Molecular Biology of the Cell
Vol. 17, 3409–3422, August 2006
Multiple Signaling Pathways Regulate Yeast Cell Death
during the Response to Mating Pheromones
Nan-Nan Zhang,* Drew D. Dudgeon,* Saurabh Paliwal,†Andre Levchenko,†
Eric Grote,‡and Kyle W. Cunningham*
*Department of Biology and†Whitaker Institute for Biomedical Engineering, Johns Hopkins University,
Baltimore, MD 21218; and‡Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg
School of Public Health, Baltimore, MD 21205
Submitted March 6, 2006; Revised May 8, 2006; Accepted May 18, 2006
Monitoring Editor: Daniel Lew
Mating pheromones promote cellular differentiation and fusion of yeast cells with those of the opposite mating type. In
the absence of a suitable partner, high concentrations of mating pheromones induced rapid cell death in ?25% of the
population of clonal cultures independent of cell age. Rapid cell death required Fig1, a transmembrane protein homol-
ogous to PMP-22/EMP/MP20/Claudin proteins, but did not require its Ca2?influx activity. Rapid cell death also required
cell wall degradation, which was inhibited in some surviving cells by the activation of a negative feedback loop involving
the MAP kinase Slt2/Mpk1. Mutants lacking Slt2/Mpk1 or its upstream regulators also underwent a second slower wave
of cell death that was independent of Fig1 and dependent on much lower concentrations of pheromones. A third wave of
cell death that was independent of Fig1 and Slt2/Mpk1 was observed in mutants and conditions that eliminate calcineurin
signaling. All three waves of cell death appeared independent of the caspase-like protein Mca1 and lacked certain
“hallmarks” of apoptosis. Though all three waves of cell death were preceded by accumulation of reactive oxygen species,
mitochondrial respiration was only required for the slowest wave in calcineurin-deficient cells. These findings suggest
that yeast cells can die by necrosis-like mechanisms during the response to mating pheromones if essential response
pathways are lacking or if mating is attempted in the absence of a partner.
Programmed cell death (PCD) occurs in metazoans as a
means of eliminating unwanted cells during development
and removing damaged, weak, infected, or malignant cells
from the organism to avoid potentially harmful conse-
quences (Danial and Korsmeyer, 2004). PCD is highly coor-
dinated and regulated at multiple levels. Inputs from a
variety of sources can impact on a core set of enzymes that
coordinate destruction of key cellular components necessary
for cell survival. Apoptosis, one form of PCD, typically
requires activation of cysteine-aspartyl proteases (caspases)
by signaling factors derived from mitochondria or the
plasma membrane. Conservation of PCD factors among all
animals (Koonin and Aravind, 2002) is consistent with a
very early origin of the PCD mechanism, potentially even
before the divergence of animals and fungi.
The occurrence of PCD in fungi has received support from
numerous studies using the budding yeast Saccharomyces
cerevisiae (reviewed in Madeo et al., 2002, 2004; Longo et al.,
2005). Several so-called “hallmarks” of apoptosis can be
observed in populations of yeast cells that have been mor-
tally wounded by environmental stresses such as high heat,
high salt, hypertonic shock, DNA damaging agents, food
preservatives, and hydrogen peroxide. Starvation and aging
also seem to induce apoptosis-like cell death in a minority of
cells in the dying population (reviewed in Longo et al., 2005).
Expression of mammalian Bax in yeast also leads to mito-
chondrial dysfunction, accumulation of reactive oxygen spe-
cies (ROS), and cell death (reviewed in Priault et al., 2003).
To date, yeast homologues of mammalian caspase (Mca1),
cytochrome c (CYC; Cyc1 and Cyc7), and several other
factors have been implicated in one or more of these forms
of apoptosis-like cell death. The molecular interactions be-
tween all these factors and the sequence of their actions have
not been thoroughly investigated.
Recently, apoptosis-like cell death was reported in yeast
cells engaged in sexual conjugation or “mating” (Severin
and Hyman, 2002). Diploid yeast cells (a/?-cells) can prop-
agate by mitosis and in certain conditions will undergo
meiosis to produce haploid cells (a-cells and ?-cells), which
are capable of mitosis as well as mating. To initiate this
process, a- and ?-cells secrete mating pheromones (a-factor
and ?-factor) that bind serpentine receptors expressed on
the opposite cell type (reviewed in Sprague and Thorner,
1992; Dohlman and Thorner, 2001). Engaged receptors acti-
vate a heterotrimeric G-protein and mitogen-activated pro-
tein (MAP)-kinase cascade, which result in induction of
mating-specific genes, arrest in G1-phase of the cell division
cycle, cell wall remodeling, and extension of a mating pro-
jection from the cell body toward the pheromone source.
After cell–cell contact and agglutination, the intervening cell
wall continues to be degraded and remodeled to permit
membrane fusion, followed by subsequent mixing of cyto-
plasmic contents, fusion of haploid nuclei, and resumption
This article was published online ahead of print in MBC in Press
on May 31, 2006.
Address correspondence to: Kyle W. Cunningham (firstname.lastname@example.org).
Abbreviations used: CN, calcineurin; CWI, cell wall integrity; CYC,
cytochrome c; MPK, mitogen-activate protein kinase; PCD, pro-
grammed cell death; ROS, reactive oxygen species.
© 2006 by The American Society for Cell Biology 3409
of cell division. Even under optimal mating conditions,
many a- and ?-cells fail to find a partner or successfully
mate. Haploid cells that fail to mate eventually become
desensitized to the mating pheromones and resume vegeta-
tive growth. In mixed cultures of a- and ?-cells, ?6% of the
nonmating cells were found dead (Severin and Hyman,
2002). In the presence of high ?-factor and in the absence of
mating partners, ?30% of a-cells died, and these cells exhib-
ited several morphological hallmarks of apoptosis (Severin
and Hyman, 2002). Because cell death in these conditions
also required CYC and an undefined target of cyclosporin A,
the authors proposed that an apoptosis-like form of PCD
occurred in populations of mating yeast cells and speculated
that PCD could benefit the species by eliminating the
weakest individuals from the mating population and re-
ducing their ability to compete with healthier cells for
mates or other resources.
However, the high levels of cell death reported in the
recent study conflicted with earlier studies that failed to
detect more than ?1% cell death in populations of wild-type
yeast cells responding to mating pheromones (Iida et al.,
1990; Cyert et al., 1991; Cyert and Thorner, 1992; Foor et al.,
1992; Iida et al., 1994; Ono et al., 1994; Moser et al., 1996;
Fischer et al., 1997; Paidhungat and Garrett, 1997; Withee et
al., 1997; Muller et al., 2001). These earlier studies also re-
ported nearly complete cell death in yeast mutants that lack
components of a calcium signaling pathway, such as the
high-affinity Ca2?influx channel (Cch1, Mid1), calmodulin
(Cmd1), or calcineurin (CN; Cnb1, Cna1-Cna2). This broadly
conserved calcium signaling pathway was also found to
prevent cell death in response to a variety of natural and
synthetic fungistatic drugs (Del Poeta et al., 2000; Marchetti
et al., 2000; Bonilla et al., 2002; Cruz et al., 2002; Edlind et al.,
2002; Bonilla and Cunningham, 2003; Onyewu et al., 2003;
Sanglard et al., 2003; Kaur et al., 2004; Steinbach et al., 2004).
In contrast to the recent report (Severin and Hyman, 2002),
compounds that directly bind and inhibit CN strongly in-
creased the observed incidence of cell death.
Here we reexamine the roles of CN, CYC, and other
factors associated with cell death in yeast during the re-
sponse to mating pheromones in order to resolve the dis-
crepancies and to define the underlying regulatory mecha-
nisms. Instead of just one wave of cell death occurring in
response to mating pheromones, three distinct waves were
distinguishable by pharmacological, genetic, and kinetic cri-
teria. None of the three waves of cell death was related to
apoptosis-like cell death or was dependent on yeast meta-
caspase, but all were preceded by accumulation of ROS. The
fastest wave of cell death involved ROS accumulation from
nonmitochondrial sources and was dependent on the
plasma membrane protein Fig1, which we identify here as
the first fungal member of the PMP-22/EMP/MP20/Clau-
din superfamily of four-spanner transmembrane proteins.
Though Fig1 promotes Ca2?influx and elevation of cytoso-
lic free Ca2?concentrations in these conditions (Muller et al.,
2003), similar to the effects of Stargazin on ionotropic gluta-
mate receptors (Letts et al., 1998), Fig1 promoted cell death
independent of Ca2?. The other two waves of cell death
were slower and observed only in cells lacking CN or the
MAP kinase Slt2/Mpk1 (or their upstream regulators).
Rather than undergoing altruistic suicide, we suggest in-
stead that yeast cells die by necrosis-like processes relating
to either the inappropriate execution of mating steps in the
absence of a mating partner (Fig1-dependent fast death) or
the failure to perform essential functions that are necessary
for cell survival in mating conditions (slow deaths in CN-
and mitogen-activate protein kinase [MPK]-less mutants).
MATERIALS AND METHODS
Media, Reagents, Yeast Strains, Plasmids, and Growth
All yeast strains were cultured in rich YPD medium or synthetic SC medium
containing 2% glucose as described (Sherman et al., 1986). Synthetic ?-factor
(U.S. Biomedical, New York, NY), cyclosporin A (Sigma-Aldrich, St. Louis,
MO) and FK506 (Fujisawa Healthcare, Deerfield, IL) were dissolved in di-
methyl sulfoxide. Antimycin and oligomycin (both from Sigma-Aldrich) were
dissolved in ethanol. d-Glucosamine (Sigma-Aldrich) and potassium-BAPTA
(Invitrogen, Carlsbad, CA) were dissolved in water, and all the reagents de-
scribed above were added to the culture medium at indicated concentrations.
All yeast strains used in this study (Table 1) were derived from wild-type
W303-1A (MATa ade2-1 can1-100 his3-1 leu2-3112 trp1-1 ura3-1; Wallis et al.,
1989) or BY4741 (MATa his3-1 Leu2-2 met15-0 ura3-0) (Brachmann et al., 1998)
parent strains using standard molecular procedures and/or genetic crosses.
Knockout mutations for CYC, Cyc3, Cpr1, Cpr3, Mca1, Cox6, Fus1, Fus2,
Rvs161, Cnb1, Lrg1, Fig1, Nuc1, Bck1, and Atp2 were generated by homolo-
gous recombination of appropriate PCR products, selection for proper drug
resistance or amino acid markers, and PCR screening of genomic loci as
described (Sambrook et al., 1989). Replacement of SST1 with sst1::URA3 was
also made by homologous recombination using the 5.6-kb fragments of the
pJGsst1 plasmid after digestion with EcoRI and SalI (Elion et al., 1993).
Table 1. List of yeast strains used in this study
Cunningham and Fink (1996)
Cunningham and Fink (1996)
Mozdy et al. (2000)
Mozdy et al. (2000)
Mozdy et al. (2000)
Gin and Clarke (2005)
Evangelista et al. (1997)
Elion et al. (1993)
Cunningham and Fink (1996)
NZY104 This study
N.-N. Zhang et al.
Molecular Biology of the Cell3410
Cell Death Assays
Yeast strains were inoculated into YPD medium, serially diluted, and cul-
tured overnight at 30°C. Log-phase cultures were selected, diluted to a
concentration of 3 ? 106cells/ml (OD600? 0.1) using fresh YPD medium and
treated with ?-factor and other compounds as described in Results. At various
times of incubation at 30°C, 100 ?l of cells were harvested by centrifugation
(1 min, 13,000 rpm, room temperature [RT]), washed with 200 ?l of SC
medium, and resuspended in 10 ?l of fresh SC medium containing 200 ?g/ml
methylene blue (Sigma), spotted onto microscope slides, imaged using bright-
field microscopy, and scored as live (unstained) or dead (blue-stained) as
described previously (Muller et al., 2001). At least 200 cells were scored for
every strain at each time point. Similar results were obtained after 5 min
staining with 0.4 mg/ml phloxine B (Sigma Chemical) in YPD medium
(unpublished data; Severin and Hyman, 2002), or after 10 min staining with
1 ?g/ml propidium iodide (PI; Sigma Chemical) in YPD medium and imag-
ing with epifluorescence illumination (Nikon Diaphot inverted microscope,
Melville, NY; 540-nm excitation and 620-nm emission).
High-throughput time-lapse imaging of individual cells was done in a mi-
crofluidic chip that allowed us to maintain chemostatic conditions for long
time periods (Groisman et al., 2005). The microfluidic device was made of a
silicon elastomer polydimethylsiloxane (PDMS; RTV 615 by General Electric,
Cleveland, OH) chip sealed to a no. 1.5 microscope coverglass, with inlets for
introduction of media and cells into the channels and test chambers of the
chip. Cells were grown in YPD medium to midlog phase, loaded into test
chambers of the chip, and grown at RT with continuous perfusion of fresh
culture medium. After a 4-h incubation, the cells were perfused with YPD
medium containing 2 ?M ?-factor and 1 ?g/ml PI and incubated an addi-
tional 8 h. Multiple fields of cells were imaged using phase-contrast micros-
copy (10-min intervals) and epifluorescence microscopy (30-min intervals)
using a Nikon Eclipse TE2000-U inverted microscope equipped with an
electronically controlled shutter and stage (X, Y, and Z directions), a 40?/0.75
objective, and a filter cube appropriate for detection of PI (540/25-nm exci-
tation and 605/55-nm emission filters with a 505-nm dichroic mirror). Time-
lapse movies of each field were generated from the stacked images and
ROS Accumulation Assays
Cells producing ROS were visualized and counted as described previously
(Madeo et al., 1999), except with slight modifications. Briefly, log-phase cul-
tures were diluted to a concentration of 3 ? 106cells/ml and exposed to
?-factor in YPD medium at 30°C. At each time point, 100 ?l of cells was
harvested by centrifugation, resuspended in 100 ?l of fresh SC medium, and
incubated with 10 ?g/ml 2?,7?-dichlorodihydrofluorescein diacetate (H2DCF-
DA, Molecular Probes, Eugene, OR) at 30°C for 10 min. Cells were concen-
trated by centrifugation and resuspended in 10 ?l of fresh SC medium. 5?L
of cells were loaded onto slides and observed immediately under epifluores-
cence microscopy (495-nm excitation and 525-nm emission). At least 200 cells
per sample were scored manually as fluorescent or nonfluorescent.
Log-phase cultures were diluted to 3 ? 106cells/ml in YPD medium and
treated with ?-factor. About 0.25 OD cells were harvested after incubation for
3, 6, and 9 h at 30°C. Cells were resuspended in 0.5 ml phosphate-buffered
saline (PBS) and fixed for 1 h at RT with 3.7% formaldehyde (J. T. Baker). After
the fixation, cells were washed with 0.5 ml PBS and resuspended in 1 ml SPM
(1.2 M sorbitol, 50 mM K-phosphate, pH 7.3, 1 mM MgCl2). After digestion
with a combination of 300 ?g/ml Zymolyase 100T (Seikagaku, Tokyo, Japan)
and 12 ?g/ml lyticase (Sigma) in SPM for 40 min at 30°C, cells were gently
harvested (5000 rpm, 1 min), washed with SPM, and resuspended in 100 ?l
SPM. Suspended cells (40 ?L) were transferred into polylysine-coated wells of
a microscope slide and allowed to settle for 20 min at RT. The slide was
washed three times by SPM to remove the enzymes. Each well was incubated
with 40 ?l fresh permeabilization solution (0.1% Triton X-100 in a 0.1%
sodium citrate solution) for 2 min at 4°C, rinsed with PBS, and incubated with
10 ?l of 5 ?g/ml DNase-free RNase (Boehringer Mannheim) for 30 min at
37°C. This last step was found to remove background staining in the cyto-
plasm that obscured nuclear staining. Each well was then rinsed three times
with PBS and incubated with 10 ?l TUNEL reaction mixture (In Situ Cell
Death Detection Kit, POD, Roche, Indianapolis, IN) for 60 min at 37°C in a
humidified container (Madeo et al., 1999). Slides were then rinsed three times
with PBS and mounted under coverslips with Prolong Gold antifade solution
(Molecular Probes). At least 200 cells per sample were scored as containing or
lacking fluorescent nuclei using a Zeiss Axioplan/Coolsnap epi-fluorescence
microscope (Thornwood, NY) with FITC channel. Though no fluorescent
nuclei were observed in cells treated with ?-factor at any time point, more
than 90% of nuclei in H2O2-treated cells and nearly 100% of nuclei in cells
exposed to DNAse I (Sigma, 0.1 ?g/?l for 10 min, RT) stained TUNEL
Aequorin Luminescence Measurements
Yeast strains were transformed with plasmid pKC147 (2 ? URA3 PMA-
aequorin; Muller et al., 2001) or pEVP11/AEQ89 (2 ? LEU2 ADH-aequorin;
Batiza et al., 1996), and independent transformants were grown to log phase
in SC medium lacking uracil or leucine. Cells were harvested by centrifuga-
tion, resuspended in fresh medium, and loaded with 25 ?g/ml coelenterazine
(Molecular Probes) for 20 min at RT. Loaded cells were washed with fresh
YPD medium and raised in YPD to OD600 ? 0.25. After recovering at 30°C
roller for 80 min, 600 ?l from each sample was transferred to an luminometer
tube, loaded with ?-factor, and monitored for luminescence in a LB9507
luminometer (EG&G Wallac, Turku, Finland). Aequorin expression and load-
ing in each strain was similar as judged by measuring total luminescence
units after cell lysis using digitonin (Sigma).
Data from several experiments were fit to a standard sigmoid equation (Y ?
min ? (max ? min)/(1 ? (mid/X) slope), where min, max, mid, and slope
are variables corresponding to the minimum value of Y, maximum value of Y,
value of X corresponding to the midpoint of Y, and slope of line as it crosses
the midpoint) using nonlinear regression. Values for min and max were
constrained to ?0 and ?100%, respectively. Additionally, some data were fit
to the sum of or difference between two sigmoid equations.
Two Waves of Cell Death Are Differentially Regulated by
CN and Concentration of ?-factor
The many discrepancies reported in studies of yeast cell
death during the response to mating pheromones might be
attributed to the 10-fold higher concentrations of ?-factor
used in the recent study (Severin and Hyman, 2002). To test
this idea, the death of yeast cells containing or lacking CN
was measured after a 10-h exposure to a wide range of
?-factor concentrations (Figure 1A). Strains lacking the se-
creted protease Sst1 were used in this experiment to avoid
extracellular degradation of the added ?-factor (Sprague
and Herskowitz, 1981; Chan and Otte, 1982). At saturating
concentrations of ?-factor, up to ?27% of the sst1 mutant
cells (●) and over 80% of the sst1 cnb1 double mutant (?)
cells died within the allotted window of time. The concen-
trations of ?-factor resulting in 50% maximal death of sst1
mutants and sst1 cnb1 double mutants were estimated at 62
and 10 nM, respectively, by nonlinear regression of the
dose–response data to standard sigmoid equations (see Ma-
terials and Methods). This sixfold difference in sensitivity to
?-factor was also observed in wild-type and cnb1 mutants
expressing the Sst1 protease, though the 50% lethal doses
were ?200-fold higher than the Sst1-deficient strains (un-
published data). However, CN-deficient mutants were not
more sensitive to ?-factor in a variety of other measurable
responses (Moser et al., 1996; Withee et al., 1997). Therefore,
CN completely prevented death at moderate concentrations
of ?-factor but not at high concentrations.
The deaths of wild-type and CN-deficient cells were also
analyzed at various times during the response to excess (60
?M) ?-factor. Approximately 25% of wild-type cells died
very rapidly (median lifespan of ?1.5 h), as determined by
nonlinear regression of the data to a standard sigmoid equa-
tion (Figure 1B). The cnb1 mutants exhibited two waves of
cell death that were described very well by the sum of two
sigmoid equations (Figure 1C). In this experiment, ?17% of
the population died in the first wave (median lifespan of
1.5 h) and the remainder died in the second wave (median
lifespan of 9.7 h). The faster wave of cell death in cnb1
mutants was decreased to undetectable levels when lower
concentrations of ?-factor were used (6 ?M; Moser et al.,
1996; Withee et al., 1997) or when certain mating genes were
eliminated (see below). Because cnb1 mutants do not die in
the absence of mating pheromones, these findings suggest
Mating and Cell Death in Yeast
Vol. 17, August 2006 3411
that low concentrations of ?-factor stimulate a slow CN-
sensitive cell death and that high ?-factor concentrations
stimulate a fast cell death in a fraction of the population that
is largely insensitive to the presence or absence of CN.
The recent study of fast cell death at high ?-factor con-
centrations demonstrated a dependence on CYC and a sen-
sitivity to cyclosporin A (Severin and Hyman, 2002) even
though cyclosporin A is known to inhibit CN. However, we
find that fast cell death did not depend on CYC and was
only slightly inhibited by cyclosporin A or the loss of CN
(Figure 1B). Cyclosporin A also stimulated a slow wave of
cell death in wild-type populations (Figure 1B) while having
no detectable effect on either the rate or extent of cell death
in cnb1 mutant populations (Figure 1C). All these effects of
cyclosporin A were mimicked by the loss of CN and by the
addition of FK506, a chemically distinct inhibitor of CN
(unpublished data), indicating that these compounds mod-
ulate cell death solely by their activity against CN. Though
CYC had no detectable effect on the fast wave of cell death
in either wild-type or CN-deficient cells, the loss of CYC
dramatically decreased the slow wave of cell death in cnb1
mutants (Figure 1C). The best-fit sigmoid equations indicate
that the loss of CYC decreases the extent of slow death by
about fourfold (from 81 to 18% of the population). Thus,
CYC specifically increased slow CN-sensitive cell death
without affecting the faster CN-insensitive type of cell death.
Collectively, these findings suggest that at least two waves
of cell death can occur during response to mating phero-
mones. The two waves differ markedly in their sensitivity to
?-factor, kinetics, penetrance in the population, sensitivity to
CN, and dependence on CYC.
Fast and Slow Waves of Cell Death are Morphologically
and Mechanistically Distinct from Apoptosis-like Cell
ROS accumulation and chromatin fragmentation, consid-
ered hallmarks of apoptosis in yeast (Madeo et al., 1999;
Frohlich and Madeo, 2001), have been associated with death
of yeast cells responding to ?-factor (Severin and Hyman,
2002). To determine whether ROS accumulation precedes
fast, slow, or both waves of cell death, CN- and/or CYC-
deficient cells were treated with high ?-factor and periodi-
cally stained for ROS accumulation using the fluorogenic
probe H2DCFDA. The frequency of ROS-positive cells in
wild-type populations peaked at ?11% during the response
to high ?-factor and then declined to very low levels (Figure
2A, ●). The CYC-deficient cyc1 cyc7 double mutants be-
haved similarly (?). Time-lapse video microscopy of wild-
type cells responding to high ?-factor demonstrated that all
of the fast cell deaths were preceded by ROS accumulation
and that the surviving cells fail to accumulate detectable
ROS (unpublished data). Therefore, fast cell death was
closely associated with accumulation of ROS from a CYC-
Slow cell death of CN-deficient mutants was also associ-
ated with ROS accumulation. The frequency of ROS-positive
cells in the population of cnb1 mutants peaked at ?20% of
the population ?3 h before the midpoint of the slow wave of
cell death (Figure 2A, f). The loss of CYC in CN-deficient
cells abolished the second wave of ROS accumulation but
had no effect on the first wave (?). These findings indicate
that CYC stimulates and CN inhibits a second wave of ROS
accumulation that precedes and contributes to CN-less
CYC may promote ROS accumulation and CN-less death
by several distinct mechanisms, such as stimulation of apop-
tosis-like processes or stimulation of mitochondrial respiration.
To discriminate between these possibilities, we measured cell
death in a panel of respiration- and apoptosis-deficient mu-
independently regulated. (A) Cell death was measured in populations
of sst1 mutants and cnb1 sst1 double mutants lacking CN activity using
methylene blue staining after 10 h of treatment with the indicated
concentrations of ?-factor. Dose–response curves for wild-type and
cnb1 mutant populations were shifted to the right by ?200-fold (un-
published data). (B and C) Populations of wild-type cells (WT) or
CN-deficient cells (cnb1) lacking cytochrome c (?CYC) or experiencing
50 ?M cyclosporin A (?CsA) were treated with 60 ?M ?-factor and
monitored for cell death at the indicated times. Averages of three
replicate experiments (?SD) are shown. Monophasic and biphasic
patterns were evident in the data, of which the maxima, minima, and
midpoints were estimated by nonlinear regression using the sum of
one or two standard sigmoid equations. A fast wave of cell death was
observed in ?18–27% of all populations, and a slow wave of cell death
was observed only in CN-deficient populations. The fast wave of cell
death required sixfold higher concentrations of ?-factor than CN-less
death. Cytochrome c was important for the slow wave but not the fast
Two waves of cell death during the response to ?-factor are
N.-N. Zhang et al.
Molecular Biology of the Cell 3412
tants after 10-h treatment with high ?-factor or high ?-factor
plus FK506 to mimic the CN-deficient state (Figure 2B). Apop-
tosis-deficient mutants lacking metacaspase (mca1), mitochon-
drial cyclophilin D (cpr3), or mitochondrial fission factors
(dnm1, fis1, mdv1) all exhibited wild-type levels of fast and slow
cell death. Respiration-deficient mutants lacking coenzyme Q
(coq1), CYC-heme lyase (cyc3), CYC-oxidase (cox6), and ATP
synthase (atp2) behaved exactly like CYC-deficient mutants,
cell death. Because respiration-deficient mutants have adapted
to the loss of respiratory function, we also examined the effects
of complex III inhibitors (antimycin) and complex V inhibitors
(oligomycin) on wild-type cells. When added at the same time
as ?-factor, all of these respiration inhibitors strongly dimin-
ished slow death of CN-deficient cells but had no effect on fast
death of wild-type cells (Figure 2B). Antimycin also abolished
the second wave of ROS accumulation in cnb1 mutants re-
sponding to ?-factor (unpublished data). Thus, functional mi-
tochondrial respiratory complexes III, IV, and V were as im-
portant as CYC and coenzyme-Q in promoting slow death of
CN-deficient cells. The data suggest that respiring mitochon-
dria directly or indirectly generate ROS in CN-deficient cells
and that ROS play a major role in their death.
To determine if chromatin fragmentation is associated
with fast, slow, or both waves of cell death, standard
TUNEL assays were performed on wild-type and cnb1 mu-
tant cells at several times during the response to high ?-fac-
tor. After 3 h of response, nearly 100% of cells in both
was measured in populations of wild-type (WT) and CN-deficient (cnb1 mutant) cells that contain or lack cytochrome c (?CYC) at various
times after treatment with 60 ?M ?-factor. (B) Mutants lacking either cyclophilin D (cpr3), metacaspase (mca1), mitochondrial fission factors
(fis1, dnm1, mdv1), cytochrome c (cyc1 cyc7), cytochrome-c heme lyase (cyc3), coenzyme-Q (coq1), complex-IV (cox6), complex-V (atp2), cell wall
degradation factors (fus1, fus2, rvs161), low-affinity Ca2?influx (fig1), membrane fusion factor (prm1), Rho-GAP of the CWI signaling pathway
(lrg1), or factors involved in pheromone signaling (fus3, far1, bni1) were treated with 60 ?M ?-factor for 10 h in the presence or absence of
CN inhibitor (2.5 ?M FK506) and assayed for cell death. Wild-type cells treated with inhibitors of complex-III (1 ?g/ml antimycin A; AM)
or complex-V (1 ?g/ml oligomycin; OM) were also analyzed. Fast cell death was estimated as the frequency of cell death occurring in the
absence of FK506. Slow cell death was estimated as the net increase of cell death occurring in the presence of FK506. (C) Chromatin
fragmentation was monitored in wild-type cells after 3-h treatment with 60 ?M ?-factor or 1 mM H2O2as indicated. Similar results were
obtained using CN-deficient cnb1 mutants and longer periods of treatment. (D) Time-lapse video microscopy was used to monitor the fate
of mother cells and buds of sst1 mutant (WT) and sst1 fig1 double mutant (fig1) populations after treatment with 2 ?M ?-factor at RT. Dead
cells were identified using PI instead of methylene blue in other experiments and the results for fig1 mothers and buds were combined. Data
were fit to standard Sigmoid equations.
Fast and slow waves of cell death are associated with ROS accumulation but not chromatin fragmentation. (A) ROS accumulation
Mating and Cell Death in Yeast
Vol. 17, August 2006 3413
populations stained TUNEL-positive (unpublished data).
However, TUNEL fluorescence was observed in the cyto-
plasm of these cells and not restricted to the nuclear DNA.
The cytoplasmic fluorescence was completely abolished by a
brief treatment of the fixed and permeabilized cells with
RNase before the TUNEL reaction, indicating the standard
TUNEL assay was subject to high background of RNA.
Using a modified TUNEL assay that included RNase, no
TUNEL-positive nuclei were detected in wild-type or cnb1
mutant populations at any time after treatment with ?-factor
(Figure 2C). As a control for sensitivity of the modified
TUNEL method, we confirmed that the vast majority of
wild-type and cnb1 mutant cells stain positive for chromatin
fragmentation after treatment with H2O2. Thus, neither fast
nor slow waves of cell death were associated with chromatin
fragmentation or stimulated by factors involved in apopto-
sis-like cell death.
Random Occurrence of Fast Cell Death in Mother Cells
and Newly Budded Daughter Cells
wild-type cells in our experimental conditions. Log-phase cul-
tures of yeast are expected to arrest in response to ?-factor with
a population structure of ?50% daughter cells, ?25% mother
cells, and ?25% grandmother cells that have produced two or
more daughters. Previous studies have shown that replica-
tively aged grandmother cells die more quickly than younger
cells (Herker et al., 2004). To test if one of these subpopulations
is more susceptible to fast cell death, the fates of 576 sst1
mutant cells responding to ?-factor were followed by time-
lapse videomicroscopy. Asynchronous log-phase cells were
placed in a microfluidic chamber with flowing YPD medium,
incubated at RT, and photographed every 10 min using phase-
contrast microscopy in order to track cell lineages. After 3 h of
incubation at RT, the medium was replaced with YPD medium
containing excess ?-factor and 1 ?M PI. Fields of cells were
photographed as before and also photographed every 30 min
using epifluorescence microscopy to detect dead (PI-positive)
cells. The resulting time-lapse movies allowed tracking of 96%
of cells in the population (44% mother and grandmother cells,
44% immature daughters, and 8% mature daughters, which
remained attached to budded mother cells). As shown in Fig-
ure 2D, immature daughters and their mother cells were ob-
served to die at slightly different rates (median lifespan at 3.5
and 4.5 h, respectively), but after 8 h the levels of cell death in
these two subpopulations was indistinguishable (20.6 and
19.1%, respectively). These frequencies of cell death were not
significantly different from each other or the overall population
(19%) but were significantly higher than those of fig1 mutants
(5% cell death in the overall population). The lifespan of cells
undergoing fast cell death was longer in this experiment, prob-
ably because of decreased incubation temperature. Impor-
tantly, the observed frequency of dead mother–daughter pairs
(3.6%) was not significantly different from the frequency ex-
pected if cell death in these two populations occurred at ran-
dom (0.191 ? 0.206 ? 3.9%). Thus, the rate and extent of fast
cell death was not correlated with any particular cell age or
lineage and such deaths appeared to occur randomly in the
population. As an alternative explanation, the low and random
incidence of fast cell death in wild-type populations may result
from stochastic variations in pro– and anti–death regulatory
Factors That Promote Fast Cell Death
To identify factors that specifically regulate fast but not slow
cell death, we first tested many factors known to participate
in the primary response to mating pheromones (receptors,
heterotrimeric G-proteins, and MAP-kinase cascades) and in
secondary responses such as cell cycle arrest and polarized
morphogenesis. All “sterile” mutants that fail to mount the
full primary response to ?-factor were completely deficient
in both fast and slow cell death (unpublished data). Mutants
lacking the ability to arrest in G1-phase of the cell cycle (fus3
and far1 mutants) or to undergo polarized morphogenesis
(bni1, pea2, and spa2 mutants) were also profoundly deficient
in both fast and slow cell death (Figure 2B and unpublished
data). Several factors involved in cell–cell fusion and low-
affinity Ca2?influx appear to function downstream of these
secondary factors (Brizzio et al., 1998; Muller et al., 2003;
Fitch et al., 2004). Remarkably, mutants lacking the down-
wall polymers. (A) Cell death was measured in wild-type, fig1
mutant, and cnb1 fig1 double mutant populations after 8.5-h treat-
ment with 60 ?M ?-factor in the presence (gray bars) or absence
(black bars) of 15 mM glucosamine. (B) Cell death was measured in
single, double, and triple mutants at various times after exposure to
60 ?M ?-factor. The averages of three replicate experiments were
plotted and fit to standard Sigmoid equations as described in Figure
1. (C) Contact-dependent lysis of wild-type, prm1 mutant, fig1 mu-
tant, and fig1 prm1 double mutant prezygotes was measured as
described previously (Jin et al., 2004).
Fast but not slow cell death requires dissolution of cell
N.-N. Zhang et al.
Molecular Biology of the Cell 3414
stream factors Fus1, Fus2, or Rvs161 were deficient in fast
cell death but were fully capable of slow cell death (Figure
2B) even at saturating ?-factor when Sst1 protease was also
eliminated (unpublished data). Thus, fast and slow waves of
cell death were genetically distinguishable on the basis of
their requirements for cell fusion factors and mitochondrial
Fus1, Fus2, and Rvs161 are localized to the tip of mating
projections and are thought to promote mating by facilitat-
ing the focal secretion of cell wall hydrolases, which remove
barriers to the membrane by locally degrading the chitin and
glucan components of the cell wall (Brizzio et al., 1998). To
test if cell wall degradation is necessary for fast cell death,
chitin biosynthesis was induced about fivefold during the
response to ?-factor by the addition of its biosynthetic pre-
cursor glucosamine (15 mM) to the culture medium (Bulik et
al., 2003). Glucosamine strongly suppressed fast death of
wild-type cells but did not suppress slow death of cnb1
mutants (Figure 3A). Therefore, cell wall degradation ap-
peared to be necessary for fast cell death.
The four-spanner transmembrane protein Fig1 functions
downstream of Fus1, Fus2, and Rvs161 as part of a low-
affinity Ca2?influx system (Erdman et al., 1998; Muller et al.,
2003). Interestingly, fig1 mutants also exhibited little or no
fast cell death whereas cnb1 fig1 double mutants exhibited
high levels of slow cell death (Figures 2B and 3B). In con-
trast, a five-spanner transmembrane protein Prm1 that pro-
motes fusion of cell membranes during mating after cell wall
removal (Heiman and Walter, 2000) had no effect on fast or
slow cell death (Figure 2B). Recently, ?10% of prm1 mutant
cells engaged in conjugation but arrested as late prezygotes
were observed to die by a process termed contact-dependent
lysis (Jin et al., 2004). Contact-dependent lysis was not de-
pendent on Fig1 (Figure 3C) and was insensitive to antimy-
(A) Fig1 orthologues from yeast and 24 other fungi were identified by BLAST searches of genome databases and aligned using CLUSTALW
algorithm. Sites of high, moderate, and poor sequence conservation are shaded black, dark gray, and light gray, respectively. White spaces
indicate gaps. The positions of four predicted transmembrane segments and a conserved G??GXC(n)C motif are indicated. (B) Cell death
was measured in sst1 mutant and sst1 fig1 double mutant populations after 3-h treatment with 60 ?M ?-factor in the presence of varying
amounts of BAPTA, a chelator of divalent metals such as Ca2?. Data were fit to standard sigmoid equations. (C) Cell death was measured
in wild-type, pmc1 vcx1 double mutant, fig1 mutant, and fig1 pmc1 vcx1 triple mutant populations after 3.5-h treatment with 60 ?M ?-factor.
(D and E) Luminescence of cytoplasmic aequorin was measured in the same strains at various times after treatment with 60 ?M ?-factor.
Fig1 is a member of the stargazin/VGCC-?/claudin superfamily but promotes fast cell death independent of [Ca2?]c elevation.
Mating and Cell Death in Yeast
Vol. 17, August 20063415
cin (unpublished data), suggesting regulation by an un-
known mechanism. Fast cell death specifically required
factors involved in cell wall degradation (Fus1, Fus2, and
Rvs161) and additionally required a function of Fig1.
Fig1 Is a Member of the PMP-22/EMP/MP20/Claudin
Superfamily and Promotes Cell Death Independent of Ca2?
BLAST searches of protein and DNA databanks revealed
orthologues of Fig1 in other species of fungi. A multiple
sequence alignment of the best homolog from 30 diverse
fungal species revealed conservation of size and transmem-
brane topology but only one strongly conserved motif (de-
noted G??GXC(n)C, where ? ? YFLM and 8 ? n ? 20)
located in the extracellular loop between the first and second
predicted transmembrane spans (Figure 4A). To identify
more distantly related proteins, the yeast Fig1 sequence was
used to seed a PSI-BLAST search of all eukaryotic proteins
(Altschul et al., 1997). Starting with the 11th iteration, meta-
zoan proteins belonging to PMP-22/EMP/MP20/Claudin
superfamily of proteins (pfam00822) were recovered with
significant E-values. Nearly all members of this diverse su-
perfamily possess four predicted transmembrane segments
of similar spacing and a highly conserved GLWXXC(n)C
motif in the same location as that of the Fig1 family. More
than 40 members of this superfamily have been described in
humans, most of which are thought to interact with them-
selves and/or other transmembrane proteins in the same or
adjacent membranes. Eight members of the superfamily in
humans are thought to bind and regulate functions of either
voltage-gated Ca2?channels (e.g., VGCC-? subunit) or glu-
tamate-sensitive ion channels (e.g., stargazin; Burgess et al.,
2001). In most neuronal cell types, excessive Ca2?influx via
either pathway can trigger excitotoxic cell death or necrosis
(Arundine and Tymianski, 2004).
Fig1 promotes Ca2?influx and elevation of cytosolic free
Ca2?concentrations ([Ca2?]c) during the response to ?-factor
by activating a low-affinity system that is independent of the
high-affinity Ca2?influx system homologous to VGCC’s (Mul-
ler et al., 2003). To test if Ca2?influx and/or [Ca2?]c elevation
are required for fast cell death, a cell-impermeant chelator with
high affinity for Ca2?(BAPTA) was added to the culture me-
dium bathing cells responding to high ?-factor, and cell death
was measured. No concentration of BAPTA was able to dimin-
ish death of wild-type cells relative to that of fig1 mutants
(Figure 4B), indicating that Fig1-dependent fast cell death was
not dependent on Ca2?influx. Instead, BAPTA concentrations
greater than 0.3 mM strongly stimulated the death of wild-type
cells and fig1 mutants. This effect can be explained by the
ability of BAPTA to prevent activation of calmodulin, which is
also necessary for activation of Ca2?/calmodulin-dependent
mutant, and bck1 fus2 double mutant populations after 3-h treatment with 60 ?M ?-factor. (B) Dead cells were measured in wild-type, bck1
mutant, fig1 mutant, and bck1 fig1 double mutant populations at various times after treatment with 60 ?M ?-factor. The average of three
independent measurements were plotted (?SD) and fit to the sum of one or two standard sigmoid equations. (C) Percentages of ROS positive
cells were measured over time in three replicate cultures of wild-type, bck1 mutant, fig1 mutant, and bck1 fig1 double mutant strains after
treatment with 60 ?M ?-factor and plotted (mean ? SD). (D) Cell death was measured in sst1 mutant, sst1 bck1 double mutant, sst1 fig1 double
mutant, and sst1 bck1 fig1 triple mutant populations after 3-h treatment with various concentrations of ?-factor. Data were fit to the sum of
one or two standard sigmoid equations.
CWI signaling inhibits fast cell death and an intermediate cell death. (A) Cell death was measured in wild-type, fus2 mutant, bck1
N.-N. Zhang et al.
Molecular Biology of the Cell 3416
protein kinases that promote cell survival in these conditions
independent of CN (Moser et al., 1996; Withee et al., 1997).
Therefore, Fig1 appeared to promote fast cell death indepen-
To test if forced [Ca2?]c elevation can restore the death of
fig1mutants, we measured cell death in mutants lacking the
vacuolar Ca2?pump Pmc1 and the vacuolar Ca2?/H?ex-
changer Vcx1, enzymes that redundantly serve to lower
[Ca2?]c in yeast (Cunningham and Fink, 1994, 1996). The
loss of Pmc1 and Vcx1 in fig1 mutants restored [Ca2?]c to
near wild-type levels and augmented [Ca2?]c in cells ex-
pressing Fig1 to even higher levels (Figure 4, D and E). The
loss of Pmc1 and Vcx1 slightly increased the death of fig1
mutants to much lower levels than those of wild-type cells
and slightly decreased the death of Fig1–proficient cells
(Figure 4C). These findings reveal no important role for
Ca2?in fast cell death but do not rule out the possibility that
Fig1 promotes influx of other toxic ions.
Factors That Inhibit Fast Cell Death
During the normal response to ?-factor, cell wall degrada-
tion activates the cell wall integrity (CWI) signaling pathway
involving the rho-type GTPase Rho1 and Pkc1, Bck1, Mkk1,
Mkk2 cascade of protein kinases that culminate with activa-
tion of the MAP kinase Mpk1/Slt2 (Buehrer and Errede,
1997). The activated CWI signaling pathway stimulates
chitin and glucan synthases, which synthesize new cell wall
polymers and compensate for the cell wall degradation
(Levin, 2005). To test the prediction that CWI signaling can
inhibit fast cell death, we first measured cell death in lrg1
mutants where CWI signaling may be hyperactivated be-
cause of the loss of Lrg1, a GTPase-activating protein for
Rho1 (Fitch et al., 2004). Indeed, similar to fus1, fus2, and
rvs161 mutants, the lrg1 mutants exhibited greatly dimin-
ished levels of fast cell death (Figure 2B).
Cells deficient in CWI signaling are known to exhibit very
high levels of cell death in response to mating pheromones
(Errede et al., 1995), but the kinetics, sensitivity to ?-factor,
and dependence on cellular factors have not been examined.
If this enhanced cell death represents enhanced fast cell
death, the death of CWI-deficient mutants should be depen-
dent on Fig1, Fus1, Fus2, and Rvs161. Remarkably, the high
level of cell death observed for bck1 mutants (lacking the
MAPKKK in the CWI pathway) was partially blocked by
glucosamine (unpublished data) and completely abolished
by the loss of Fus2 (Figure 5A). However, the death of bck1
mutants was not blocked by the loss of Fig1 (Figure 5B).
Careful inspection of the data indicated two waves of cell
death in bck1 mutants and only one wave of cell death in
bck1 fig1 double mutants. The monophasic death curve of
bck1 fig1 double mutants was described very well by a
sigmoid equation where all cells died at an intermediate rate
(median lifespan of ?4.5 h). The death of bck1 mutants was
best described by the sum of two sigmoid equations where
40% of the population died rapidly (median lifespan of 1.4 h)
and the remainder of the population died at or near the
intermediate rate (median lifespan of ?3.6 h). Two peaks of
ROS accumulation corresponding to the two waves of cell
death were observed in bck1 mutants (Figure 5C). The loss of
Fig1 abolished the first peak of ROS and the gain of Bck1
abolished the second peak (Figure 5C). These findings are
consistent with two waves of cell death occurring in CWI-
deficient cells, a faster wave that is dependent on Fig1 and a
slower one that is independent of Fig1 and dependent on
cell wall degradation. The slower wave in bck1 mutants and
bck1 fig1 double mutants was triggered by about sevenfold
lower concentrations of ?-factor than the faster wave in
wild-type cells (Figure 5D) and was mechanistically distinct
from that of cnb1 and cnb1 fig1 mutants in that it could not
inhibited by antimycin A (unpublished data). The findings
that Fig1-dependent fast cell death increased about threefold
in bck1 mutants and decreased more than fivefold in lrg1
mutants relative to wild-type cells (?13% in this strain back-
ground) indicates that this process may be negatively regu-
lated by CWI signaling.
The findings described here support a working model where
at least three genetically independent regulatory pathways
contribute to the death of yeast cells responding to mating
pheromones (see Figure 6). The CN and CWI signaling
pathways become activated during the response to low and
high mating pheromones and independently perform func-
tions essential for long-term cell survival. CN- and CWI-
deficient cells appear to die by two distinct processes be-
cause only the former can be suppressed by blockade of
mitochondrial respiration and only the latter can be sup-
pressed by glucosamine-enhanced cell wall biosynthesis.
Even in wild-type cells that are protected by the CN and
CWI pathways, Fig1 promotes a third, fast wave of death in
response to high concentrations of mating pheromones. It is
possible that all three forms of death involve common path-
ways. However, the different kinetics and genetic require-
death in response to mating pheromones. Low concentrations of ?-fac-
tor (?F) activate regulatory pathways leading to cell wall degradation
(CW deg.) and stimulation of the high-affinity Ca2?influx system
(Cch1, Mid1) but not the Fig1-dependent low-affinity Ca2?influx sys-
tem. The resulting rise in [Ca2?]c activates CN, which prevents ROS
accumulation by either inhibiting ROS production by the mitochon-
drial respiratory chain or stimulating ROS dissipation. The diminished
cell wall strength may lead to increased ROS accumulation and inter-
mediate lifespan of cells were it not for the activation of the CWI
signaling pathway, which increases biosynthesis of new cell wall ma-
terial by increasing the activities of chitin and glucan synthases (CHS
and GLS). Because CN and CWI signaling pathways operate in wild-
type cells, low concentrations of ?-factor fail to induce significant
amounts of cell death. High concentrations of ?-factor activate the
above processes but also induce Fig1-dependent Ca2?influx, early
ROS accumulation, and rapid cell death in a fraction of the population.
Glucosamine (GlcN) supplement and deficiencies of Fus1, Fus2, or
Rvs161 block all these responses and additionally prevent activation of
CWI signaling. Fig1 deficiency does not affect cell wall degradation or
Working model of regulatory mechanisms controlling cell
Mating and Cell Death in Yeast
Vol. 17, August 2006 3417
ments we documented are most easily accounted for by
distinct prodeath or antisurvival mechanisms playing a role.
Because it is not yet known if any of these pathways regulate
cell death in normal development or physiological matings
in the wild, we cannot conclude that any of them represent
physiological mechanisms of PCD. Nevertheless, endoge-
nous mating pheromones acting through their G-protein–
coupled receptors are frequently considered physiological
stimuli in yeasts. For the sake of clarity, we will first discuss
independently each of the three regulatory pathways and
their possible evolutionary relationships to cell death mech-
anisms operating in mammalian cells and later consider the
possibility that all three pathways might be interrelated.
Death of CN-Deficient Mutants (“CN-less death”)
CN-less death was first discovered after chelating extracel-
lular Ca2?during the response to ?-factor and later was
found to be mimicked by mutations that eliminate the high-
affinity Ca2?influx channel, Ca2?/calmodulin, and CN (Iida
et al., 1990; Cyert et al., 1991; Cyert and Thorner, 1992; Foor
et al., 1992; Iida et al., 1994; Ono et al., 1994; Moser et al., 1996;
Fischer et al., 1997; Paidhungat and Garrett, 1997; Withee et
al., 1997; Muller et al., 2001). This pathway does not desen-
sitize cells to mating pheromones or promote recovery after
withdrawal of mating pheromones (Moser et al., 1996) and
therefore implicates CN and its upstream regulators as key
components of an antideath or prosurvival pathway in cells
responding to mating pheromones. Here we demonstrate
that many cells undergoing CN-less death transiently accu-
mulate ROS before death. ROS accumulation may occur in
all cells undergoing CN-less death, and the brief appearance
of ROS-positive cells may reflect some combination of a
transient ROS production, active ROS dissipation, and the
loss of ROS and fluorescent probes upon cell death. There-
fore, CN can be firmly positioned as an upstream inhibitor of
ROS accumulation in these conditions. CYC, coenzyme Q,
and complexes III, IV, and V of the mitochondrial respira-
tory chain were all required for efficient CN-less death and
for accumulation of ROS. Whether CN regulates these fac-
tors or other factors involved in ROS production or dissipa-
tion remains to be elucidated.
CN-less death was not prevented by cyclosporin A or
dependent on mitochondrial cyclophilin D (Figures 1 and 2),
suggesting that the mitochondrial permeability transition
pore may not have as strong a role as previously suggested
(Severin and Hyman, 2002). CN-less death also did not
require Fus1, Fus2, Rvs161, Lrg1, or Fig1 but did require
Bni1, Spa2, and Pea2 for maximum effectiveness (Figure 2B).
The latter factors are all components of the polarisome, a
regulator of actin dynamics and cell polarity during yeast
mating (Chenevert et al., 1994; Valtz and Herskowitz, 1996;
Evangelista et al., 1997; Bidlingmaier and Snyder, 2004).
Recently, polarisome function was shown to be important
for maximum activation of the pheromone signaling path-
way and for mating (Qi and Elion, 2005), so its roles in
CN-less death and Fig1-dependent death are not unex-
pected. A dominant-negative fragment of Spa2 recently iso-
lated as a high-dosage suppressor of CN-less death (Noma et
al., 2005) may also depress pheromone signaling. Because of
the pleiotropic effects of the polarisome on mating and mul-
tiple death pathways, it seems an unlikely target of CN
regulation. Obviously, more work will be needed to deter-
mine precisely how CN and the other pheromone-respon-
sive pathways regulate CN-less death.
Interestingly, the dependence of CN-less death on mito-
chondrial respiration closely resembles “Bax-death,” which
arises in yeast as a consequence of heterologous overexpres-
sion of human Bax (Harris et al., 2000). Both CN-less death
and Bax-death are sensitive to respiration inhibitors but
insensitive to other factors involved in apoptosis-like death
such as Mca1 (Guscetti et al., 2005). It is therefore reasonable
to speculate that a Bax-like factor endogenous to yeast might
be activated in response to mating pheromones and coun-
teracted by CN. In mammals, CN dephosphorylates at least
one regulator of mammalian apoptosis (Bad), which then
stimulates Bax-induced apoptosis by interfering with Bax
inhibitors (Wang et al., 1999). However, no obvious homo-
logues of Bax or Bad are evident in the genomes of modern
fungi. Fis1, a strongly conserved protein involved in fission
of yeast and mammalian mitochondria (Okamoto and Shaw,
2005), has been proposed as a surrogate of Bcl-2 or Bax in
yeast cells undergoing apoptosis-like cell death (Fannjiang et
al., 2004). Though Fis1 can regulate apoptosis-like cell death
in other conditions (Ivanovska and Hardwick, 2005), Fis1
did not detectably modulate CN-less death in response to
mating pheromones (Figure 2B). Therefore, CN-less death in
yeast seems mechanistically distinct from apoptosis in ani-
mals and apoptosis-like cell death in fungi.
Death of CWI-Deficient Mutants
CWI-deficient mutants responding to low concentrations of
?-factor also die, but their manner of death appears quite
distinct from that of CN-deficient mutants. Unlike CN-less
death, the death of CWI-deficient cells did not depend on
respiration and did depend on the functions of Fus1, Fus2,
and Rvs161, which promote localized degradation of cell
wall polymers at the tips of mating projections (Fitch et al.,
2004). In wild-type cells, activation of CWI signaling during
the response to ?-factor leads to increased biosynthesis of
new cell wall polymers (Bulik et al., 2003) and prevention of
cell death. As expected for an inducer of cell wall chitin
biosynthesis, glucosamine suppressed death of CWI-defi-
cient cells (but not CN-deficient cells). FK506 increased the
rate of death of bck1 mutants and bck1 fig1 double mutants
(unpublished observations), and independent effects of
MPK and CN have been noted previously (Zhao et al., 1998).
Although the death of CWI-deficient mutants responding to
?-factor was originally considered to be a consequence of
lysis due to cell wall failure (Errede et al., 1995), we observed
a peak of ROS-positive cells in the dying population, and
their death was not avoided by environmental osmotica
such as sorbitol and salt (unpublished observations). There-
fore, CWI signaling may prevent death during the response
to ?-factor, primarily by inhibiting a nonmitochondrial
source of ROS. It may be impossible to prove that “MPK-less
death” and CN-less death are completely independent until
their direct and indirect targets are fully elucidated.
High concentrations of ?-factor triggered rapid death of
wild-type cells, in striking contrast to low concentrations
that induced much slower cell deaths in CN-deficient and
CWI-deficient mutants but not wild-type cells. The fast high-
?-factor cell death was further distinguished from the slow
low-?-factor cell deaths (CN- and MPK-less deaths) by its
complete dependence on Fig1, a plasma membrane protein
that is strongly induced by the response to mating phero-
mones (Erdman et al., 1998; Muller et al., 2003). A Fig1-
dependent fast wave of cell death was detected even in
CN-deficient and CWI-deficient cells responding to high
concentrations of ?-factor, suggesting that Fig1 can promote
cell death independent of CN and CWI signaling. However,
mathematical deconvolution of the biphasic death curves
indicated a mild stimulation of Fig1-dependent death by CN
N.-N. Zhang et al.
Molecular Biology of the Cell3418
signaling and a strong inhibition of Fig1-dependent death by
CWI signaling. The effect of CN may be insignificant because
it prevents a large majority of cells from succumbing to
CN-less death and therefore may increase the number of
cells susceptible to Fig1-dependent death. On the other
hand, the effect of CWI signaling appeared much more
robust: the loss of CWI signaling in bck1 mutants increased
Fig1-dependent death to ?40% of the population, whereas
the gain of CWI signaling in lrg1 mutants decreased Fig1-
dependent death to 2% or less. Factors that promote cell wall
degradation (Fus1, Fus2, and Rvs161) were required for
Fig1-dependent death and factors that prevent cell wall
degradation (glucosamine) also prevented Fig1-dependent
death. Because all these factors are interconnected in a neg-
ative feedback loop (see Figure 6), the incidence of fast cell
death in a population may reflect the outcome of a race be-
tween the Fig1-dependent prodeath pathway and a CWI-de-
pendent antideath pathway (that probably involves cell wall
repair). The apparently random occurrence of fast cell death
can be explained by cell-to-cell variations in either the phero-
mone signaling pathways (Colman-Lerner et al., 2005) or the
CWI signaling pathway as observed for other MAP kinase
pathways (Ferrell and Machleder, 1998). Small strain-to-strain
variations in either pathway also may be responsible for the
varying degrees of fast cell death in different strain back-
grounds. Therefore, the negative feedback loop provides pos-
sible explanations for why only a fraction of yeast cells die by
the Fig1-dependent process, why this fraction differs in various
strain backgrounds, and why the cell death appears random in
the population with respect to cell age. Because Fig1 also per-
forms functions that promote cell fusion during mating (Erd-
man et al., 1998), fast cell death may reflect a lethal outcome of
attempting late steps of the mating program in the absence of
a mating partner. These findings provide an alternative to the
view of altruistic PCD triggered by mating pheromones.
The most conserved region of Fig1 and its closest homo-
logues in fungi mapped to the G??GXC(n)C motif in the
first extracellular loop. A similar motif in the PMP-22/EMP/
MP20/Claudin superfamily frequently mediates homophilic
and heterophilic interactions with transmembrane proteins
in the same or adjacent cell membrane (Van Itallie and
Anderson, 2006). Fig1 does not function like a VGCC-?
subunit in yeast because Fig1 was not required for normal
activation of the VGCC-related high-affinity Ca2?influx
channel (Muller et al., 2003). Although Fig1 resembles star-
gazin in its ability to promote low-affinity Ca2?influx and
Ca2?influx may contribute to yeast cell death in other
circumstances (Courchesne, 2002; Gupta et al., 2003; Poznia-
kovsky et al., 2005), Ca2?influx was not required for fast cell
death in yeast. However, we cannot rule out other possible
ion fluxes as the cause of Fig1-dependent death. Other mem-
bers of the PMP-22/EMP/MP20/Claudin superfamily
termed PERP have been shown to induce apoptosis of mam-
malian cells by unknown mechanisms (Ihrie et al., 2003).
Overexpression of PMP-22 or EMP1,2,3 proteins stimulates
apoptosis in HEK293 cells, possibly through interactions
with P2X7 ion channels (Wilson et al., 2002). Identification of
factors that function together with or downstream of Fig1
may provide additional insights into its roles and possible
conservation in mammals.
Apoptosis and PCD in Yeast?
Apoptosis is a well-characterized form of programmed cell
death occurring in all animals. Orthologues of the key pro-
apoptosis factors caspase/Ced-3, Apaf-1/Ced-4, and Bax/
Ced-9 are all absent from all fungal genomes sequenced to
date. Some “apoptosis-like” cell deaths in yeast may be
regulated by a distantly related caspase-like protease termed
Mca1 (Madeo et al., 2004) and certain mitochondrial factors
(Fannjiang et al., 2004). We found no detectable role of these
factors and no evidence for chromatin fragmentation (a hall-
mark of apoptosis-like cell death in yeast) during any of the
three waves of cell death reported here. Reports to the
contrary (Severin and Hyman, 2002) utilized a TUNEL assay
method that we find susceptible to background from an
RNase-sensitive factor. Previous findings that cyclosporin A
can prevent fast cell death during the response to mating
pheromones (Severin and Hyman, 2002) were not reproduc-
ible in our hands. All three manners of cell death studied
here result in uptake of methylene blue and PI, suggesting
the cells become metabolically inactive and lose their integ-
rity, which seems very different from apoptosis and apop-
tosis-like cell death in which only cell viability/proliferation
is lost. Finally, death of CN-deficient and CWI-deficient
mutants appears morphologically dissimilar from apoptosis
(Errede et al., 1995; Withee et al., 1997). Collectively, these
findings argue against the idea that mating pheromones
trigger apoptosis-like cell death in yeast.
PCD in animals, whether apoptotic or not, implies an
altruistic behavior that benefits the surviving cells. It has
been speculated that death of yeast cells responding to
mating pheromones may represent an altruistic behavior
where, for example, older or weaker cells sacrifice them-
selves to preserve resources (nutrients and/or mating
partners) for younger or healthier cells (Severin and
Hyman, 2002). We have directly tested one aspect of that
idea and determined that mother cells and newborn
daughter cells are equally likely to undergo Fig1-depen-
dent death and that death of mother-daughter pairs is not
more common or less common than that expected by
chance. Thus, Fig1-dependent death was apparently inde-
pendent of cell aging. One might argue that only weak
cells undergo Fig1-dependent death to eliminate their
inferior genomes from the mating pool. However, genet-
ically weakened mutants lacking CN or CYC did not show
higher levels of Fig1-dependent death. Fig1-dependent
death in a-cells also required much higher concentrations
of ?-factor than other types of cell death, conditions that
might occur naturally only when potential mates are in
abundance or when mating cells are closely apposed.
However, Fig1-dependent death was not detectable in
mixed populations of a- and ?-cells undergoing mating or
in prezygotes arrested at the membrane fusion step of
mating. Thus, Fig1-dependent death may be too infre-
quent in normal conditions to provide a significant ad-
vantage to the surviving kin.
As an alternative to altruistic cell suicide, Fig1-dependent
death may represent the execution of an ordinary event in
yeast cell mating at an inappropriate time or place, resulting
in lethal consequences. Low concentrations of mating pher-
omones trigger early events in mating such as agglutination
of a- and ?-cells and formation of pre-zygotes while high
concentrations of mating pheromones are necessary for dis-
solution of the cell wall material that prevents close apposi-
tion of cell membranes before membrane fusion (Brizzio et
al., 1996; Dorer et al., 1997). Mutants lacking Fig1 exhibit
mild but detectable defects in dissolution of cell walls of
prezygotes and zygotes (Erdman et al., 1998). Therefore,
high mating pheromones may trigger Fig1-dependent dis-
solution of cell wall material, which promotes fusion of
prezygotes and possibly lysis of cells that lack a mating
partner. Thus, there is no need to speculate the existence of
altruistic behavior of yeast cells in these circumstances.
Mating and Cell Death in Yeast
Vol. 17, August 20063419
CN-less death and MPK-less death did not occur at ap-
preciable levels in populations of wild-type cells but occur in
nearly 100% of cells responding to low concentrations of
?-factor (unless they have already undergone Fig1-depen-
dent death). Although these putative cell death pathways
have the potential to eliminate certain types of weakened
cells from the mating pool, the time required for death is
longer than the time required for successful conjugation, and
mutants lacking CN can mate as efficiently as wild-type cells
(Cyert et al., 1991; Cyert and Thorner, 1992). One interpre-
tation of these findings is that CN and MPK each perform
functions that are essential for adaptation of cells exposed to
mating pheromones for long periods of time. The essential
function of CWI signaling may be the repair of damaged cell
walls or suppression of nonmitochondrial ROS production,
though other pathways have not been excluded. In the case
of CN, its essential function appears to be suppression of
mitochondrial ROS production or enhancement of ROS de-
toxification. Therefore, CN-less death and MPK-less deaths
may be viewed more accurately as prosurvival pathways
than antideath pathways, though such a view may change if
bona fide prodeath pathways were identified in the future.
Independent of their potential relationships to mamma-
lian processes, CN-less death and MPK-less death merit
further study because similar processes may occur broadly
in the fungal kingdom during the response to commonly
prescribed antifungal antibiotics. Azole-class and echinocan-
din-class antibiotics do not kill the fungal species in diverse
yeasts and fungi (Del Poeta et al., 2000; Marchetti et al., 2000;
Bonilla et al., 2002; Cruz et al., 2002; Edlind et al., 2002; Bonilla
and Cunningham, 2003; Kraus et al., 2003; Onyewu et al.,
2003; Reinoso-Martin et al., 2003; Sanglard et al., 2003; Kaur
et al., 2004; Steinbach et al., 2004). Like mating pheromones,
the natural antifungal compound tunicamycin stimulates
both CN-less death and MPK-less in yeast and probably
other species (Bonilla et al., 2002; Chen et al., 2005). There-
fore, chemical inhibitors of CN, MPK, or their targets in
fungi are expected to vastly improve the fungicidal activity
of most clinically relevant antibiotics and potentially dimin-
ish the acquisition of antibiotic resistance. Identification of
the factors and processes downstream of CN, MPK, and Fig1
involved in each type of fungal cell death pathway should
provide insights into the origins and diversity of cell death
mechanisms in multicellular organisms.
We are grateful to Janet Shaw, Charlie Boone, Cathy Clarke, and Lymarie
Maldonado-Baez for yeast strains used in this study. We also thank Christian
Martin and Hui Jin for excellent technical assistance. This work was sup-
ported by grants from the National Science Foundation (MCB-0331306 to
A.L.), the American Cancer Society (RSG-05-205-01-MBC to E.G.), and the
National Institutes of Health (GM072024 and RR020839 to A.L.; GM053082
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