From the Department of Biochemistry II, Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan
Received for publication, November 26, 2000, and in revised form, March 19, 2001
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ABSTRACT |
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The mechanism of the regulatory
degradation of ornithine decarboxylase (ODC) by polyamines was studied
in fission yeast, Schizosaccharomyces pombe. To regulate
cellular spermidine experimentally, we cloned and disrupted
S-adenosylmethionine decarboxylase gene (spe2)
in S. pombe. The null mutant of spe2 was devoid
of spermidine and spermine, accumulated putrescine, and contained a
high level of ODC. Addition of spermidine to the culture medium
resulted in rapid decrease in the ODC activity caused by the
acceleration of ODC degradation, which was dependent on de
novo protein synthesis. A fraction of ODC forming an inactive
complex concomitantly increased. The accelerated ODC degradation was
prevented either by knockout of antizyme gene or by selective
inhibitors of proteasome. Thus, unlike budding yeast, mammalian type
antizyme-mediated ODC degradation by proteasome is operating in
S. pombe.
Polyamines (putrescine, spermidine, and spermine) are biologically
ubiquitous compounds that have been implicated in many aspects of
growth and development in a wide range of organisms (1-3), but their
precise function is largely unknown. The biosynthesis of polyamines in
yeast and most of animals depends on the decarboxylation of ornithine
to putrescine by ornithine decarboxylase
(ODC,1 EC 4.1.1.17).
Subsequent attachment of an aminopropyl moiety forms spermidine, and
the second aminopropyl transfer to spermidine yields spermine.
Decarboxylated S-adenosylmethionine serves as the
aminopropyl donor, which is produced by S-adenosylmethionine decarboxylase (AdoMetDC, EC 4.1.1.50). Both the decarboxylating enzymes
are highly regulated and subject to control by cellular polyamines.
In mammalian cells, ODC is negatively regulated in response
to the increase in cellular polyamines mainly through a unique mechanism mediated by a protein termed antizyme (4, 5). Synthesis of
antizyme requires translational frameshifting, which occurs at the end
of the initiating frame or open reading frame 1 (ORF1) (6, 7). The
ribosome shifts its reading frame to the +1 frame and continues to
decode the second ORF to synthesize the entire antizyme protein. The
second ORF of antizyme encodes all the known functions of the protein
(8-10). Polyamines stimulate the frameshifting and thus control the
level of antizyme. Antizyme binds to ODC monomer preventing formation
of the homodimeric active enzyme. The ODC associated with antizyme is
rapidly degraded by the 26 S proteasome without ubiquitination
(11-13). In addition, antizyme negatively regulates the polyamine
transporter (14, 15). Mammalian cells contain another regulatory
protein, antizyme inhibitor, a homolog of ODC without the
decarboxylating activity (16). It binds to antizyme with a higher
affinity than ODC and releases active ODC from the inactive
antizyme-ODC heterodimer.
At least in two lower eukaryotes, polyamine-induced destablization of
ODC has been studied in detail. In Saccharomyces cerevisiae, spermidine-induced ODC destabilization seems to be dependent on protein
synthesis (17, 18), and the 26 S proteasome is responsible for the
proteolysis of ODC (18, 19). However, computer search of an antizyme
homolog in the completed genome of S. cerevisiae has been
negative (20). In a filamentous fungus, Neurospora crassa,
spermidine and putrescine accelerate the turnover of ODC protein (21).
Recently, a presumed antizyme homolog has been noted in the N. crassa genome (AF291578, MIPS Neurospora data base).
Although S. cerevisiae and the fission yeast,
Schizosaccharomyces pombe, are both ascomycetous fungi, they
are almost as divergent each other as between these and mammals (22).
S. pombe is often more similar to mammalian cells than
S. cerevisiae in various aspects. Interestingly, the
frameshift signal of mammalian antizyme directs the correct +1 shift in
S. pombe, but aberrant In the present paper, we studied the mechanism of ODC repression by
spermidine in S. pombe. In order to be able to regulate cellular spermidine experimentally, we cloned and disrupted S. pombe gene for AdoMetDC (spe2). We show that the null
mutant of spe2 ( Materials--
Proteasome inhibitors,
clasto-lactacystin Yeast Strains and Culture--
S. pombe strain JY745
(ura4-D18 leu 1-32 ade6-M210 h Cloning and Sequencing of Spe2--
Degenerated oligonucleotide
primers (sense (5'-TTYGARGGNYHNGARAARYTNYTNGA-3') and antisense
(5'-GTNGTNCCRCANGTNWTNARDAT-3'), where R = A or G; Y = T or
C; W = A or T; D = A, G, or T; H = A, C, or T; N = A, G, C, or T), were designed based on multiple alignment of AdoMetDC
from several eukaryotic organisms on the data base and used to amplify
a fragment of spe2 gene from S. pombe genomic DNA
with PCR. The product was labeled with [ Disruption of spe2--
One-step gene replacement was performed
to disrupt spe2 as follows (Fig. 1B). S. pombe ura4 gene was taken from pREP2 (28) as a HindIII
fragment and cloned into pBluescript SK ( Enzyme and Polyamine Assays--
For AdoMetDC assay, cells were
grown as described above and harvested at an
A600 of 1.0. Cells from 3 ml of culture were
collected with centrifugation at 1,000 × g, 4 °C
for 10 min, and were suspended in 0.3 ml of the extraction buffer
containing 50 mM Tris-HCl (pH 7.2), 1 mM DTT,
0.1 mM EDTA, and 2.5 mM putrescine. The
suspensions were transferred to 2-ml screw-cap tubes (Sarstedt)
together with 0.4 g of zirconia/silica beads (0.5 mm diameter,
Biospec, Tokyo). The cells were broken by a Bead Beater Mini (Biospec)
with three 1.5-min bursts. The cell lysate was centrifuged at
12,000 × g, 4 °C for 30 min with a microcentrifuge
to collect the supernatant (extract). The reaction mixture included 40 mM Tris-HCl (pH 7.2), 1 mM DTT, 0.1 mM EDTA, 0.3 mg/ml bovine serum albumin, 0.1 mM putrescine, and 8 µM
S-adenosyl-4-[carboxyl-14C]methionine
(59 mCi/mmol, Amersham Pharmacia Biotech), and 50 µl of cell extract
in a total volume of 125 µl. The 14CO2
released during the reaction at 37 °C for 1 h was trapped onto
a filter paper disc soaked with 10 µl of 10% KOH and counted with scintillant.
ODC activity was measured using
L-[14C]ornithine as a substrate essentially
as described (30). The cells were harvested at the indicated time and
disrupted as above in extraction buffer containing 50 mM
Tris-HCl (pH 7.5) and 1 mM DTT. One unit of ODC activity is
defined as the amount releasing 1 nmol of 14CO2
from ornithine/h at 37 °C.
To measure the polyamines, S. pombe cells were collected
from 2 ml of the culture with centrifugation at 1,000 × g, 4 °C for 10 min and washed twice with
phosphate-buffered saline (20 mM sodium phosphate, 140 mM NaCl, pH 7.4). Each pellet was resuspended in 0.2 ml of
phosphate-buffered saline, and a part (50 µl) was mixed with the same
volume of 8% perchloric acid. The mixture was vortexed, kept on ice
for 5 min, and centrifuged at 12,000 × g, 4 °C for
5 min. The supernatant (10 µl) was subjected to polyamine analysis on
high performance liquid chromatography and fluorometry as described
(31).
Protein concentration of the cell extracts was measured by the Bradford
method (32). Protein content of the cells in the suspension for
polyamine analysis was assayed by BCA kit (Pierce). Bovine serum
albumin was used as standard in both cases.
Expression of ODC in E. coli--
We have isolated the cDNA
and genomic DNA of S. pombe
ODC.2 Details of bacterial
expression constructs of ODC are available from the authors upon
request. Briefly, the 1296-bp fragment containing the ORF of S. pombe ODC cDNA was amplified with PCR and cloned into either
pET3a (Novagen) at the NdeI-BamHI sites or
pGEX-3T (Amersham Pharmacia Biotech) at the
BamHI-EcoRI sites. The constructs were verified
by sequencing. The resultant expression plasmids, pET3a-SPO and
pGEX-SPO, were used for transformation of E. coli strains
BL21 (DE3) and BL21, respectively.
Production of Antibody against ODC and Western
Blotting--
E. coli BL21 carrying pGEX-SPO was grown in
the presence of 0.5 mM
isopropyl-1-thio- Measurement of Inactive ODC Complex--
The untagged ODC was
induced by 0.3 mM
isopropyl-1-thio- Cloning of S. pombe spe2 Gene--
A region of spe2
containing the proenzyme cleavage site was amplified with degenerated
PCR. The product (249 bp) was used in screening of an S. pombe cDNA library as the probe. Several clones with the
identical cDNA insert of 1,421 bp were isolated. The cDNA
contained the entire ORF that encodes a polypeptide of 378 amino acids
corresponding to the AdoMetDC proenzyme with a calculated molecular
mass of 42,711Da (Fig. 1A).
The amino acid sequence deduced from the cDNA was 36% and 35%
identical to the S. cerevisiae and human counterparts,
respectively. The highly conserved proenzyme cleavage sequence
YLLSE
Screening of the S. pombe genomic DNA library identified
seven overlapping clones encompassing an 8.2-kb region that covers the
entire ORF. Comparison of the genomic and the cDNA sequences revealed that spe2 gene contains no intron (Fig.
1B). Searching the data base showed that spe2 is
located at the close 3' vicinity of the cut14 gene on the
S. pombe genome (39).
Disruption of spe2--
The spe2 gene was disrupted by
the single-step gene replacement (Fig. 1B). Southern
hybridization confirmed the replacement of the single-copy
spe2 gene with homologous recombination (data not shown).
AdoMetDC activity in the extracts of exponentially growing cells was measured.
Changes in ODC Activity in Wild Type and Protein Synthesis-dependent Degradation Accounts for
Accelerating ODC Decay--
Spermidine (100 µM) was
added to the Detection of Complex Form of ODC and Increase in Its Ratio by
Spermidine--
In mammalian cells, antizyme binds to ODC, forming an
inactive heterodimer from which active ODC can be released by adding an
excess amount of DFMO-ODC (30) or antizyme inhibitor (16). To test if a
similar ODC-antizyme complex exists in S. pombe, we prepared
S. pombe ODC inactivated with DFMO and used it in the
competitive assay. A significant increase in ODC activity was observed
when DFMO-ODC was added to the cellular extracts (Fig.
6A). Addition of spermidine to
the culture medium increased the ratio of the gain over the total ODC
activity. Cycloheximide blocked the increase. A virtually identical
result was obtained by the use of rat antizyme inhibitor instead of
DFMO-ODC (Fig. 6B). Spermidine thus increased the fraction
of S. pombe ODC that forms an inactive complex. The ratio of
the complex form to the total ODC was up to 25% after spermidine
treatment, although 98% of the initial ODC activity disappeared in
6 h. It is therefore likely that the ODC associated with antizyme
is degraded rapidly.
Involvement of Antizyme in ODC Degradation--
To confirm that
antizyme is the protein factor that is induced by spermidine and
promotes the degradation of ODC, we employed a knockout strain of
S. pombe antizyme ( Effects of the Proteasome Inhibitors on ODC Degradation--
The
26S proteasome has been shown to catalyze ODC degradation in both
animals and S. cerevisiae (11, 18, 19). We tested if the
proteasome is also involved in the accelerated ODC decay in S. pombe using inhibitors of proteasome.
clasto-Lactacystin Mammalian antizyme represents a unique regulatory protein with a
number of novel features, namely (i) expression and induction through
translational frameshifting, (ii) the function as a non-ubiquitin stimulator for proteolysis of ODC by the proteasome, and (iii) dual
activity on ODC and polyamine transporter. The frameshift induction
serves as a polyamine sensor. The feedback system allows both the
maintenance of the cellular polyamines within a certain range and their
appropriate fluctuation. The molecular mechanisms of antizyme
functions, however, have not been fully understood. In addition to the
availability of genetics, recent identification of antizyme in S. pombe (25) makes the organism particularly an attractive system to
study the mechanisms of antizyme functions.
The wild type S. pombe cells contain a high endogenous level
of spermidine, which is not readily changed by exogenous spermidine. To
study the feedback control of ODC by exogenous polyamines, use of
mutants lacking spermidine synthesis appears to be useful as shown in
S. cerevisiae (17, 18). Therefore, we intended to clone and
disrupt S. pombe gene for AdoMetDC, spe2.
The Wild type S. pombe cells contain very low basal level of
ODC. It was induced by changing medium, but subject to rapid repression due probably to increase in the endogenous polyamines (Fig.
4A). In The half-life of ODC activity was greatly shortened by spermidine in
Using an S. pombe mutant lacking antizyme and AdoMetDC
(spa::Leu2
spe2::Ura4), we show requirement of antizyme
for the accelerated ODC decay. In the absence of functional antizyme
gene, the initial ODC activity was slightly higher and the decay of ODC
activity after spermidine addition was much retarded than in
SPA+ cells (Fig. 7). It is noted, however, that spermidine
still promoted ODC decay to a certain extent in the absence of the
antizyme in a protein synthesis-dependent manner. This
observation may suggest that spermidine not only induces de
novo synthesis of the antizyme, but some other protein(s) or
additional form(s) of antizyme to causes the slower decay of ODC. The
presence of multiple forms of antizyme has been reported from mammalian
systems (for review, see Ref. 44) and a lower vertebrate (45).
We show that S. pombe ODC is subject to the
polyamine-stimulated degradation catalyzed by the 26 S proteasome as
previously observed in S. cerevisiae and the higher animals.
The mechanism by which ODC is targeted for the degradation is very
similar between S. pombe and mammals, but substantially
different between the two yeast species. S. pombe will serve
as a suitable model system to study both the mammalian-type polyamine
regulation, and the molecular bases of the difference in ODC regulation
among the yeast, which may reflect the difference in the critical
features of ODC and the proteasome.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 shift in S. cerevisiae
(23, 24). Very recently, Ivanov et al. (25) identified an
antizyme gene in S. pombe demonstrating its frameshift induction by polyamines, involvement in the cellular polyamine control,
and inhibitory activity on ODC. Deletion of antizyme gene (SPA) from
the fisson yeast genome did not bring about apparent phenotypes, but
resulted in the increase in putrescine, spermidine, and cadaverine,
which was more prominently observed in the cells at stationary phase
than in exponentially growing cells, whereas overexpression of SPA led
to large reduction in the cellular level of polyamines (25). A role for
antizyme in ODC degradation in S. pombe cells, however, was
not demonstrated.
spe2) is devoid of spermidine
and spermine and contains a high ODC activity, which is rapidly
repressed in response to addition of spermidine through
antizyme-dependent ODC degradation catalyzed by proteasome.
To our knowledge, this is the first experimental evidence of antizyme
being involved in the regulation of ODC degradation in single-cellular eukaryotes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactone and MG132 (Z-LLL-CHO), were
purchased from Boston Biochem (Cambridge, MA) and Peptide Institute
(Osaka, Japan), respectively. Restriction enzymes were obtained from
New England Biolabs. Oligodeoxynucleotides were from Amersham Pharmacia
Biotech.
-Difluoromethylornithine (DFMO) was kindly provided by
Merrell Dow Research Institute (Cincinnati, OH). Media components were
from Difco. Other chemicals and reagents were obtained from Sigma
unless otherwise mentioned.
) was a kind
gift from Dr. M. Yamamoto. An antizyme knockout strain,
spa (ura4-D18 leu1-32
spa::Leu2 ade6-M216 h
) (25) was
generously provided by Drs. J. F. Atkins and I. P. Ivanov.
Polyamine-free liquid MM (minimal medium) (26) was prepared in
acid-washed glass containers and filter-sterilized with plastic disposable units. URA selection was performed on synthetic dextrose plates minus uracil. To measure cell growth rates in the polyamine-free condition, S. pombe cells were precultured in YES medium
(0.5% yeast extract, 3% dextrose, and amino acid supplements), washed twice with, and diluted 1:100 in, polyamine-free MM. After growing for
24 h, the cells were again washed twice and diluted in fresh polyamine-free MM to the A600 of 0.01. A
part of the culture was taken for measuring
A600. The cells for polyamine assay were taken from the same cultures 20 h after the second dilution. For the use
in the other experiments, cells were pre-cultured overnight in YES,
washed twice with, and diluted in polyamine-free MM to A600 = 0.2 unless otherwise mentioned. All the
yeast culture was carried out at 30 °C with shaking.
-32P]dCTP
(3,000 Ci/mmol, PerkinElmer Life Sciences) using Random Primer
DNA Labeling kit (Takara). An S. pombe cDNA library in pCD2 vector (a gift from Dr. H. Nojima) was screened with the standard
colony hybridization protocol (27). Positive clones were isolated,
subcloned in the BamHI site of pBluescript SK (
) (Stratagene), and sequenced using Prism cycle sequencing kit (ABI) with
T3 and T7 primers. Additional primers were designed to sequence the
entire cDNA in both directions. The cDNA was then used in screening an S. pombe genomic DNA library constructed in
pBluescript KS (
) vector (a gift from Dr. H. Nojima). One of the
positive clones (R7A, 5.2 kb) was digested with EcoRI,
subcloned into pBluescript SK (
), and sequenced as above. Sequence
comparison was carried out with GENETYX-MAC program (Software Development).
) that had been modified by
reversing the positions of the EcoRI and PstI
sites with an oligonucleotide pair. The 3'-flanking region of
spe2 (453 bp) was then inserted downstream of the
ura4 gene at the PstI-EcoRI sites. The
5'-flanking region (840 bp) was first subcloned in pBluescript SK (
)
at the EcoRI site, and then KpnI (on the
vector)-BsaAI fragment (containing the 740-bp
EcoRI-BsaAI fragment) was ligated upstream of
ura4 at the KpnI-HincII sites of the
earlier construct. In each step, subclones with the proper orientation
were selected with restriction analysis and confirmed by sequencing.
Thus in the final construct, almost the entire coding region of
spe2 was replaced by ura4. The construct was digested with EcoRI followed by gel purification and used to
transform JY745 or
spa strains with modified lithium
acetate method (29). The stable URA+
transformants were selected. Homologous recombination
(spe2::ura4) was confirmed first by
colony PCR, and then by Southern analysis of genomic DNA digested with
HindIII using the standard techniques (27). The
PstI- EcoRI fragment of S. pombe
genome at the 5' vicinity of the disruption construct (1.0 kb) was used
as a probe.
-D-galactopyranoside at 18 °C
overnight to induce glutathione S-transferase (GST)-ODC
fusion protein. The expression product was purified on a
glutathione-Sepharose 4B-affinity column (Amersham Pharmacia Biotech)
according to the manufacturer's instructions. Rabbits were injected
with 0.5 mg of the protein mixed with Freund's complete adjuvant. Two
booster doses were given with the same amount of protein using
Freund's incomplete adjuvant 1 and 2 months later. The antisera were
taken 7 days after the second booster and absorbed by an extract of ODC
(spe1) gene-disrupted S. pombe
cells.2 The same S. pombe extracts (20 µg)
that were used for ODC assay were fractionated on 10% SDS-PAGE.
Immunoblotting was performed according to a standard protocol using
Immobilon-P membrane (Millipore). Immunodetection was carried out with
the primary antibody (one of the antisera) against GST-ODC at a
dilution of 1:5,000 and secondary antibody, alkaline-phosphatase
conjugated anti-rabbit IgG (
-chain-specific, Sigma), at a dilution
of 1:10,000 essentially following the method of Mierendorf et
al. (33).
-D-galactopyranoside in BL21 (DE3)
carrying pET3a-SPO and purified from the extract by DEAE-Cellulofine
(Seikagaku Kogyo) chromatography as described previously (34). The
partially purified ODC (7,500 units) was incubated with 25 µM DFMO, in a mixture containing 10 µM
pyridoxal phosphate, 5 mM DTT, 40 mM Tris-HCl
(pH 7.4), and 0.01% Tween 80 in a total volume of 100 µl at 37 °C
for 2 h. The residual free DFMO was removed by gel filtration
through a NAP 5 column (Amersham Pharmacia Biotech). The resultant
preparation did not contain detectable level of ODC activity. For each
ODC assay, 15 units of DFMO-ODC (as initially determined) was mixed and
kept on ice for 30 min before enzyme reaction. The amount of ODC
complex was calculated from the gain of ODC activity as described
previously (30). Alternatively, 0.1 µg of recombinant rat antizyme
inhibitor, expressed as GST fusion protein and purified as described,
was mixed with each sample before enzyme reaction (16). The amount of
ODC complex was assumed to be equal to the gain of ODC activity.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SSMFV was noted (where the arrow indicates the cleavage
site). S. pombe AdoMetDC also contains the residues known to
be important for its activity, substrate binding, and
putrescine-mediated activation (35). The 5'- untranslated region of
S. pombe spe2 mRNA contained a small upstream ORF
(uORF), which encodes a tetrapeptide, MTIF, located closely to
the main ORF with only one nucleotide gap (Fig. 1A). There is no common
feature in the peptide sequence specified by uORFs between S. pombe and other organisms (see Refs. 36 and 37 and references
therein). S. cerevisiae spe2 transcript lacks uORF (38).
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Fig. 1.
Structure and disruption of
Schizosaccharomyces pombe Ado- MetDC gene
(spe2). A, the nucleotide sequence of
the cloned spe2 gene is shown with the deduced amino acid
sequence. The nucleotides of the ORF are depicted in
uppercase. Numbers of nucleotide and amino acid residues are
indicated on the left and right, respectively.
The tetrapeptide uORF is boxed. B, diagrammatic
representation of the genomic organization of spe2 and
cut14 genes, and the construct for spe2
disruption. The genomic DNA is represented with a gray
transversal line. The boxes on the
top represent open reading frames of spe2 and
cut14 (partial) with arrows in the 5' to 3'
direction. A genomic clone that was used in the subsequent study is
indicated with a bold transversal
line. Some of the used restriction sites are shown:
H, HindIII; P, PstI;
E, EcoRI; BA, BsaAI. The
EcoRI-BsaAI and PstI-EcoRI
fragments indicated with the bold lines at the
bottom were used to flank the ura4 gene to make
the disruption construct.
spe2 mutant cells contained no detectable activity. Wild
type cells exhibited 423 pmol of CO2/h/mg of AdoMetDC
protein.
spe2 mutant and wild type cells were grown in
the polyamine-free medium for two passages. Twenty hours after the
second passage, the mutant cells contained no detectable levels of
spermidine and spermine and accumulated 11.7 times putrescine and 6.0 times cadaverine over those detected in wild type cells (Fig.
2). Thereafter, the mutant cells ceased
growing without addition of spermidine or spermine (Fig.
3). When spermidine or spermine was added
to the culture medium at 100 µM concentration, they
restored growing at a doubling time comparable to that of wild type
cells after several hours of time lag. Addition of putrescine (Fig. 3),
cadaverine, or diaminopropane (data not shown) had no supportive effect
on the growth of
spe2 cells.
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Fig. 2.
Polyamine contents in wild type and
spe2 mutant cells. Cells were
cultured in the polyamine-free medium for two passages. The whole cells
were subjected to the polyamine measurement on high performance liquid
chromatography. Polyamine contents of wild type (open
bars) and
spe2 (solid
bars) cells are shown. Put, putrescine;
Cad, cadaverine; Spd, spermidine; Spm,
spermine.
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Fig. 3.
Growth of spe2
mutant cells in the presence or absence of polyamines. The
cells were grown in the polyamine-free medium over night and then
diluted in the same medium to 0.01 of A600. The
growth was monitored with A600 in the presence
or absence of polyamines.
,
spe2 without polyamine;
,
spe2 with putrescine;
,
spe2 with
spermidine; ×,
spe2 with spermine;
, wild type
without polyamine.
spe2 Cells--
Wild
type cells showed virtually no ODC activity after growing in
polyamine-free medium overnight (Fig.
4A). At this time, the cells
were collected and resuspended in the same volume of fresh
polyamine-free medium. ODC activity was rapidly induced reaching a peak
in 4 h, and then a sharp decay was observed. Addition of
spermidine (100 µM) 4 h after changing the medium
caused faster ODC decay. On the other hand,
spe2 cells
contained a high ODC activity after overnight culture in the
polyamine-free medium (Fig. 4B). Changing medium resulted in
a much larger and more prolonged ODC induction in
spe2
cells than in wild type cells. Addition of spermidine caused rapid
decay of ODC activity also in
spe2 cells. Thus, in
spe2 cells, ODC was derepressed despite a large
accumulation of putrescine. The mechanism of the accelerated ODC decay
by spermidine was studied further in
spe2 cells.
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Fig. 4.
ODC induction after changing medium and
repression by spermidine. A, changes in ODC activity in
wild type cells. B, changes in ODC activity in
spe2 mutant cells. For each cell type, duplicate cultures
were grown overnight in the polyamine-free medium and the medium was
replaced by fresh medium at time 0. After 4 h of medium change,
spermidine (100 µM) was added to one of the cultures.
Aliquots were removed from the culture at indicated times, and ODC
activity was measured as described in the text.
, without
spermidine;
, with spermidine.
spe2 culture 4 h after the medium
change, and cells were harvested at the indicated time to measure ODC
activity. In Fig. 5A the total
ODC activities (the sum of free ODC and complex form ODC activities
measured in the presence of DFMO-ODC, see below) are plotted. Addition of spermidine reduced 98% of ODC activity in 6 h with a half-life of 65 min. This decay was much faster than that after addition of
cycloheximide (50 µg/ml), the half-life of which was more than 6 h. When cycloheximide was added together with spermidine, the acceleration of ODC decay was completely prevented. Actinomycin D (2.5 µg/ml), in contrast, did not change the effect of spermidine. Western
blot analysis of the same cell extracts depicted that the decrease in
ODC activity was associated with decrease in the amount of ODC protein
(Fig. 5B). Addition of cycloheximide, but not actinomycin D,
prevented this accelerated decrease of the protein. These results are
consistent with a model that a short-lived protein that is induced by
spermidine at a posttranscriptional level mediates the accelerating
decay of ODC. Since it has been shown earlier that S. pombe
antizyme is expressed through polyamine-inducible translational
frameshifting (25), the protein is very likely to be antizyme.
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Fig. 5.
Accelerated degradation of ODC by
spermidine. A, decay in ODC. ODC had been induced by
medium change, and spermidine (100 µM) was added to the
culture alone, in combination with cycloheximide (50 µg/ml), or with
actinomycin D (2.5 µg/ml). Control cells were maintained with
addition of cycloheximide alone or without any addition. Aliquots were
removed from the culture at indicated times, and ODC activity was
measured as described in the text. The total ODC activity (sum of free
ODC plus complex ODC) is plotted. Additions are: , spermidine; ×,
spermidine and cycloheximide;
, spermidine and actinomycin D;
,
cycloheximide alone;
, none. B, Western blot analysis
showing changes in ODC protein. Cellular extracts (20 µg of protein)
from the above experiment were loaded in each lane of SDS-PAGE, blotted
onto a membrane, and detected with antibody against S. pombe
ODC. Cont, control; Spd, spermidine;
CHX, cycloheximide; ACD, actinomycin D.
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Fig. 6.
Increase in antizyme-ODC complex in
spermidine-treated cells. A, changes in antizyme-ODC
complex as determined by DFMO-ODC. ODC activity of the cellular
extracts used in Fig. 5 was assayed with or without addition of
DFMO-ODC (15 units). Amounts of antizyme-ODC complex were calculated
from gain of ODC activity by adding DFMO-ODC and expressed as
percentage over the total ODC activity. B, changes in
antizyme-ODC complex as determined by rat antizyme inhibitor. ODC
activity was assayed with and without addition of recombinant rat
antizyme inhibitor (0.1 µg). Amounts of antizyme-ODC complex were
assumed to be equal to the gain of ODC activity and expressed as
percentage over the total ODC activity. , spermidine; ×, spermidine
and cycloheximide;
, cycloheximide alone;
, none.
spa) (25). The
spe2 gene in the mutant strain was further disrupted by the
single-step gene replacement. Addition of spermidine to the culture
medium of
spa-
spe2 double knockout cells
caused only a very slow ODC decay (Fig.
7). Approximately 90% of the activity
remained even 6 h after addition of spermidine in the double
knockout mutant compared with 2% in the
spe2 single
mutant. The result clearly indicates that antizyme is necessary for a
major part of ODC decay that is stimulated by spermidine. It was also
noted that about 10% of the initial ODC activity reproducibly
disappeared in 6 h after the addition of spermidine in the absence
of antizyme. Almost no ODC reduction was observed in the double mutant
to which cycloheximide was added together with spermidine. Simultaneous addition of actinomycin D did not block the ODC decay.
View larger version (17K):
[in a new window]
Fig. 7.
Antizyme is required for accelerated ODC
decay. Changes in ODC activity after spermidine addition to
spa-
spe2 double mutant cells and
spe2 single mutant cells are shown. ODC had been induced
by medium change. Spermidine was added to
spa-
spe2 double mutant alone (
), in
combination with cycloheximide (×), or with actinomycin D (
).
Control culture was maintained without addition (
). In parallel
experiments,
spe2 single mutant was treated with
spermidine (
) or none (
).
-lactone or MG132, both at 50 µM, was added 4 h before spermidine. Both did not
affect the cell growth at this concentration. As shown in Fig.
8, the rapid decay of ODC caused by
spermidine was effectively inhibited by both inhibitors. This result
strongly suggests that the 26 S proteasome catalyzes spermidine
-induced degradation of ODC in S. pombe.
View larger version (15K):
[in a new window]
Fig. 8.
Effects of proteasome inhibitors on
accelerated ODC decay induced by spermidine. spe2
cells were cultured for 4 h in the polyamine-free medium
containing 50 µM MG132 (×),
clasto-lactacystin
-lactone (
), or none (
).
Spermidine (100 µM) was then added to the culture and ODC
activity was measured at indicated times. Control culture was
maintained without any addition (
).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
spe2 mutant
(spe2::ura4) cease dividing after
several generations in polyamine-free medium. The mutant contained a
large excess of putrescine and cadaverine, indicating indispensability of spermidine and spermine for growth in the organism. The essential role of spermidine and spermine was also demonstrated in S. cerevisiae and N. crassa (40, 41).
spe2
mutant of the budding yeast showed an increase in cell size, a decrease
in budding, accumulation of vesicle-like bodies, and abnormal
distribution of actin-like material (40). In the presence of oxygen, a
rapid cessation of cell growth and associated cell death were observed
(42). Similar studies in S. pombe are yet to be performed.
spe2 cells ODC is elevated (Fig.
4B) despite a large accumulation of putrescine (Fig. 2).
Spermidine added to the culture medium could rapidly repress ODC (Fig.
4B). It is of interest to note that S. pombe ODC
is repressed by spermidine, but not effectively by putrescine, in
connection with the observation that spermidine or spermine, but not
putrescine, supports cellular growth. In S. cerevisiae, ODC
decay is accelerated by putrescine to much less extent than by
spermidine (43), whereas N. crassa ODC is strongly
destabilized by both spermidine and putrescine (21).
spe2 cells (Fig. 5A). The ODC decay is mostly
attributable to a change in ODC protein (Fig. 5B). The
accelerated ODC decay was prevented by the inhibitor of protein
synthesis, but not by the inhibitor of RNA synthesis. In addition, it
was found that a part of ODC existed as an inactive complex, from which
active ODC is released by the competitors such as DFMO-ODC or antizyme inhibitor (Fig. 6). Although the fraction of ODC forming the inactive complex was increased by spermidine, it only accounts for a part of ODC
decay in the activity. All these results are compatible with a model
that antizyme is induced through the stimulation of translational
frameshifting by spermidine, bound to ODC forming inactive complex, and
promotes ODC protein for rapid degradation. The half-life of ODC
activity in the spermidine-treated cells, 65 min (Fig. 5A),
is not as short as those observed in mammalian cells, but comparable to
those in S. cerevisiae in the repressed conditions (17,
43).
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ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge Dr. H. Nojima of Osaka University for providing the S. pombe cDNA and genomic DNA libraries, Dr. M. Yamamoto of Tokyo University for JY745 strain of S. pombe, Drs. J. F. Atkins and I. P. Ivanov of University of Utah for the spa knockout strain, and Drs. R. H. Davis of University of California, Irvine, and M. A. Hoyt of University of California, San Francisco for valuable comments on the manuscript. We also gratefully acknowledge advice from Dr. N. Murai of Jikei University and of Drs. T. Tani and Y. Habara of Kyushu University.
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FOOTNOTES |
---|
* This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan (to S. M. and Y. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession numbers AB045110 (cDNA) and AB045111 (genomic DNA).
Postdoctoral fellow of the Japan Society for the Promotion of
Science. Present address: Laboratory of Biochemical Genetics, NIDDK,
National Institutes of Health, Bethesda, MD 20892-0830.
§ To whom correspondence should be addressed. Tel.: 81-3-3433-1111; Fax: 81-3-3436-3897; E-mail: senya@jikei.ac.jp.
Published, JBC Papers in Press, March 30, 2001, DOI 10.1074/jbc.M010643200
2 M. K. Chattopadhyay, K. Mita, and S. Matsufuji, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are:
ODC, ornithine
decarboxylase;
AdoMetDC, S-adenosylmethionine decarboxylase;
ORF, open reading frame;
MM, minimal medium;
DTT, dithiothreitol;
GST, glutathione S-transferase;
PAGE, polyacrylamide gel
electrophoresis;
DFMO, -difluoromethylornithine;
uORF, upstream
open reading frame;
kb, kilobase(s);
bp, base pair(s);
PCR, polymerase
chain reaction;
SPA, Schizosaccharomyces pombe
antizyme gene.
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