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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.
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