase by which both
L-proline and AZC bind to ATP, thereby only
L-proline is incorporated into proteins, leading to cell
growth even in the presence of AZC. The structural determination of
N-acetyl AZC is needed to prove this hypothesis and is
currently in progress.
1278b possesses the MPR genes, although a hypothetical
protein with significant sequence similarity to Mpr1p was found in
fission yeast (16). We consider the following two possibilities for the
physiological role of MPR genes. One is that the S. cerevisiae strain 1278b in nature made its habitat on the
Lilaceae or in soils around these plants. To avoid the toxicity of AZC,
the strain may have successfully acquired the MPR1 gene
involved in AZC resistance from the Lilaceae or some unknown organism
during the evolutionary process. It would be of interest to seek the
common ancestor of the MPR1 gene to further understanding of
the mechanism of AZC resistance. Therefore, this work might contribute
to our understanding of the yeast in the natural habitat. Another
possibility is that Mpr1p has a bona fide substrate(s)
(i.e. not AZC) in the S. cerevisiae strain
1278b. To identify the natural substrate for Mpr1p, we are now
analyzing the growth phenotype on media containing various carbon and
nitrogen sources, and examining the intracellular contents of the
metabolites and the genome-wide transcriptional response to AZC in the
wild-type strain and the mpr1 mpr2 disruptant.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Drs. Gerald R. Fink and Chris A. Kaiser for providing yeast strains, Dr. Satoshi Inouye for the gift of plasmid pGreenscript A, and Drs. Masaru Wada and Masakazu Takahashi for helpful comments on this work.
| |
FOOTNOTES |
|---|
* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (to H. T.).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.
To whom correspondence should be addressed: Dept. of Bioscience,
Fukui Prefectural University, 4-1-1 Kenjojima, Matsuoka-cho, Fukui
910-1195, Japan. Tel.: 81-776-61-6000; Fax: 81-776-61-6015; E-mail:
hiro@fpu.ac.jp.
Published, JBC Papers in Press, September 12, 2001, DOI 10.1074/jbc.C100487200
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
AZC, L-azetidine-2-carboxylic acid;
NAT, N-acetyltransferase;
IPTG, isopropyl--D-thiogalactopyranoside;
DHP, 3,4-dehydro-DL-proline;
TAC, L-thiazolidine-4-carboxylic
acid;
PCR, polymerase chain reaction;
kb, kilobase pair;
GFP, green fluorescent protein;
bp, base pair;
TNB, 5-thio-2-nitrobenzoic
acid;
DTNB, 5,5'-dithiobis(2-nitrobenzoic acid).
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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