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 sc
Trang 1Leucine aminopeptidase during meiotic development
Takashi Ishizaki’, Aki Tosaka"*, Takayuki Nara"*, Narumichi Aoshima’, Satoshi Namekawa’,
Kei Watanabe’, Fumika Hamada’, Akira Omori? and Kengo Sakaguchi’
‘Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Noda, Chiba, Japan; Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan
We found a leucine aminopeptidase (LAP; EC 3.4.11.1) to
be abundant in meiotic prophase tissue of a basidiomycete,
Coprinus cinereus After direct purification of the amino-
peptidase component from meiocytes, we cloned the gene
by degenerate PCR using partial amino-acid sequences
of the purified 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 leucine aminopeptidase (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 factors using
meiotic cells in a basidiomycete, Coprinus cinereus [1-12] In
meiosis, chromosomes condense from the dispersed state
typical of interphase during early meiotic prophase, to form
long thin threads in leptotene, and each acquires a
proteinaceous axial core to which the two sister chromatids
are attached Then, homologous chromosomes become
aligned during zygotene, 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 studies 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 karyogamy,
when the two nuclei fuse, for the next 5 h the cells are at the
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: leucine aminopeptidase (LAP; EC 3.4.11.1)
* Present address: Nagoya University School of Medicine, Chikusa-ku,
Nagoya 466-8550, Japan
t Present address: Department of Food Science and Human Nutrition,
University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,
USA
(Received 11 October 2001, revised 26 November, accepted 29
November 2001)
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 it possible to purify the meiosis-related protein factors to near homogeneity [1-12]
According to DeGuzman & Riggs [15], proteolytic activities intensified 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 efficiently 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- fied an aminopeptidase to near homogeneity through five columns The purified component showed aminopeptidase activity with a molecular mass of 50 kDa, but its involve- ment in meiosis was not clear Therefore, we attempted to determine its partial amino-acid sequences, so as to clone the gene through degenerate PCR methods We subse- quently found that the gene sequence has homology with leucine aminopeptidases (LAP) in mammals, plants, and bacteria The meiosis-specific aminopeptidase was concluded
to be a Coprinus alternative of LAP (CoLAP) Consideration should now be given to the possibility that CoLAP has roles
in the progression and development of the meiotic cell cycle There must be some coordination between CoLAP and meiosis Analysis of the proteins that are required for these processes provides insight into the mechanism of this coordination
Trang 2In this report, we have focused on the LAP that is
associated with the meiotic development of C cinereus, and
characterized the enzyme in relation to meiotic events
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 x 60 mm)
containing sterile horse manure were inoculated with a
dikaryotic stock culture of C cinereus, then incubated for
7 days in an incubator at 37 °C in total darkness, before
photo induction of fruitification with a light cycle regime of
16 hlight/8 h dark at 28 °C The light cycle began at 05 : 00
local time Karyogamy, defined as the time at which 5% of
all basidia have fused nuclei, starts at 04 : 00, | 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 assay, | mm L-leucine-p-nitro-
anilide in 50 mm Tris/HCl pH 7.6, was incubated at 37 °C
Reactions were terminated by adding sodium acetate The
absorbance of the liberated p-nitroanilide was measured
with Bio-Rad’s microplate reader at 405 nm
Purification of aminopeptidase from tissues
at meiotic prophase
The TMG buffer contained 50 mm Tris/HCl pH 7.5, 5 mm
2-mercaptoethanol, 15% (v/v) glycerol, and three protein
inhibitors, pepstatin A(1 mgmL"'), leupeptin (1 mgmL7‘),
and 1 mm phenylmethanesulfonyl fluoride All procedures
were performed at 4 °C
The tissues of Coprinus fruiting bodies (20 g) at pachytene
to diplotene were homogenized in 10 vol TMG buffer
containing 0.8 m NaCl using a French press and 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 TMG buffer
The elution profile 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 TMG buffer The elution was performed with
60 mL of a linear NaCl gradient (0-1.0 m) in TMG buffer
The component with significant aminopeptidase activity
was present at 0.4m The fractions from the HiTrap- Heparin-agarose column chromatography were collected together, and then after being 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 (0O-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 mm NaCl as a single peak The fractions were analysed further by SDS/PAGE and Superose 6 gel filtration 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 band was purified 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 membrane Peptides released from the membrane were fractionated by reversed-phase HPLC using a C8 column (1.0 x 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 partial cDNA sequence was obtained with two reverse transcription (RT)/PCR degenerate primers derived from two determined amino-acid sequences: C-67 sense primer (ŠS-GGCACCGCCCGCACNTTYTAYAA-3) and S-2009 antisense primer (5-GGACGTTGGGGATGCCNGCR AAYTC-3’/) (N = A, C, G, T, R = AG, Y = CT) Cycling conditions were: 95 °C for 5 min; 95 °C for 1 min;
60 °C for 1.5 min; 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, GSPI (5-GACAACCTCGGTCGTCTCTT T-3’) and GSP2 (5’-CCTCAAGACTTCTCCCCCTTC-3’) were designed using Primer3 (MIT Whitehead Institute) For 5’ RACE, A-GSP1 (5’-GGAGAAGTCTTGAGGGT GAAC TT-3’), A-GSP2 (5’ TCCTAGCAAGGTTCTGG GACT-3’) and A-GSP3 (5-GGAGAAGTCTTGAGGG TGA ACTT-3’) were used Downstream 1300-bp and upstream 400-bp products were cloned and sequenced The DDBJ/EMBL/GenBank accession number of the CoLAP nucleotide sequence reported in this paper is ABO052095
Genomic DNA isolation and Southern hybridization analysis
Genomic DNA was isolated from Coprinus mycelium tissue and digested with four restriction enzymes: EcoRV, Sall,
Trang 3Smal or XhoI 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 probes were gel-purified 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 prophase
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] Total RNA
(25 ug) from the caps at each meiotic stage and the methyl
methanesulfonate-treated tissues harvested at 1-h intervals
were loaded in each lane The agarose gel was stained with
ethidium bromide and blotted overnight onto Hybond-N +
membranes (APB) The membranes were fixed by alkali
reagents, rinsed with 2 x NaCl/P;/EDTA, and hybridized
with *°P-labelled probe for hybridization analysis
Over-expression and purification of a CoLAP protein
The CoLAP coding region was amplified using N- and
C-terminus open reading frame primers with EcoRI and
Xhol sticky ends The amplified product was gel-purified,
digested with EcoRI and XholI, and cloned into the pET2la
expression vector (Novagen) to generate pET21-CoLAP-
(his)s 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, containing 50 ugmL7 ampicillin and
| mm isopropyl thio-B-p-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 profile of 50 mL of a linear gradient
(0-1 m) imidazole buffer [20 mm Tris/HCl pH 8.0, 500 mm
NaCl, 10% (v/v) glycerol, 0.02% NP-40] at a flow rate of
0.75 mLmin'Ì, followed by a Mono Q column (APB) with
15 mL ofa linear gradient of 0.05—-1 M NaClin TMG buffer
at 0.5mLmin™! The protein, identified by assay of
aminopeptidase activity and SDS/PAGE, was pooled and
stored in aliquots at 4 °C (data not shown)
Immunological analysis and immunofluorescence
microscopy
A polyclonal antibody against CoLAP protein was raised in
a rabbit Western blot analysis was carried out according to
the method of Towbin e¢ 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 paraffin
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 SugmL! 4’,6-diamino-2-phenylindole dihydrochloride (DAPI) for 5 min The specimens were examined under a light or fluorescence microscope (OLYMPUS BH-2)
RESULTS
Purification 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- specific events, crude extracts were generated from the caps
at different stages of meiotic prophase in a basidiomycete,
C cinereus, and partially purified through HiPrep-DEAE sepharose column chromatography They were then assayed for various protease activities The aminopeptidase activity bound to HiPrep-DEAE sepharose was greatest 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 forming the chiasmata which become visible during diplotene It is interesting that the aminopeptidase activity increases as meiotic development proceeds 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 the Coprinus meiosis-specific aminopeptidase to near homogeneity, and succeeded through five rounds of column chromatography
as described in Materials and methods The active fraction from the final (single-stranded DNA sepharose) column chromatography was purified 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 filtration (Fig 1B), suggesting that the protein probably forms a homohexamer
This study represents the first purification and character- ization of an aminopeptidase, which might have a role in the meiotic cell cycle, especially at meiotic prophase However, the amount of enzyme isolated was not sufficient 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 using a set of degenerate primers
Isolation and characterization of cDNA of the meiotic aminopeptidase in Coprinus meiocytes
To isolate cDNA of the meiotic aminopeptidase in Copri- nus, two degenerate PCR primers (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 template An ~ 500-bp fragment was obtained and sequenced Downstream 3’ sequences and upstream 5’ sequences were extended by RACE methods The Coprinus cDNA sequence contains 489 amino acid residues, with a calculated molecular mass of 52.4 kDa
Trang 4Fig 1 SDS/PAGE of the purified C cinereus
meiosis-specific aminopeptidase component and
determination of its molecular mass by gel
filtration chromatography (A) The final pre-
paration of the metosis-specific aminopepti-
dase was analysed 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 on Superose 6 (APB) The activity
was detected in the 340-kDa fraction
(Fig 2), which contained three obtained amino-acid
sequences (see underlined sequence in Fig 2) Interestingly,
the amino-acid sequence was highly homologous to that of
LAP Database searches with the BLASTx program [19]
revealed that the CoLAP gene has identity 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 1s common to LAPs from other organisms
(see box in Fig 2) The meiotic aminopeptidase appears to
be a counterpart of LAP from mammals, plants and yeast
We temporarily designated 1t CoLAP (Coprinus leucine
aminopeptidase)
The Coprinus genomic DNA was digested using the
restriction enzyme EcoRV, Sall, Smal or Xhol Southern
hybridization analysis revealed that, as each of the digested
products had only a single band, it 1s a single-copy gene
(data not shown)
Isolation and characterization of the recombinant
CoLAP homologue protein
To characterize CoLAP in detail, the histidine-tagged
recombinant protein was over-expressed and purified by
Ni" affinity and Mono Q chromatography (see Materials
and methods) SDS/PAGE and Sephacryl S-300 gel
filtration chromatography of the Mono Q fraction re-
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 filtration (data
not shown) The molecular mass of CoLAP was slightly
smaller than that of the originally purified 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 filtration of the native
enzyme might be consistent with it being modified post-
translation in its native state These properties are
consistent with the results for the originally purified
aminopeptidase The pH dependence and optimum tem-
perature toward leucine-p-nitroanilide were quite similar
to those of previously reported LAPs
< 29
Bf = } a er
` TOU he
> ke
NG
Hetlerflon Volươne (ml)
f17/ +
HÀ
Retention Volurne imi!
1 aatctcttcgactccaaccatcacgatcccettttctcgccecgtccaATGTCGTCCGCAATCGTCGTTC 69
MS S A TY VP
70 CCTTTGACCACCAGGCCTCAGCAAAGTCGGTTGCTGGCGTCGACCCAGCCAAGCTCTGGGCTCTGACCC = 138 FDHQAS AK S VAGYVYODP A KL WAL TP
139 CTTCTGGCGAAAAACCACCAAAGGCGGGCACCGCCCGCACGTTTTACAACACCCCCGAGTCAAAGACAA 207
$S 6 E K PP KAÁA 6T A RTF Y NT PE 5 KT TT
208 CCTCGGTCGTCTCTTTGGGCGAAGGCTTTGCCAGCAAGCCTGCAGAGGTCAAGCGAGAGATCGTCAGGA 276
SV VS L GEGF AS K P AE VK REI VR &«K
277 AGGCTGTCGGTAGCGCTGTCAAGGACCTCAAGGGCTACGACGGCGTCAAGGACGTTGCCATTGACGCGT 345 AYVYGS AY KODLKGYODGYKODY ATI ODA S
346 CTTTG6ACCCCCATGCTGCTGCTGTCGCTGCTCACTTGGCCTTGTACAAGTTCACCCTCAAGACTTŒTC 414 LDP HA A AÁA Ý A A HL AL YKPF P
415 CCCCTTICGCCTTTCGACCCCAACCTCAAGGAGCCCATCCCACCCAAGCTCCAGTTCTCGCCCATCGAAG 483
PS P F DPNLK EP IT PPK LQF S PI EA
484 CTTCAAAAGAATGGGACCGCGGTGTCATCTACGCCGAGTCCCAGAACCTTGCTAGGACTTTGATGGAAT 552
$ K E WÝWD R6 Y Il YÝ A E Š5 QN L À R TL MF ŸY
553 ACUCG(CAACATGATGACCCCTACTCTCTTCACCGAACUTGTCAAGACAGAGTTTUCTUGCATCUCCA 021 PPA N M MT PTL FT ER VY K TCE F ÀA 6L PN
622 ACGTCGAAATCATTGTGCGAGACGAGGCATGGGCTGCTGAGAAGGGAATGAACGTCTTCTTGTCTGTCA 690
ŸÝ E II VYRODEAWA AE K GM NY FL S Ý T
691 CCCGTGGAACCTCAGAACCAGCCAAGTTCTTGGAAATCCACTACAAGGGTGCTGCTGACAAGAACGCTC =759
R GTS EP AK FL ETI HY KGAAOD KN A Q
760 AGCCTCTTGCCTTTGTTGGCAAGGGTATCACCTTCGACACTGGAGGAATCAGCTTGAAGCCCGGCGCTG 828 PLAFYVGkKG#tITFODTGGtIS LK PGA G
829 GCATGAAGTT GATGAGGGGAGACATGGGCGGTGCTGCTACCGTCGTCTCTGCTGCGCTTGCTATCGCCA 897 MKLMRGoODMGGAATY YS AAT Ad A XK
898 AGCTCCAACTCCCCATCAACTTGGTTGTCACTACTCCTTTGACGGAGAACATGCCAGGCCCCAGCGCTA 966
L QLPINLYVYVTTPLTENMP GPS AT
967 CCAAGCCCGGTGATATCATCTATGCCATGAACGGCAAGTCCGTCGAGGTCGATAACACTGATGCTGAGG =1035 KPGDIIYAMNGKSVEVODW TDAEG
1036 GTCGCCTCGTTCTCTCCGATGCCATCTACTACACCTCGACTGAGTACAAGCCTCACACTTIGATCGACG 1104
V~LS DA IY YTS TE Y K PH TCT DY
1105 TT CTTGACTGGTGCCATGGTCATCGCCCTCGGAGAGGTCTACTCCGGCGTCTTTGCTTICCTCCG 1173
A TLTG&AM”’YVYIAL GEV YS GY F AS S D
1174 ATGAATTGTGGCAACAACTCTACGAAGCCGGCCAAATCGAGCACGACAGGATGTGGAGAATGCCCCTCG 1242
EL WQaQqtLY EAGQtI EHD RM WRM PL D
1243 ACGATGAGTTTGGACCTCAGATCCACTCTTCGAATGCCUACTTGCAGAACACTGGTGGACGACCTGCGG 1511
D E F 6 P.QIH š5 S5 NA DL QN T06 06 RP À 6õ
1312 GAAGCGCTACCGCCGCCTTGTTCTTGAAGCCCTTCGTTAACGGATTGGAGCCCAAGGAAGGAGAGCCTA 1380
S ATA AL FL K P F VY N GUL EP K EGE P T
1381 CCATCAAGTGGGCTCACCTTGATATCGCTGGTTCCATGGAGGCCACTCGACCTTCTCCTTACCAGGATA 1449 [ K ÝŸWA H L D IAG S M E AT RPS PY QOD K
1450 AGGGCATGACTGGGCGACCTGTCAGGGCCCTCGTCGAGTTCACTCGCCGACTCGCCAACAGCGCTTAAL 1518 GMTGRPVRAL Y EF TRRIAN S A *
1519 tecaaatgtcgegctttgattttettccgagaggtctctccgatggaaggagtgattgatgatatatcgc 1587
l6o7 cctttttggataattcaaaaaa aaaa_ T891
Fig 2 Nucleotide and deduced amino-acid sequences of CoLAP and its flanking regions Amino acids derived from peptide sequencing are underlined; the cytosol aminopeptidase signature 1s boxed
Northern hybridization of CoLAP
To examine whether the CoLAP gene 1s expressed at meiotic prophase as the aminopeptidase was originally purified at
Trang 5pachytene, 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 mycelium tissues (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 fluorescent microscopic observa-
tion of the monokaryonic nuclei, it was concluded that
CoLAP was expressed throughout the meiotic prophase
when the homologous chromosomes pair and recombine
Z
Š 8
Fig 3 CoLAP expression analysis in various phases of meiotic devel-
opment Northern blot analysis of total RNA (25 ug) from the caps at
leptotene, L, zygotene, Z, pachytene, P, and diplotene, D, probed with
°P_labelled CoLAP cDNA 26 S and 18 S rRNA were stained with
ethidium bromide as a loading control
MMS treatment
Fig 4 CoLAP expression analysis in methyl methanesulfonate-treated
tissue Northern blot analysis of total RNA (25 pg) from 0.01%
methyl methanesulfonate (MMS)-treated somatic tissue (hyphae) at
different times probed with *’P-labelled CoLAP cDNA
and then the pachytene-recombined chromosomes separate and form the chiasmata
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 detected 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 methanesulfonate, and expression was analysed by Northern blotting We detected strong expres- sion of CoLAP mRNA immediately after treatment The induction of expression peaked within | 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 role 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 affinity-purified 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) Intense signal for CoLAP was detected from leptotene to 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 are 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 cells 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 confirm it further, in situ immunofluorescence staining using the antibody and stan- dard epifluorescence 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 in a basidiomycete, C cinereus,
a LAP (CoLAP) is specifically 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 first report to indicate that the LAP gene is expressed at meiotic prophase, and to imply
Trang 6
Fig 5 Analysis of CoLAP expression in meiotic tissue by immuno-
chemiluminescence staining Meiotic tissue from leptotene, L, zygotene,
Z, pachytene, P, and diplotene, D, were sectioned, and the sections
were incubated with CoLAP antiserum or pretmmune serum Detec-
tion of antigen-antibody complex was facilitated by the use of anti-
rabbit IgG alkaline phosphatase-conjugated secondary Ig Arrows
marked M indicate meiotic cells and S indicate supporting cells
(Bars = 0.2 mm)
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 in DNA repair in the mycelial cells
Recently, it has become evident that the intracellular
selective degradation of proteins is important as part of the
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 of 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 meiotic development proceeds The results were
quite similar to those studied in microsporogenesis in a
higher plant, Lilium longiflorum, which is another organism
used for this type of study [15], although their enzymes were
in classes of serine and aspartate proteases In lily, protease
activities were correlated with the morphological and
biochemical events of late microsporogenesis [15] The
most dramatic of these was the programmed cell death of
CoLAP
Fig 6 Analysis of CoLAP expression in meiotic tissue by immuno- fluorescence staining The sections were stained with DAPI or incu- bated with CoLAP antiserum Detection of antigen-antibody complex was facilitated by the use of Alexa Fluor® 488 goat anti-rabbit IgG (H + L) conjugate secondary antibody Arrows marked M indicate meiotic cells and S indicate supporting cells (Bar = 0.2 mm)
tapetal cells and anther wall cells which precedes dehiscence [15] It is possible that in Coprinus, as the somatic cells neighbouring the meiotic cells correspond to the Liliun tapetal cells, and as CoLAP is expressed markedly in the somatic cells at zygotene and pachytene, the LAP as 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 specific 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 regulation of the levels of hormones [21—23] It has also been proposed that the enzymes are involved in regulating rates of hydrolysis of proteins that are degraded
by the ubiquitin-dependent pathway [22] Recent assess- ments have suggested that the ubiquitin-dependent path- ways are responsible for degradation of a significant amount
of damaged or obsolete protein On the other hand, PepA reportedly functioned as a DNA-binding protein in Xer site- specific recombination and in transcriptional control of the carAB operon in E coli [25—27], although CoLAP does not appear to show such activities
Trang 7According 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 synthesis, homologous chromosome pairing
at zygotene and recombination at pachytene According to
Hotta and Stern [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 neighbouring somatic cells In the meiotic
cells, CoLAP was transcribed efficiently only at the
diplotene stage or later, when DNA is no longer synthe-
sized, suggesting that CoLAP hydrolyses the obsolete
proteins related to DNA synthesis
The roles of LAPs are of interest and pose a problem to
be solved in the future CoLAP-deficient mutants are
required for further information, and a detailed investiga-
tion of the phenotype of such mutants is necessary
Attempts to knock out the gene are being made
ACKNOWLEDGEMENTS
We thank all the people who support us
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