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TheSaccharomyces cerevisiae early secretion mutanttip20 is synthetic lethal with mutants in yeast coatomer and the SNARE proteins Sec22p and Ufe1p

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
. 14: 617–622 (1998)
An Extracellular Meiosis-promoting Factor in
Saccharomyces cerevisiae
MICHIO HAYASHI, KENTARO OHKUNI AND ICHIRO YAMASHITA*
Center for Gene Science, Hiroshima University, Kagamiyama 1–4-2, Higashi-Hiroshima 739, Japan
Received 10 November 1997; accepted 23 December 1997
Meiosis and sporulation in the yeast Saccharomyces cerevisiae has been classically viewed as an example of
unicellular, eukaryotic differentiation that occurs in response to nutritional starvation. We present evidence that
S. cerevisiae produces an extracellular factor(s), called meiosis-promoting factor (MEP), that is required, in addition
to starvation conditions, for efficient meiosis and sporulation. This factor is secreted and accumulates in a cell
density-dependent fashion such that cells at a low density sporulate poorly under conditions in which cells at a high
density sporulate efficiently. Conditioned medium from sporulating cells at a high density contains a small anionic
molecule that has cytostatic activity and stimulates sporulation of cells at low density under a normal starvation
condition. These results indicate that MEP-mediated social communication between cells is required for meiosis and
sporulation. 1998 John Wiley & Sons, Ltd.
Yeast 14: 617–622, 1998.
  — Saccharomyces cerevisiae; extracellular factor; meiosis; sporulation
INTRODUCTION
Regulation of cell fate decision between proliferation and differentiation is a basic issue of biology.
In animal and plant cells, such a decision is
commonly regulated by intercellular communications. Differentiation in prokaryotes also often
involves communication between cells (Kaiser,
1996). These communications are mediated by a
variety of chemical signals or pheromones. In a
unicellular eukaryote such as yeast, however, it is
generally believed that cells grow exponentially
when supplies of essential nutrients are plentiful,
and that, in the reverse way, limitation of nutrients
such as nitrogen allows cells to arrest proliferation
at a specific cell cycle stage and to enter the
so-called ‘stationary’ or meiotic phase (Esposito
and Klapholz, 1981; Hartwell, 1974).
Growing diploid cells of Saccharomyces cerevisiae with the mating-type a/á can be induced to
*Correspondence to: Ichiro Yamashita, Center for Gene
Science, Hiroshima University, Kagamiyama 1–4-2, HigashiHiroshima 739, Japan. Tel: (+81) 0824 24 6271; fax: (+81) 0824
22 7184.
Contract/grant sponsor: Ministry of Education, Science, Sports
and Culture of Japan
CCC 0749–503X/98/070617–06 $17.50
1998 John Wiley & Sons, Ltd.
arrest division and to differentiate into meiosis and
sporulation upon shift to a sporulation medium
which essentially contains only a non-fermentable
carbon source such as acetate. Cell division arrest
at G1 phase may be a prerequisite for entry into
meiosis. The meiotic process is characterized
by numerous alterations in gene expression and
a variety of physiological and morphological
changes. Initiation of meiosis requires at least two
genes called IME1 and IME2, which are specifically expressed early in meiotic development
and required for premeiotic DNA synthesis and
subsequent spore formation (Foiani et al., 1996;
Kassir et al., 1988; Mitchell, 1994; Yoshida et al.,
1990).
While studying the effects of nutritional deprivation on gene expression, we observed that
sporulation was inefficient at low cell densities,
indicating that starvation conditions per se were
not sufficient to cause efficient sporulation. From
previous reports, we found that this observation
had been reported many times (Fowell, 1969;
Jakubowski and Goldman, 1988), but its molecular basis remained unsolved. Jakubowski and
Goldman (1988) reported that yeast cells secrete
.   .
618
adenine (and other degradation products from
RNA) during sporulation, and that adenine auxotrophs cannot sporulate at low cell density in the
sporulation medium (SPO) not supplemented
with adenine but can when adenine is added
exogenously. They concluded that the adenine
secreted is required for sporulation of adenine
auxotrophs. They also reported, however, that
most prototrophs were not induced by adenine to
sporulate at low cell density, and that sporulation
of a certain prototrophic strain (AP3-R1) was
stimulated by adenine, although the effect of
adenine was much less pronounced than the auxotrophs: in fact, this strain was able to sporulate
relatively efficiently even at low cell density. It
should be concluded that the cell-densitydependent sporulation of adenine auxotrophs is an
artefact because these strains should require
adenine for sporulation as well as for growth and
that communication between prototrophic cells (or
auxotrophic cells incubated in SPO supplemented
with auxotrophic requirements) during sporulation
is not mediated by adenine. In this report, we show
that S. cerevisiae produces an extracellular factor
(MEP) that inhibits proliferation and stimulates
entry into meiosis and sporulation, indicating that
there is indeed communication between cells of
S. cerevisiae during sporulation and that this
extracellular signalling is required for efficient
sporulation.
MATERIALS AND METHODS
Strains, plasmids and media
The S. cerevisiae strains used and their relevant
genotypes are as follows: YIY808 (MATa leu2
ura3 ade6) transformed with a LEU2 plasmid
YEp51B (Akada et al., 1997); YIY1014 (MATa
leu2 ura3 trp1 ade6 lys2); and YIY1018 (MATa/
MATá) isogenic to YIY1014. The plasmids
pIY134 and pIY281 (Yamashita, 1993) contain
lacZ (Escherichia coli â-galactosidase) fusions,
sga1 (sporulation-specific glucoamylase)-lacZ and
ime2-lacZ, respectively, and both URA3 markers.
The fusion genes contain intact upstream regulatory sequences for transcription that lead to regulated expression. The plasmid pIY134 was
constructed by replacing the KpnI-XhoI SGA1
fragment of plasmid pSGA (Kihara et al., 1991)
with EcoRI-SalI lacZ of plasmid pMC1587
(Casadaban et al., 1983), in which the 26th codon
of SGA1 (Yamashita et al., 1987) was fused
in-frame to lacZ.
1998 John Wiley & Sons, Ltd.
Rich media contained 1% yeast extract, 2%
peptone, and as a carbon source 2% glucose (YPD)
or 1% potassium acetate (YPA). Synthetic medium
(SD) contained 0·67% yeast nitrogen base without
amino acids (Difco) and 2% glucose. Sporulation
medium (SPO) contained 1% potassium acetate.
SD and SPO were supplemented with auxotrophic
requirements. Plates contained 2% agar.
Sporulation
Cells were precultured on plates or in liquid
media. Approximately 1107 cells of strain
YIY1018 freshly grown on YPD plates were
spread on YPD, YPA and SD plates (diameter,
8·5 cm), each containing 40 ml of medium, and
cultured for 40 h at 28C, yielding stationary cells.
Transformed cells (YIY1018 with plasmid pIY134
or pIY281) were cultured on SD plates as above.
Cells were also grown in liquid YPA and YPD to
logarithmic and early stationary phases, respectively. These cells were collected, washed with
deionized water, and shifted into SPO. The culture
was shaken at 28C. Sporulated cells were counted
under a microscope after a brief sonication. Meiosis was examined after staining DNA with DAPI
(Yoshida et al., 1990). Cells at low density were
concentrated, before counting, by successive
centrifugations, during which loss of cells was less
than 20% in three references.
Conditioned medium
Cells of strain YIY1018 were grown in YPA to
log phase (at an optical density at 600 nm of 0·5),
collected, washed with deionized water, and shifted
into SPO at an initial density of 6106 cells/ml.
The culture was shaken at 28C. For a large-scale
preparation, the culture was centrifuged twice and
the upper portion of the final supernate was carefully removed to avoid contamination of cells
(usually less than 1103 cells/ml). For a smallscale preparation, the supernate was further
filtrated with a syringe. Conditioned medium was
prepared by mixing equal volumes of the supernate
(or the filtrate) and fresh SPO. SPO was also
diluted two-fold with deionized water, and used as
a control.
Assay for MEP activity
MEP activity was assayed biologically based on
its cytostatic activity. The filtrate from sporulating
cells was diluted successively, two-fold each, with

. 14: 617–622 (1998)
 - 
619
Table 1. Effects of preculture conditions on sporulation efficiencies at high and low densities.
Preculture condition
Sporulation (%)b
Medium
Growth phasea High
Low
YPA
YPD
SD
Log
Stationary
Early stationary
Stationary
Stationary
82
26
58
27
46
28
13
15
19
4
a
Log and early stationary cells were prepared by cultivation of
cells in liquid media to densities of 7·5106 and 3·4107
cells/ml, respectively. Stationary cells were prepared by cultivation on plates.bCells were incubated for sporulation at high
(4106 cells/ml) and low (4104) densities.
deionized water in wells of a 96-well multititer
plate. To the diluted solution (100 ìl) was added
an equal volume of melted 2% agar containing a
two-fold concentration of SPO. The plate was
dried for 30 min under air currents. Cells of the
tester strain (YIY808), grown on an SD plate at
28C for 2 days, were suspended in deionized
water at a density of 2104 cells/ml, and 10 ìl
of the cell suspension was spotted onto an agar
block in each well. The multititer plate was
incubated at 28C for 4 days, then visually
inspected for colony formation. MEP activity was
presented as a maximal dilution fold showing no
colony formation.
Reproducibility
Experiments were repeated at least twice with
similar results.
examined for meiosis after staining DNA with
DAPI, and obtained similar results (data not
shown). We selected and used, for further study,
the condition, shifting stationary cells grown on
synthetic glucose (SD) plates into SPO, because
these cells showed the lowest efficiencies of meiosis
and sporulation at low cell density. The percentage
for meiosis and sporulation varied from experiment to experiment and especially among
strains, but the large effect of cell density on
meiosis and sporulation was reproducible
(data not shown).
Expression of sporulation-induced genes in cells at
high and low density
We measured the effect of cell density on the
expression of three genes that are induced during
sporulation. These are IME1 and IME2, which are
induced at an early stage during sporulation, and
SGA1, which is induced late during sporulation
(Yamashita and Fukui, 1985). The expression of
each gene was measured using lacZ fusions or by
RNA blotting. Because cells at low density were
not sporulating, expression of genes normally
induced during sporulation was expected to be
blocked. This was the case for all the genes tested.
Following the shift to SPO, expression of these
genes was induced only in cells at high density and
not efficiently in cells at low density (Figure 1).
Thus, the sporulation-specific events required
to induce expression of these genes must be
blocked at low cell density. Because the induction
of IME2 and SGA1 is dependent on IME1
(Kihara et al., 1991; Smith and Mitchell, 1989;
Yoshida et al., 1990), the primary event blocked
at low cell density should be related to induction
of IME1.
RESULTS AND DISCUSSION
Inefficient sporulation at low cell density
There are several experimental protocols for
sporulation, in which preculture conditions may
affect the kinetics and efficiency of sporulation. To
find the condition that shows the highest dependency of sporulation efficiency on cell density, cells
of the strain YIY1018 were grown in several media
to different growth phases, shifted into sporulation
medium (SPO) at high (4106 cells/ml) and low
(4104) cell densities, and examined for sporulation efficiency (Table 1). For all the conditions
tested, sporulation occurred more efficiently at
high cell density than at low cell density. We also
1998 John Wiley & Sons, Ltd.
Proliferation of cells at low density
When stationary cells were shifted into SPO at
high density, no cell division occurred: cells did not
form buds or increase in cell number (data not
shown). However, cells at low density initiated to
bud and increased in number about four-fold by
48 h, generating smaller cells upon each division
(Figure 2A). Although this abortive cell division
might be caused by a trace amount of nutrients
present in SPO or by nutritional deposits within
cells, it could result in the failure of cells to induce
IME1 expression or to sporulate at low cell density, because division arrest at G1 is a prerequisite

. 14: 617–622 (1998)
.   .
620
Figure 2. Proliferation of cells at low density in SPO, and
cytostatic activity of conditioned medium. (A) Phase-contrast
micrograph of budding cells cultured at low density (4104
cells/ml) in SPO for 30 h. Bar, 10 ìm. (B) Plate assay for
cytostatic activity present in conditioned medium. Haploid cells
(YIY1014) were spread as single cells on SPO (top) and
conditioned (bottom) plates, cultured for 3 days, and photographed. Cells spread on a sporulation plate (SPO diluted
two-fold with deionized water) formed the same size of microcolonies as shown on the SPO plate (data not shown). Bar,
50 ìm.
Figure 1. Expression of sporulation-induced genes at high
and low densities. (A) Diploid cells (YIY1018) containing lacZ
fusions to the indicated gene were cultured in SPO at high
(4106 cells/ml) and low (4104) cell densities. Samples were
taken periodically for determination of â-galactosidase specific
activity (Miller, 1972). (B) Diploid cells (YIY1018) were cultured in SPO and conditioned medium (prepared from the 5-h
supernate) at the same cell densities as above. Samples were
taken for RNA blot analysis (Kawaguchi et al., 1992; Yoshida
et al., 1990) of transcripts from IME1, IME2 and ACT1 (yeast
actin). ACT1 transcripts were shown as loading controls.
for IME1 induction (Kawaguchi et al., 1992;
Sherman et al., 1993).
Cytostatic and sporulation-stimulating activities
present in conditioned medium
The efficiency of sporulation of cells at
low density could be stimulated by inducing
sporulation in conditioned medium (Figure 3).
Conditioned medium was prepared by incubating
cells in SPO at high density, removing the cells by
centrifugation or filtration, and mixing equal
volumes of the supernate or the filtrate and
fresh SPO. The stimulation of sporulation by
1998 John Wiley & Sons, Ltd.
Figure 3. Conditioned medium contains sporulationstimulating activity. Diploid cells (YIY1018) were cultured at
high (2·6106 cells/ml) and low (2·6104) cell densities in
SPO and in the conditioned media which had been prepared
from the supernates obtained at the indicated times. After 2
days, sporulation was scored.
conditioned medium indicated that during incubation of cells at high density in SPO an extracellular factor required for efficient sporulation

. 14: 617–622 (1998)
 - 
had accumulated in the medium. During the incubation of cells at low density in conditioned media
(prepared from the supernates obtained after 3-h
incubation), bud formation was absent and cell
number was constant (data not shown), indicating
that conditioned medium had a cytostatic activity.
The cytostatic and sporulation-stimulating activities were not due to dilution of SPO medium,
because cells, incubated at low density in a sporulation medium (SPO diluted two-fold with deionized water), formed buds and proliferated but did
not sporulate (data not shown). The cytostatic
activity present in conditioned medium was verified by examining the growth inhibition of single
cells on plates (Figure 2B.). Conditioned medium
also stimulated expression of IME1 and IME2 in
cells at low density (Figure 1B).
Characterization of the extracellular factor
The factor, or factors, present in conditioned
medium is called meiosis-promoting factor (MEP).
We characterized MEP by treating the filtrate from
sporulating culture in a variety of ways and then
assaying for MEP activity, based on its cytostatic
activity (Table 2). The molecular weight of MEP
was roughly estimated by using dialysis tubes with
varying values of molecular weight cut off ranging
from 100 to 2000. Dialysis experiments were done
in two ways. First, the filtrate was inside the
dialysis tubes and was dialysed against fresh SPO,
which resulted in the loss of MEP activity. Second,
fresh medium was inside the dialysis tubes and was
dialysed against the filtrate, which resulted in the
recovery of MEP activity inside the tubes. These
results indicate that MEP is a small molecule with
a molecular weight of approximately 100 or less.
MEP activity was adsorbed on anion exchange
resins (QAE- and DEAE-Sephadexes) but not on a
cation exchange resin (CM-Sephadex), indicating
the anionic nature of MEP. We also examined for
stability of MEP against heat and pH. MEP
activity was heat-stable (boiling 10 min). It was
also stable at high pH (pH 11) but unstable at low
pH (pH 5).
In summary, our results confirm the previous
observations that nutritional limitation itself does
not induce sporulation and indicate that cells
secrete a small anionic molecule with cytostatic
and sporulation-promoting activities, which is
required for efficient meiosis and sporulation. Cells
at low density could not accumulate sufficient
amounts of this molecule, providing the basis for
1998 John Wiley & Sons, Ltd.
621
Table 2.
Characterization of MEP.
Treatmenta
MEP activity
Control
Dialysis against SPO: MWCO 100
MWCO 500
Dialysis against filtrate: MWCO 100
MWCO 500
QAE-Sephadex
DEAE-Sephadex
CM-Sephadex
Heat
pH 5
pH 11
8
4
0
4
8
2
4
8
8
2
8
a
The filtrate from sporulating cultures was prepared as described. Dialysis was done at 4C for 2 days using sterile dialysis
tubes (Spectrum) with approximate molecular weight cutoff
(MWCO) of 100, 500, 1000, and 2000. One milliliter of the
filtrate was inside the dialysis tube and was dialysed against 1
liter of SPO. On the other hand, one ml of SPO was inside the
tube and was dialysed against 150 ml of the filtrate. After
dialysis, the medium inside the tube was tested for MEP
activity. Similar results were obtained with dialysis tubes having
MWCO of 500, 1000 and 2000. Adsorption of MEP activity
onto anion (QAE- and DEAE-Sephadexes) and cation (CMSephadex) exchange resins was examined. One ml of the filtrate
was mixed with 0·2 ml of resins equilibrated with SPO, shaken
at 28C for 1 h, and centrifuged. The resulting supernate was
filter-sterilized and assayed for MEP activity. Heat stability was
examined by boiling the filtrate for 10 min. We also examined
for stability against pH. The filtrate (pH 8·3) was adjusted to
pH 5 and 11 by addition of HCl and KOH, respectively,
filter-sterilized, and incubated at 28C for 24 h. The solution
was neutralized back to pH 8·3, filter-sterilized, and assayed for
MEP activity.
the reduced efficiency of sporulation at low cell
density.
ACKNOWLEDGEMENTS
We thank A. Kiso for technical assistance. This
work was supported in part by Grants-in-Aid
for Scientific Research from the Ministry of
Education, Science, Sports, and Culture of Japan.
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coatomer, ufe1p, sec22p, thesaccharomyces, mutanttip20, mutant, early, cerevisiae, synthetic, snarf, lethal, protein, secretion, yeast
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