Báo cáo Y học: Leucine aminopeptidase during meiotic development doc

Leucine aminopeptidase during meiotic 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 leucine aminopeptidase (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 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 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: leucine aminopeptidase (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 meiotic development 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 Leucine aminopeptidase 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 meiotic development 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 Leucine aminopeptidase 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 dierent 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 meiotic development 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 Leucine aminopeptidase 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 during meiotic 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