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MICROBIOLOGY LETTERS ELSEVIER FEMS Microbiology Letters 143 (1996) 4145 Autocrine response of Schizosaccharomyces pombe haploid cells to mating pheromones Kenji Kitamura ‘, Tomohiro Departmentof Biology, Nakamura Faculty of Science. 2, Futaba Miki 3, Chikashi Osaka City University, Sumiyoshi-ku. Shimoda * Osaka 558, Japan Received 14 May 1996; revised 15 July 1996; accepted 15 July 1996 Abstract The mating response of the fission yeast Schizosaccharomyces pombe is mediated by mating pheromones, M-factor and Pfactor, produced by h- and h+ cells, respectively. When the M-factor receptor (Map3) was ectopically expressed in h- cells lacking the P-factor receptor (MamZ), they acquired mating competence in response to M-factor which they secreted. The autocrine response to P-factor in h+ cells was so weak that mating competence was not acquired, although expression of the pheromone-responsive gene matl-Pm was detected. These observations support the notion that the intensity of cellular response to mating pheromones is different between h- and h+ cells, although downstream pathways of the pheromone receptors are shared by the two mating types. Keywords: Fission yeast; M-factor; P-factor; Pheromone receptor; 1. hltroduction Schizosaccharomyces proteins pombe for P-factor and M-factor are encoded by [3,4]. Pheromone-activated receptor proteins transmit signals to the trimerit GTP-binding protein whose a subunit is encoded by gpal+ [5]. This pheromone signal is further transferred through the MAP kinase cascade to nuclei [6-S] and finally induces expression of matl-Pm [9] and sxu+?+ [lo]. The structural genes encoding M-factor and P-factor are transcribed only in h- and h+ cells, respectively [1,2] and their expression is strictly controlled in a mating-type-specific manner [3,4]. Such strict transcriptional control might guarantee the specificity of the cellular response to mating pheromones. In this study we demonstrated the autocrine response of S. pombe haploid cells to their own pheromones by ectopically expressing the receptor protein of the mam2+ and map3+, respectively The mating reaction between opposite mating types (h- and h+) of the fission yeast Schizosaccharomyces pombe is regulated by diffusible mating pheromones, M-factor and P-factor [ 1,2]. Receptor * Corresponding author. Tel.: +81 6605 2576; Fax: +81 6605 3158; E-mail: [email protected] ’ Present address: Center for Gene Science, Hiroshima University, Higashi-Hiroshima, Hiroshima 739, Japan. 2 Present address: Department of General Education, Osaka Institute of Technology, Omiya, Asahi-ku, Osaka 535, Japan. 3 Present address: Kazusa DNA Research Institute, Kisarazu, Chiba 292, Japan. 0378-1097/96/ $12.00 Copyright 0 1996 Federation of European Microbiological Societies. Published by Elsevier Science B.V. iWSO378-1097(96)00289-3 42 K. Kitamura et al. I FEMS Microbiology LetterA 143 (1996) 4145 opposite mating type and that the autocrine was stronger in h- cells than in h+ cells. response 2. Materials and methods 2.1. Yeast strains and media The strains of S. pombe used in this study are listed in Table 1. Mating and sporulation were induced on MEA and SSA media [l 11. SSL+N is a liquid synthetic minimal medium and SSL-N is its nitrogen-free version used as a sporulation medium lkb B 1 --- 18S- A 3.8kb Hind111 fragment carrying the map3+ open reading frame (ORF) was cloned into the yeast multicopy plasmid pDB248’ [13] and the resultant plasmid was designated pTN21 (Fig. 1A). The same Hind111 fragment was inserted into the same vector in the reverse direction to construct pTN22 (Fig. 1A). A 1.4-kb DNA fragment carrying the mam2+ ORF was inserted into the expression vector pCMV-LX [14] to make mam2’ express under the control of the cytomegalovirus promoter (pCV2L). In plasmid pCV2R, the mam2+ ORF was present in the reverse orientation. The mam2-A84 mutation was fully complemented by the former plasmid but not by the latter. 2.3. Northern analysis Northern blot analysis was performed according to the method of Kitamura and Shimoda [3]. Poly(A)+ RNA was prepared using oligo(dT)-latex beads according to the manufacturer’s instructions (Nihon 3 25S- [121. 2.2. Plasmid construction 2 *i i., _, Fig. I. Overexpression of map3’. (A) Restriction map of map3’. Top line indicates the chromosomal region and restriction enzyme sites surrounding the map3+ locus. Thick black arrow indicates the map3’ ORF. The leftward dotted arrow represents the hybridization probe used in Northern analysis. Shaded boxes in the center and at the bottom represent genomic DNA fragments isolated in this study. Flanking vector sequences are indicated by the thick black line. (B) Northern analysis of plasmid-derived map3+ transcripts. Cells of C534-13C transformed with three different plasmids were cultured in SSL+N medium. 1 lg of poly(A)+ RNA prepared from these cells was blotted onto nylon membranes and then hybridized with the probe of map3. Localization of the 18s and 25s rRNAs used as size markers is also indicated. Lane 1, pDB248’ (vector); lane 2, pTN21; lane 3, pTN22. Roche, Tokyo, Japan). The 0.8-kb XbaIIXhoI fragment containing map3 was cloned into pBluescript (Stratagene, La Jolla, CA) and the map3 riboprobe was labeled with 32P using a T7 in vitro transcription system (Nippon Gene, Tokyo, Japan). 3. Results and discussion 3.1. map3+ was cloned as the multicopy suppressor oj’ mam2- A84 Table I List of S. pombe strains used in this study Strains Genotype Cl 14-2D C534-13C C534-13D C526-1D L975 hgo h-S h-s his htN mam2-A84 mam2-A84 mam2-A84 leul ura4-Dl8 ade6-M216ieul ade6-M216leul ade6-M216leul uraQ-DIX mam2+ encodes the membrane-integrated receptor protein for P-factor secreted by h+ cells [3]. In order to isolate genes which function downstream of the mam2+ gene, we screened for multicopy suppressors of the mam2-A84 mutation by transforming the homothallic strain C114-2D with an S. pombe genomic library [15]. A few plasmids which complemen- K. Kitamura et al. IFEMS Microbiology Letters 143 (1996) 41-45 ted the mam2-A84 mutation were isolated. Most of the positive clones contained mam2+. One plasmid in the rest, pTN21, was further analyzed. Restriction analysis and Southern blotting of pTN21 revealed that it contained map3+ which encodes the M-factor receptor. Comparison of the restriction map of the DNA fragment cloned in pTN21 with that reported for map3+ [4] suggested that the cloned gene lacked its transcriptional promoter (Fig. 1A). Thus, map3+ in pTN21 was expressed from the cryptic promoter in the vector, pDB248’ [16]. To confirm ectopic expression of map3+ in h- cells, Northern analysis was conducted (Fig. 1B). map3+ transcripts were detected on autoradiographs of blots as two major bands in cells harboring pTN21. These bands may have represented RNA products transcribed from different start sites, although we did not clarify this further. No corresponding bands were found on autoradiographs of blots of RNA from control transformants or transformants harboring pTN22. h- cells harboring pTN21 exhibited characteristic shmooing (Fig. 2B), as previously reported by Tanaka et al. [4]. We then investigated whether h- cells autocrinely stimulated with pheromones acquired mating competence. Map3-expressing cells carrying the mar&-A84 mutation were mixed on a sporulation medium with wild-type h+ cells. As shown in Fig. 2D, normal zygotic asci were formed, though the same strain carrying vector plasmid alone did not mate with h+ cells (Fig. 2C). These observations indicate that autocrine stimulation of h- cells by ectopic expression of M-factor receptors was sufficient for acquisition of mating competence by these cells. Fig. 2. Effect of ectopic expression of map3+ on mating behavior of a mar&-A84 mutant. Cells were cultured on SSA medium. Micrographs were taken using a phase-contrast microscope. (A,C) C534-13D harboring pDB248’; (B,D) C534-13D harboring pTN21. (A,B) Single cultures; (C,D) C534-13D transformants mixed with L975 (h+ strain). t 0 43 1 200 fs-Gahct&be I 600 400 aclhitg I 800 (unit@ Fig. 3. Autocrine response to P-factor of h+ cells expressing Mam2. Wild-type h+ cells (C526-1D) were cotransformed with the matl-Pm/lad fusion plasmid, pAUZ1, and pCV2L. As a negative control, the same strain was cotransformed with pAUZ1 and pCV2R in which the coding region of mam2+ was inserted in an antisense direction to the promoter. Activity of B-galactosidase was measured at 0 (open bar) and 8 h (closed bar) after cells were transferred to SSL-N medium. 3.2. Autocrine response to P-factor of h+ cells expressing Mam2 We determined whether the aforementioned autocrine response occurs in h+ cells. An h+ strain was transformed with pCV2L to effect ectopic expression of mam2+ encoding the P-factor receptor. The transformed cells exhibited very poor &mooing under nitrogen-free conditions (data not shown). Furthermore, ectopic expression of mam2+ in a map3 mutant failed to complement the mating deficiency (data not shown). Expression of the mating-type gene mad-Pm is dependent upon pheromone signaling [9]. We then investigated whether this gene is induced in h+ cells harboring pCV2L which are overexpressing mam2+. The strain was cotransformed with the multicopy plasmid pAUZ1, which carries the mad-Pm gene fused to the E. coli 1acZ gene [9]. The expression level of mad-Pm was assessed in terms of the level of B-galactosidase activity in the cells. As shown in Fig. 3, P-galactosidase activity was markedly increased after the transformants were transferred to a nitrogen-free medium, suggesting that the transformants exhibited an autocrine response to P-factor which they were secreting. On the other hand, B-galactosidase activity was very low, even in nitrogenthe plasmid free medium, in h+ cells harboring pCV2R in which mar&+ was inserted so as to generate antisense mRNA. We may conclude that hi cells ectopically expressing the P-factor receptor pro- K. Kitamura et al. I FEMS 44 Microbiology tein showed an autocrine response to their secreted pheromone. In the present study, we demonstrated that cells ectopically expressing pheromone receptors showed an autocrine response to their secreted pheromones resulting in full or partial mating reaction. Similar results have been reported for the budding yeast Succharomyces cerevisiae [ 171. Therefore, mating specificity in yeasts is determined by the expression of mating pheromone by cells of one mating type and the expression of the corresponding pheromone receptor by cells of the other mating type. The pheromone-responsive genes, mad-Pm in h+ cells and sxu2+ in h- cells, have been reported to be transcribed only in cells of the respective mating types [ 10,171, suggesting the involvement of unknown mating-type-specific factors in addition to pheromone signaling in the control of gene expression. We also observed that h- and h+ cells were not equal in the extent of their autocrine response. Why is the autocrine response in h+ cells weaker than that in h- cells? One plausible explanation is that the level of secreted M-factor in a culture of h- cells is higher than that of secreted P-factor in a culture of h+ cells. A previous report that P-factor was detected at extremely low levels in the culture filtrate of a wild-type h+ strain may support this assumption [2]. Sensitive bioassay would require the use of mutants of sxu2’ which is potentially relevant to the proteolysis of secreted P-factor [lo]. We assume that the production and/or secretion of P-factor might be stimulated by M-factor signaling. Our observation that the autocrine response in h+ cells was very weak has prompted us to test this hypothesis. Acknowledgments The present study was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan to C.S. References [I] Davey, J. (1992) Mating pheromones zosaccharomyces pombe: purification of the fission yeast Schiand structural character- Letters 143 (19961 4145 isation of M-factor and isolation and analysis of two genes encoding the pheromone. EMBO J. 11, 951-960. [2] Imai, Y. and Yamamoto, M. 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