Leucineaminopeptidaseduringmeiotic development
Takashi Ishizaki
1
, Aki Tosaka
1,
*, Takayuki Nara
1,²
, Narumichi Aoshima
1
, Satoshi Namekawa
1
,
Kei Watanabe
1
, Fumika Hamada
1
, Akira Omori
2
and Kengo Sakaguchi
1
1
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan;
2
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan
We found a leucineaminopeptidase (LAP; EC 3.4.11.1) to
be abundant in meiotic prophase tissue of a basidiomycete,
Coprinus cinereus. After direct puri®cation of the amino-
peptidase component from meiocytes, we cloned the gene
by degenerate PCR using partial amino-acid sequences
of the puri®ed enzyme and 5¢ and 3¢ RACE. It was
homologous to the eukaryotic leucine aminopeptidase
gene. The recombinant protein possesses the characteristic
activities of a Coprinus leucineaminopeptidase (CoLAP)
with a molecular mass of 52.4 kDa, and forms a homo-
hexamer. Northern blot and spatial distribution analysis
by immunohistochemical staining indicated CoLAP to be
abundant in meiotic prophase cells and the supporting cells
around meiocytes, but scarce in mycelium cells. Interest-
ingly, from zygotene to pachytene, CoLAP was mostly
present in supporting cells around meiocytes, but from
diplotene onwards, it was plentiful in meiocytes them-
selves, suggesting that its expression is required to control
some of the biochemical events at meiotic prophase.
Moreover, the strong expression of CoLAP mRNA
immediately after treatment with methyl methanesulfonate
in mycelium implies that CoLAP has a role in somatic
DNA repair.
Keywords:CoLAP;Coprinus cinereus; leucine amino-
peptidase; meiotic prophase.
We have investigated meiosis-related protein facto rs using
meiotic cells in a b asidiomycete, Coprinus cinereus [1± 12]. In
meiosis, chromosomes condense from the dispersed state
typical of interphase during early meiotic p rophase, to form
long thin threads in leptotene, a nd each acquires a
proteinaceous axial core to which the two sister chromatids
are attached. Then, homologous chromosomes become
aligned during z ygotene, forming the synaptinemal complex
and, at pachytene, nonsister chromatids of the completely
paired chromosomes recombine forming the chiasmata
which become visible during diplotene. Two cell divisions
follow, reductional and equational, resulting in four
gametes.
C. cinereus is well suited for s tudies of meiosis, because its
meiotic cell cycle is long and naturally synchronous [9±14].
The dikaryonic cells are at the premeiotic stage from
S-phase to leptotene. From the beginning of the k aryogamy,
when the two nuclei fuse, for the next 5 h the cells are at t he
zygotene stage, when homologous chromosomes pair.
Later, the chromosomes recombine at pachytene. We were
able to obtain plenty of meiotic tissues at leptotene,
zygotene, pachytene or diplotene at any time. This made i t
possible to purify the meiosis-related protein factors t o near
homogeneity [1±12].
According to DeGuzman & Riggs [15], proteolytic
activities intensi®ed as the development of Lilium anther
proceeded and these activities were temporally correlated
with events crucial for the maturation of viable pollen, as
well as with the apoptotic events that precede dehiscence.
In this connection, we focused on the fact that tissues which
proliferate ef®ciently exhibit protease activity in meiotic
prophase. Experiments using various protease substrates
revealed that not only proteases, but also aminopeptidases
are responsible for proteolysis in meiosis (T. Ishizaki and
K. Sakaguichi, unpublished data). Based on this result, we
screened for major aminopeptidase components in the
meiotic development of C. cinereus, and successfully puri-
®ed an aminopeptidase to near homogeneity through ®ve
columns. The puri®ed component showed aminopeptidase
activity with a molecular mass of 50 kDa, but its involve-
ment in meiosis was not clear. Therefore, we atte mpted to
determine its partial amino-acid sequences, so as to clone
the g ene through degenerate PCR methods. We subse-
quently found that the gene sequence has homology with
leucine aminopeptidases (LAP) in mammals, plants, and
bacteria. T he m eiosis-speci®c aminopeptidase was concluded
to be a Coprinus alternative o f LAP (CoLAP). Consideration
should now be given to the possibility that CoLAP has roles
in the p rogression and development of the meiotic cell cycle.
There must be some c oordination between CoLAP and
meiosis. Analysis of the p roteins t hat are required for these
processes p rovides insight into the mechanism of this
coordination.
Correspondence to K. Sakaguchi, Department of Applied Biological
Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba
278-8510, Japan. Fax: +81 471 23 9767, Tel.: +81 471 24 1501
(extn 3409), E-mail: kengo@rs.noda.sut.ac.jp
Abbreviations: LAP, leucine aminopeptidase; CoLAP, Coprinus
leucine aminopeptidase; DAPI, 4¢,6-diamino-2-phenylindole dihydro-
chloride.
Enzyme: leucineaminopeptidase (LAP; EC 3.4.11.1).
*Presen t address: Nagoya University School of M edicine , Chikusa-ku,
Nagoya 466-8550, Japan.
Present address: Dep artmen t of F ood Science and Human Nutrition,
University of Illinois at Urbana-Champaign, Urbana, I llinois 61801,
USA.
(Received 11 October 2001, revised 26 N ovember, accepted 29
November 2001)
Eur. J. Biochem. 269, 826±832 (2002) Ó FEBS 2002
In this report, we have focused on the LAP that is
associated with the meioticdevelopment of C. cinereus,and
characterized the e nzyme in re lation to meiotic e vents.
MATERIALS AND METHODS
Culture of
C. cinereus
and collection of the fruiting
bodies
A basidiomycete , C. cinereus (#5026+5132) was used. The
culture methods used here were identical to those described
in our previous study [10]. Culture dishes (90 ´ 60 mm)
containing sterile horse manure were inoculated with a
dikaryotic stock culture of C. cinereus, then incubated for
7 d ays in an incubator at 37 °C in total darkness, before
photo induction of fruiti® cation with a light cycle regime of
16 hlight/8 hdarkat28 °C. The light cycle began at 05 : 00
local time. Karyogamy, de®ned as the time at which 5% of
all basidia have fused nuclei, starts at 04 : 00, 1 h before
lights on. Fruiting bodies that appeared between 04 : 00 and
07 : 00 were assigned to leptotene, 07 : 00±09 : 00 to
zygotene, 10 : 00±11 : 00 to pachytene, and 12 : 00 through
14 : 00 to diplotene or later. The time course of the meiotic
events in Coprinus was depicted in our previous report [10].
Under these conditions, meiotic cells all in the same stage of
prophase could be readily obtained. The fruiting caps
harvested were immediately frozen in liquid nitrogen and
stored at )80 °C.
Aminopeptidase activity assays
For aminopeptidase activity a ssay, 1 m
ML
-leucine-p-nitro-
anilide in 50 m
M
Tris/HCl pH 7.6, was i ncubated at 3 7 °C.
Reactions were terminated by adding sodium acetate. The
absorbance of the liberated p-nitroanilide was measured
with Bio-Rad's microplate reader at 4 05 nm.
Puri®cation of aminopeptidase from tissues
at meiotic prophase
The TMG buffer contained 50 m
M
Tris/HCl pH 7.5, 5 m
M
2-mercaptoethanol, 15% (v/v) glycerol, and three protein
inhibitors, pepstatin A (1 mgámL
)1
), leupeptin (1 mgámL
)1
),
and 1 m
M
phenylmethanesulfonyl ¯uoride. All procedures
were performed at 4 °C.
The tissues of Coprinus fruiting bodies (20 g) at p achytene
to diplo tene were homogenized in 10 vol. TMG buffer
containing 0.8
M
NaCl using a French press a nd centrifuged
at 15 000 g for 20 min. The supernatant, precipitated using
30% ammonium sulfate, was centrifuged, and the superna-
tant was further saturated with 75% ammonium sulfate.
The 75% ammonium sulfate precipitate was collected by
centrifugation, and the pellet was resuspended in 30 mL
TMG buffer. After being dialysed, it was loaded onto
HiPrep-DEAE sepharose equilibrated with T MG buffer.
The elution pro®le using 200 mL of a linear gradient from
zero to 0.6
M
KCl in TMG buffer showed a peak at 0 .2
M
KCl.
The fractions from the HiPrep-DEAE chromatography
were loaded onto a HiTrap-Heparin-agarose column equi-
librated with T MG buffer. The elution was p erformed with
60 mL of a linear NaCl gradient (0±1.0
M
)inTMGbuffer.
The component with signi®cant aminopeptidase activity
was present at 0.4
M
. The fractions from the HiTrap-
Heparin-agarose column chromatography were collected
together, and then after b eing dialysed, were loaded onto a
Mono Q column (1 mL) equilibrated with TMG buffer.
The elution was performed with 20 mL of a linear NaCl
gradient (0±1.0
M
) in TMG buffer.
Finally, the fractions from the Mono Q column chroma-
tography were loaded onto a single-stranded DNA sepha-
rose column (1 mL) equilibrated with TMG buffer. The
elution was performed with 20 mL of a linear gradient
(0±1.0
M
) of NaCl in TMG buffer. The active component
was eluted at 370 m
M
NaCl as a single peak. The fractions
were analysed further by SDS/PAGE and Superose 6 gel
®ltration chromatography.
Internal amino acid microsequencing
About 10 mg of the aminopeptidase component from the
single-stranded DNA sepharose column chromatography
was subjected to SDS/PAGE, and the band was cut out.
The b and was puri®ed again by a second SDS/PAGE. The
protein eluted from the band was blotted on a PVDF
membrane, and digested with lysylendoprotease (Wako
Pure Chemical Industries, Osaka, Japan ) on the m embrane.
Peptides released from the m embrane were fractionated by
reversed-phase HPLC using a C8 column (1.0 ´ 100 mm),
and sequenced using a pulse-liquid phase protein sequencer
(Procise cLc, Applied Biosystems). The three fragmented
peptides were designated C-67 (AGTARTFYNTPE), C-69
(LWALTP), and S-2009 (TEFAGIP).
CDNA and gene cloning of CoLAP
The p artial cDNA sequence was obtained with two reverse
transcription (RT)/PCR degenerate primers derived from
two determined amino-acid sequences: C-67 sense primer
(5¢-GGCACCGCCCGCACNTTYTAYAA-3 ¢) and S-2009
antisense primer (5¢-GGACGTTGGGGATGCCNGCR
AAYTC-3¢)(N A, C, G, T, R AG, Y CT).
Cycling c onditions were: 95 °Cfor5min;95°Cfor1min;
60 °Cfor1.5min;72°C for 2 min; 40 cycles, followed by a
10-min extension at 72 °C. The major 500 bp PCR product
was subcloned into the pGEM-T Easy vector (Promega)
and sequenced.
To lengthen the 3¢ and 5 ¢ ends, 3 ¢ and 5¢ RACE
were performed with SuperScript (Invitrogen). For
3¢ RACE, GSP1 (5¢-GACAACCTCGGTCGTCTCTT
T-3¢)andGSP2(5¢-CCTCAAGACTTCTCC CCCTTC-3¢)
were designed using Primer3 (MIT Whitehead Institute).
For 5¢ RACE, A-GSP1 (5¢-GGAGAAGTCTTGAGGGT
GAAC TT-3¢), A-GSP2 (5¢-TCCTAGCAAGGTTCTGG
GACT-3¢)andA-GSP3(5¢-GGAGAAGTCTTGAGGG
TGA ACTT-3¢) were u sed. Downstream 13 00-bp and
upstream 400-bp products were cloned and sequen ced.
The DDBJ/EMBL/GenBank accession number of the
CoLAP nucleo tide sequence reported in t his p aper is
AB052095.
Genomic DNA isolation and Southern hybridization
analysis
Genomic DNA was isolated from Coprinus mycelium t issue
anddigestedwithfourrestrictionenzymes:EcoRV, SalI,
Ó FEBS 2002 Leucineaminopeptidase and meiosis (Eur. J. Biochem. 269) 827
SmaIorXhoI. The DNA fragments were resolved on 1 .0%
agarose gel, and transferred to Hybond-N+ membrane
(Amersham Pharmacia Biotech, or APB) according to the
manufacturer's instructions. The DNA fragments used as
the p robes were gel-puri®ed and labelled using a Multiprime
DNA labelling system ( APB) (data not shown ).
RNA extraction and Northern hybridization analysis
Total RNA was prepared from the caps at meiotic p rophase
and the methyl methanesulfonate-treated tissues (described
previously in [10]) of C. cinereus according to the TRIzol
(Invitrogen) manufacturer's protocol.
RNA samples were separated on 1.2% agarose/formal-
dehyde gels as described by Ausubel et al.[16].TotalRNA
(25 lg) from t he caps a t each m eiotic stage and the methyl
methanesulfonate-treated tissues harvested at 1-h intervals
were loaded in each lane. The agarose ge l was st ained with
ethidium bromide a nd blotted overnight onto Hybond-N +
membran es (APB) . The memb ranes were ®xed by alkali
reagents, rinsed with 2 ´ NaCl/P
i
/EDTA, and hybridized
with
32
P-labelled probe for h ybridization analysis.
Over-expression and puri®cation of a CoLAP protein
The CoLAP coding region was ampli®ed using N- and
C-terminus open reading frame primers with EcoRI and
XhoI sticky ends. The ampli®ed product was gel-puri®ed,
digested with EcoRI and XhoI, and cloned into the pET21a
expression vector (Novagen) to generate pET21-CoLAP-
(his)
6
. The vector was transformed into Escherichia coli
BLR for protein induction. The cells were incubated for 4 h
in Luria±Bertani medium with 50 mL of a culture preincu -
bated overnight, c ontaining 50 lgámL
)1
ampicillin and
1m
M
isopropyl thio-b-
D
-galactoside, and centrifuged at
15000 g for 20 min The pellet was resuspended in ice-cold
binding buffer, and sonicated for extraction. The extract
was loaded on to a Ni+ charged FPLC chelating column
(APB) with the elution pro®le o f 50 mL of a linear gradien t
(0±1
M
) imidazole buffer [ 20 m
M
Tris/HCl pH 8.0, 500 m
M
NaCl, 10% (v/v) glycerol, 0.02% NP-40] at a ¯ow rate of
0.75 mLámin
)1
, followed by a Mono Q column (APB) with
15 mL of a linear gradient of 0.05±1
M
NaCl in TMG buffer
at 0.5 mL ámin
)1
. The protein, identi®ed by assay of
aminopeptidase activity and SDS/PAGE, was pooled and
stored in aliquots at 4 °C (data not shown).
Immunological analysis and immuno¯uorescence
microscopy
A polyclonal antibody against CoLAP protein was raised in
a rabbit. Western blot analysis was carried out according to
the method of Towbin et al. [17]. Anti-rabbit IgG conju-
gated with alkaline phosphatase (Cell Signaling Technol-
ogy, Inc.) was used as a secondary antibody with nitroblue
tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate as
substrates of alkaline phosphatase (data not shown).
Immunostaining of Coprinus fruiting caps was carried out
as described by Hasezawa and Nagata [18]. The paraf®n
sections of the fruiting caps described above for the in situ
hybridization were used. The cells were incubated for 3 h
with the antibody against CoLAP protein. The antibody
was diluted 1 : 500 before use. The cells were then treated
for 1 h with anti-rabbit IgG together with alkaline phos-
phatase and Alexa Fluor 488 goat anti-rabbit IgG (H + L )
conjugate ( Molecular Probes), diluted 1 : 1000 as secondary
antibodies. The cells were also stained with a solution of
5 lgámL
)1
4¢,6-diamino-2-phenylindole dihydrochloride
(DAPI) for 5 min. The specimens were examined under a
light or ¯uorescence microscope (OLYMPUS BH-2).
RESULTS
Puri®cation and characterization of an aminopeptidase
in basidia of
C. cinereus
at meiotic prophase
To screen for a protease that might play a role in meiosis-
speci®c even ts, crude extracts were generated from t he caps
at different stages of meiotic prophase in a basidiomycete,
C. cinereus , and partially puri®ed through HiPrep-DEAE
sepharose column chromatography. They were then
assayed f or various protease activities. The am inopeptidase
activity bound to HiPrep-DEAE sepharose was g reatest in
the fruiting caps harvested at meiotic prophase, and
markedly reduced at the tetrad stage, the end of meiosis
(data not shown). Subsequently, we found relatively strong
aminopeptidase activity during the zygotene to diplotene
stages, when the homologous chromosomes pair and
recombine f orming the chiasmata which become visible
during diplotene. It is interesting that the aminopeptidase
activity increases as meioticdevelopment procee ds. This
enhanced activity could be correlated with morphological
and biochemical events of meiotic prophase which require
proteolytic enzymes.
In this connection, we tried to purify t he Coprinus
meiosis-speci®c aminopeptidase to near homogeneity, and
succeeded through ®ve rounds of column chromatography
as described in Materials and methods. The active fraction
from the ®nal (single-stranded DNA sepharose) column
chromatography was puri®ed 17500-fold. The component
was indicated to be a single band of molecular mass 50 kDa
on SDS/PAGE (Fig. 1A), but a 340-kDa molecule by
Superose 6 gel ®ltration (Fig. 1B), suggesting that the
protein probably forms a ho mohexamer.
This study represents the ®rst puri®cation and character-
ization o f an aminopeptidase, which might have a role i n the
meiotic cell cycle, e specially at meiotic prophase. However,
the amount o f enzyme i solated was not suf®cient for further
analysis, and the peptide sequences obtained, C-67, C-69
and S-2009, were so small that homology to any known
proteins was not demonstrated. For that reason, we tried to
clone the cDNA encoding the enzyme by RT-PCR u sing a
set of degenerate p rimers.
Isolation and characterization of cDNA of the meiotic
aminopeptidase in
Coprinus
meiocytes
To isolate cDNA of the m eiotic aminopeptidase in Copri-
nus, t wo degen erate PCR p rimers (see Materials and
methods) were used in reactions with Coprinus cDNA
created from poly(A)+ RNA isolated from fruiting bodies
at meiotic prophase as the t emplate. An 500-bp fragment
was obtained and sequenced. Downstream 3 ¢ sequences and
upstream 5¢ sequences were exten ded by RACE methods.
The Coprinus cDNA sequence contains 489 amino acid
residues, with a calculated molecular m ass of 52.4 k Da
828 T. Ishizaki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
(Fig. 2), which contained three obtained amino-acid
sequences (see underlined sequence in Fig. 2). Interestingly,
the a mino-acid sequence was highly homologous to that of
LAP. Database searches with the
BLASTX
program [19]
revealed that the CoLAP gene has i dentity with human LAP
(42%), bovine lens LAP ( 42%), E. coli PepA (39%),
Schizosaccharomyces pombe putative LAP (39%), Pseudo-
monas PhpA (36%) and Arabidopsis LAP (35%). The
consensus region is common to L APs f rom other organisms
(see box in Fig. 2). The meio tic a minopeptidase appears t o
be a counterpart of LAP from mammals, p lants an d yeast.
We temporarily designated it CoLAP (Coprinus leucine
aminopeptidase).
The Coprinus genomic DNA was digested using the
restriction enzyme EcoRV, SalI, SmaIorXhoI. Southern
hybridization a nalysis revealed that, as each of the digested
products had only a single band, it is a single-copy gene
(data not shown).
Isolation and characterization of the recombinant
CoLAP homologue protein
To characterize CoLAP in detail, t he h istidine-tagged
recombinant protein was over-expressed and puri®ed by
Ni
+
af®nity and Mono Q chromatography (see Materials
and methods). SDS/PAGE and Sephacryl S-300 gel
®ltration chromatography of the M ono Q fraction r e-
vealed the molecular mass of the recombinant CoLAP
protein monomer to be 50 kDa; CoLAP was found to
be present as a 310-kDa hexamer by gel ®ltration (data
not shown). The molecular mass of CoLAP was slightly
smaller than that of the originally puri®ed aminopeptidase
(340 kDa). As the recombinant protein should have a
greater mass because of the addition of the histidine-tag,
the increase in size found on gel ®ltration of the native
enzyme might be consistent with it being modi®ed post-
translation in its native state. These properties are
consistent with the results for t he originally puri®ed
aminopeptidase. The pH dependence and optimum tem-
perature toward leucine-p-nitroanilide were quite similar
to those of previously reported LAPs.
Northern hybridization of CoLAP
To examine w hether the CoLAP gene is expressed at meiotic
prophase as the aminopeptidase was originally puri®ed at
Fig. 2. Nucleotide and deduced amino-acid s equences o f CoLAP and its
¯anking regions. Amino acids derived from peptide sequencing are
underlined; the cytosol aminopeptidase sign ature is boxed.
Fig. 1. SDS/PAGE of the puri®ed C. cinereus
meiosis-speci®c aminopeptidase component and
determination of its molecular mass by gel
®ltration c hromatography. (A) The ®n al pre-
paration of the meiosis-speci®c aminopepti-
dase was a nalysed by SDS/PAGE. Proteins
were stained with CBB. Relative mobility
measurements showed the major band to be
50 kDa. (B) The 50-kDa aminopeptidase
was loaded o n Superose 6 (APB). The activity
was detected in the 340-kDa fraction.
Ó FEBS 2002 Leucineaminopeptidase and meiosis (Eur. J. Biochem. 269) 829
pachytene, total RNA was extracted from the basidia taken
from the synchronous culture every hour after induction of
meiosis, and hybridization with a CoLAP cDNA probe was
performed (Fig. 3). The transcript was detected faintly in
the m ycelium t issues (the mitotic cells, 0 h in Fig. 4) and at
premeiotic S (PreS in Fig. 3) when the genomic DNA
replicates. In meiosis, the transcript was detected faintly in
the basidia at leptotene, began to accumulate dramatically
after karyogamy, reached a maximal level at pachytene, and
disappeared gradually after diplotene (Fig. 3). Because the
majority of the basidia signal was detected from zygotene to
diplotene, as judged from ¯uorescent microscopic observa-
tion of the monokaryonic nuclei, it was concluded that
CoLAP was expressed throughout the m eiotic prophase
when the homologous chromosomes pair and recombine
and then the pachytene-recombined chromosomes s eparate
and form the ch iasmata.
As shown in Fig. 4, the CoLAP gene was expressed only
faintly in the somatic cells (see 0 h in Fig. 4). However,
eukaryotic LAP genes were detec ted widely in somatic cells,
and their roles in these cells have been discussed [20±24].
Some of the transcript of the CoLAP gene might be
involved in the events occurring in the somatic cells. To
determine whether the CoLAP gene is transcribed in
somatic cells, the mycelium was treated with an alkylating
reagent, methyl methanesulfo nate, and expression was
analysed by Northern blotting. We detected strong expres-
sion of CoLAP mRNA immediately after treatment. The
induction of expression peaked within 1 h , and then
disappeared gradually over 5 h (Fig. 4). In the mycelium,
the CoLAP gene is expressed in response to DNA damage,
suggesting that CoLAP has a r ole in the repair of DNA.
Immunohistochemical localization of CoLAP
during meiosis
We raised a polyclonal antibody against recombinant
CoLAP protein in rabbits. The immunoblot signals coin-
cided with the molecular weight of CoLAP (50 kDa). The
af®nity-puri®ed antibody recognized the CoLAP protein
species (data not shown). As the fruiting caps we used as
meiotic tissue contain some somatic cells, the assumption
that all or some CoLAP is present in somatic cells is valid.
Therefore, to prove that CoLAP comes from meiotic cells,
the distribution of CoLAP was investigated by in situ
immunohistochemical staining using the antibody (Figs 5
and 6). Intens e signal for CoLAP was d etected from
leptotene t o diplotene and diakinesis, indicating that
CoLAP was transcribed and translated in the meiotic cells
during meiotic prophase. The tissues densely stained by
DAPI on the surface of the gillus a re meiotic tissues (DAPI
in Fig. 6). Densely DAPI stained tissues from premeiotic S
to leptotene (L in Fig. 5), from early to late zygotene (Z), at
pachytene (P) and from diplotene to diakinesis (D) were
selected. Strangely, until pachytene, the CoLAP staining
appeared not in the meiotic cells, but in the c ells which
support them. From pachytene, however, the signals
occurred in the meiotic cells themselves strongly as well as
in the cells which support them. To con®rm it further, in situ
immuno¯uorescence staining using the antibody and stan-
dard epi¯uorescence microscopy were also performed in the
meiotic cells (Fig. 6). The signal was clearly visible in the
meiotic cells at diplotene. These results indicated that from
leptotene to zygotene, CoLAP is mostly transcribed in the
cells neighbouring the meiotic cells, and at pachytene or
later, begins to be present in the meiotic cells.
DISCUSSION
We have reported here that i n a basidiomycete, C. cinereus,
a LAP (CoLAP) is speci®cally expressed in meiotic
prophase at the stages in which homologous chromosomes
pair (zygotene), recombine ( pachytene) and disjunct (diplo-
tene or later). Until pachytene, CoLAP is present in the
somatic cells next to the meiotic cells; however, from
diplotene CoLAP occurs in the meiotic cells themselves. To
our knowledge, this is the ®rst report to indicate that the
LAP gene is expressed at meiotic prophase, and to imply
Fig. 3. CoLAP expression analysis in various phases of m eiotic devel-
opment. Northern blot analysis of total RNA (25 lg) from the caps at
leptotene, L, zygotene, Z, pachytene, P, and diplotene, D, probed wit h
32
P-labelled CoLAP cDNA. 26 S and 18 S rRNA were stained with
ethidium bromide as a loading control.
Fig. 4. CoLAP expression analysis in methyl methanesulfonate-treated
tissue. Northern blot analysis of total RNA (25 lg) from 0.01%
methyl methanesu lfonate (MMS)-treated somatic tissue (hyphae) at
dierent times probed with
32
P-labelled CoLAP cDNA.
830 T. Ishizaki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
that the meiosis-related events require the LAP protein
especially at diplotene or later stages. Moreover, we found
that CoLAP gene expression is low in the mycelium cells,
but strongly induced by DNA damage caused by an
alkylating agent, methyl methanesulfonate, suggesting that
CoLAP has a role i n DNA re pair in the mycelial cells.
Recently, it has become evident that the intracellular
selective d egradation of proteins is important as part of t he
primordial regulation process in many metabolic pathways,
especially where timing control is concerned [20]. The
selective degradation of proteins in eukaryotes is carried out
by the ubiquitin-ATP system, and LAP is a protein that
catalyses the cleavage o f amino acids from the N terminus
of protein [21±23]. LAP might be able to modify the
terminus region differentially, as recognized by the ubi-
quitin system [24]. It is interesting that LAP expression
increases as meioticdevelopment proceeds. The results were
quite similar to those studied in microsporogenesis in a
higher plant, Lilium longi¯orum, which is anoth er organism
used for this t ype of study [15], although their enzymes were
in classes of s erine and aspartate proteases. In lily, protease
activities were correlated with the morphological and
biochemical events o f late m icrosporogenesis [15]. The
most dramatic of these w as the programmed cell death of
tapetal cells and anther wall cells which p recedes dehiscence
[15]. It is possible that in Coprinus, as the somatic cells
neighbouring the meiotic cells correspond to the Lilium
tapetal cells, and as CoLAP is expressed markedly in the
somatic cells at zygotene and pachytene, the LAP a s a kind
of protease may have a similar role to the lily meiotic
protease, promoting the maturation of meiotic cells from
supporting cells in the caps at zygotene and pachytene.
However, LAP may play not only a general role in the
breakdown of the tissues, but also more speci®c roles in
cleaving particular proteins in the meiotic cells during
meiotic development, and in the DNA repair process in the
mycelium (somatic) cells.
For example, many aminopeptidases including LAP are
essential for digestive and intracellular protein metabolism,
including r egulation of the levels of hormones [21±23]. It has
also been proposed that the e nzymes are i nvolved in
regulating rates of hydrolysis o f proteins that a re degrad ed
by the ubiquitin-dependent pathway [22]. Recent assess-
ments have suggested that the ubiquitin-dependent path-
ways are r esponsible for degradation of a signi®cant amount
of damaged or obsolete protein. On the other hand, PepA
reportedly functioned as a DNA-binding protein in Xer site-
speci®c recombination and in transcriptional control of t he
carAB operon in E. coli [25±27], although CoLAP does not
appear to show such activities.
Fig. 5. Analysis of CoLAP expression in meiotic tissue by immuno-
chemiluminescence staining. Meiotic t issue from leptotene, L, z ygotene,
Z, pachytene, P, and diplotene, D, were s ectioned , and the section s
were incubated with C oLAP antiserum or preimmune serum. Detec-
tion of antigen±antibody complex was facilitat ed by the use of anti-
rabbit IgG alkaline phosphatase-conjugated secondary Ig. Arrows
marked M indicate meiotic cells and S i ndicate supporting cells.
(Bars 0.2 mm).
Fig. 6. Analysis of CoLAP expression in meiotic tissue by immuno-
¯uorescence staining. The sections were stained with DAPI or incu-
bated with C oLAP antiserum. De tection of antigen±antibody complex
was facilitated by the use of Alexa F luorÒ 488 goat anti-rabbit IgG
(H + L) conjugate se condary an tibody. A rrows m arked M indicate
meiotic cells and S indicate supporting cells. (Bar 0.2 mm).
Ó FEBS 2002 Leucineaminopeptidase and meiosis (Eur. J. Biochem. 269) 831
According to biochemical studies of lily meiosis [28±30], a
small amount of DNA is replicated at zygotene, and repair
synthesis of DNA occurs at pachytene. Both DNA synthe-
ses occur in nonsense DNA regions of the chromosomal
DNA, and the regions differ from each other, nonrepeti-
tious sequences at zygotene and middle repetitious sequences
at pachytene [28±30]. There are therefore two possible
events in DNA synthes is, homologous chromosome pairing
at zygotene and recombination at pachytene. According t o
Hotta and S tern [28], a small amount of DNA synthesis at
zygotene was required for the homologous chromosome
pairing and the recombination. As these functions occur
only in meiotic cells, they must be shut off from the
neighbouring somatic cells. That may be why CoLAP is
abundant in the neighb ouring somatic cells. In the meiotic
cells, C oLAP was transcribed ef®ciently only at the
diplotene stage or later, when DNA is no longer synthe-
sized, suggesting t hat Co LAP hydrolyses the obsolete
proteins related to DNA synthesis.
The roles of LAPs are of interest and pose a problem to
be solved in the f uture. CoLAP-de®cient mutants a re
required for further information, and a detailed investiga-
tion of the phenotype of such mutants is necessary.
Attempts to knock ou t the gene are being made.
ACKNOWLEDGEMENTS
We thank all the p eople who support us.
REFERENCES
1. Sakaguchi, K. & L u, B.C. (1982) Meiosis in Coprinus:character-
ization and activities of two forms of DNA polymerase during
meiotic stages. Mol. Cell. Biol. 2, 752±757.
2. Sakaguchi, K. (1987) DNA polymerases used in siste r-chromatid
exchanges or meiotic chromo some recombin ation. (in Japanese )
Tanpakushits u Kakusa n Koso 32, 1321±1328.
3. Lu, B.C. & Sakaguchi, K. (1991) An endo-exonuclease
from meiotic tissues of the basidiomycete Coprinus cinereus.Its
puri®cation and c haracterization. J. Biol. C hem. 266, 21060±
21066.
4. Matsuda, S., Takami, K., S ono, A. & Sakaguchi, K. (1993)
A meiotic D NA polymerase from Coprinus cinereus:furtherpuri-
®cation and chara cterization. Chromosoma 102, 6 31±636.
5. Matsuda, S., Sakaguchi, K., Tsukada, K. & Teraoka, H. (1996)
Characterization of DNA ligase from the fungus Coprinus cine-
reus. Eur. J. Biochem. 237, 691±697.
6. Gomi, K. & Sakaguchi, K. (1994) A new m eiotic protein factor
which enhances activity of meiotic DNA polymerase from
Coprinus cinereus. Biochem. Biophys. Res. Comm. 198, 1232±1239.
7. Takami, K., Matsuda, S., S ono, A. & Sakaguchi, K. (1994)
AmeioticDNApolymerasefromamushroom,Agaricus bisporus.
Biochem. J. 299, 335±340.
8. Kitamu ra, A., Kouroku, Y., Onoue, M., Kimura, S., Takenouchi,
M. & Sakaguchi, K. (1997) A new meiotic endonuclease from
Coprinus meiocytes. Biochim. Biophys. Acta 1342, 205±216.
9. Sawado, T. & Sakaguchi, K. (1997) A DNA polymerase alpha
catalytic subunit is puri®ed independently from the tissues at
meiotic prometaphase I of a basidiomycete, Coprinus cinereus.
Biochem. Biophys. Res. Comm. 232, 454±460.
10. Nara,T.,Saka,T.,Sawado,T.,Takase,H.,Ito,Y.,Hotta,Y.&
Sakaguchi, K. (1999) Isolation of a LIM15/DMC1 homolog from
the basidiomycete Coprinus cinereus and its expression in relation
to meiotic chromosome pairing. Mol. Gen. Genet. 262, 781±789.
11. Nara, T., Yamamoto, T. & Sakaguchi, K. (2000) Characterization
of interaction of C- and N-terminal domains in LIM15/DMC1
and RAD51 from a basidiomycetes, Coprinus cinereus. Biochem.
Biophys. Res. Comm. 275, 97±102.
12. Nara, T., Hamada, F., Namekawa, S. & Sakaguchi, K. (2001)
Strand exchange re action in vitro and D NA-dependent ATPase
activity of recombinant LIM15/DMC1 and RAD51 proteins
from Coprinus cinereus. Biochem. Biophys. Res. Commun. 285,
92±97.
13. Raju, N .B. & Lu, B .C. (1970) Meiosis in Coprinus. III. T iming of
meiotic e vents in C. lagopus (sensu Buller). Can. J. Bot. 48, 2183±
2186.
14. Pukkila, P.J., Yashar, B .M. & Binninger, D.M. (1984) Analysis o f
meiotic developm ent in Coprinus cinereus. Symp Soc. Exp. Biol.
38, 177±194.
15. DeGuzman, R. & Riggs, C.D. (2000) A survey of proteinases
active duringmeiotic development. Planta 210, 921±924.
16. Au subel, F.M., Brent, R. & Kingston , R.E. (1987) Current
Protocols i n Mole cular Bio logy . Green Pu blishing Asso ciates &
Wiley-Interscience, New York.
17. Towbin, H., Staehelin, T. & Gordon, J. (1979) Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc. Natl Acad. Sci.
USA 76, 4350±4354.
18. Hasezawa, S. & Nagata, T. ( 1991) Dynamic organization of plant
microtubules at the three dis tinct transition points du ring the cell
cycle progression of synchroniz ed toba cco BY-2 ce lls. Bot. Acta
104, 206±211.
19. Altschul, S.F., Gish, W ., Miller, W ., Myers, E.W. & Lipman, D. J.
(1990) Basic local alignment search tool. J. Mol. Biol. 215,
403±410.
20. Mcdonald, J.K. (1986) Mammalian Proteases (Mcdonald, J.K. &
Barrett, A.J., e ds), Vol. 2, pp. 7±19. A cademic Press, New York.
21. He rshko, A. (1991) The ubiquitin pathway for protein degrada-
tion. Trends Biochem. Sc i. 16, 265±268.
22. Taylo r, A. (1993) Aminopeptidases: structure and function.
FASEB J. 7, 290±298.
23. Hochstrasser, M. (1995) Ubiquitin, proteasomes, and the regu-
lation of intracellular protein degradation. Curr.Opin.CellBiol.7,
215±223.
24. Bachmair, A., Finley, D. & Varshavsky, A . (1986) In vivo half-life
of a protein is a function of its amino-terminal residue. Science
234, 179±186.
25. Stirling, C.J., Colloms, S.D., Collins, J.F., Szatmari, G. &
Sherratt, D.J. (1989) xerB, an Escherichia coli gene required for
plasmid ColE1 site-speci®c recombination, is identical to pepA,
encoding aminopeptidase A, a protein with substantial similarity
to bovine lens leucine aminopeptidase. EMBO J. 8, 1623±1627.
26. Charlier, D., Hassanzadeh, G., Kholti, A., Gigot, D., Pierard, A.
& Glansdor, N. (1995) carP, involved in pyrimidine regulation of
the Escherichia coli carbamoylphosph ate synthetase operon en-
codes a sequ ence-spe ci®c DN A-bind ing p rotein ide ntical to X erB
and PepA, also required for resolution of ColEI multimers. J. Mol.
Biol. 250, 392±406.
27. Strater, N., Sherratt, D .J. & Colloms, S.D. (1999) X-ray structure
of aminopeptida se A from Escherichia coli and a model for the
nucleoprotein c om plex in Xer site-sp eci®c recombination. EMBO
J. 18, 4513±4522.
28. Hotta, Y. & Stern, H. (1971) Analysis of DNA synthesis during
meiotic prophase in Lilium. J. Mol. Biol. 55, 337±355.
29. Hotta, Y., Tabata, S. & Stern, H. (1984) Replication and nick ing
of zygotene DNA sequences. Control by a meiosis-speci®c protein.
Chrmosoma 90, 243±253.
30. Hotta, Y., Tabata, S., Bouchard, R.A., Pinon, R. & Stern, H.
(1985) General r ecombination m echanisms in extracts of meiotic
cells. Chromosoma 93, 140±151.
832 T. Ishizaki et al. (Eur. J. Biochem. 269) Ó FEBS 2002
. CoLAP, Coprinus leucine aminopeptidase; DAPI, 4¢,6-diamino-2-phenylindole dihydro- chloride. Enzyme: leucine aminopeptidase (LAP; EC 3.4.11.1). *Presen t address: Nagoya University School of M. because its meiotic cell cycle is long and naturally synchronous [9±14]. The dikaryonic cells are at the premeiotic stage from S-phase to leptotene. From the beginning of the k aryogamy, when the. identi®ed by assay of aminopeptidase activity and SDS/PAGE, was pooled and stored in aliquots at 4 °C (data not shown). Immunological analysis and immuno¯uorescence microscopy A polyclonal antibody against