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 (email@example.com).
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
Dorer, R., Boone, C., Kimbrough, T., Kim, J., and Hartwell, L. H. (1997).
Genetic analysis of default mating behavior in Saccharomyces cerevisiae. Ge-
netics 146, 39–55.
Edlind, T., Smith, L., Henry, K., Katiyar, S., and Nickels, J. (2002). Antifungal
activity in Saccharomyces cerevisiae is modulated by calcium signalling. Mol.
Microbiol. 46, 257–268.
Elion, E. A., Satterberg, B., and Kranz, J. E. (1993). FUS3 phosphorylates
multiple components of the mating signal transduction cascade: evidence for
STE12 and FAR1. Mol. Biol. Cell 4, 495–510.
Erdman, S., Lin, L., Malczynski, M., and Snyder, M. (1998). Pheromone-
regulated genes required for yeast mating differentiation. J. Cell Biol. 140,
Errede, B., Cade, R. M., Yashar, B. M., Kamada, Y., Levin, D. E., Irie, K., and
Matsumoto, K. (1995). Dynamics and organization of MAP kinase signal
pathways. Reprod. Dev. 42, 477–485.
Evangelista, M., Blundell, K., Longtine, M. S., Chow, C. J., Adames, N.,
Pringle, J. R., Peter, M., and Boone, C. (1997). Bni1p, a yeast formin linking
cdc42p and the actin cytoskeleton during polarized morphogenesis. Science
Fannjiang, Y., Cheng, W. C., Lee, S. J., Qi, B., Pevsner, J., McCaffery, J. M., Hill,
R. B., Basanez, G., and Hardwick, J. M. (2004). Mitochondrial fission proteins
regulate programmed cell death in yeast. Genes Dev. 18, 2785–2797.
Ferrell, J. E., Jr., and Machleder, E. M. (1998). The biochemical basis of an
all-or-none cell fate switch in Xenopus oocytes. Science 280, 895–898.
Fischer, M., Schnell, N., Chattaway, J., Davies, P., Dixon, G., and Sanders, D.
(1997). The Saccharomyces cerevisiae CCH1 gene is involved in calcium influx
and mating. FEBS Lett. 419, 259–262.
Fitch, P. G., Gammie, A. E., Lee, D. J., de Candal, V. B., and Rose, M. D. (2004).
Lrg1p is a Rho1 GTPase-activating protein required for efficient cell fusion in
yeast. Genetics 168, 733–746.
Foor, F., Parent, S. A., Morin, N., Dahl, A. M., Ramadan, N., Chrebet, G.,
Bostian, K. A., and Nielsen, J. B. (1992). Calcineurin mediates inhibition by
FK506 and cyclosporin of recovery from alpha-factor arrest in yeast. Nature
Frohlich, K. U., and Madeo, F. (2001). Apoptosis in yeast: a new model for
aging research. Exp. Gerontol. 37, 27–31.
Gin, P., and Clarke, C. F. (2005). Genetic evidence for a multi-subunit complex
in coenzyme Q biosynthesis in yeast and the role of the Coq1 hexaprenyl
diphosphate synthase. J. Biol. Chem. 280, 2676–2681.
Groisman, A., Lobo, C., Cho, H., Campbell, J. K., Dufour, Y. S., Stevens, A. M.,
and Levchenko, A. (2005). A microfluidic chemostat for experiments with
bacterial and yeast cells. Nat. Methods 2, 685–689.
Gupta, S. S., Ton, V. K., Beaudry, V., Rulli, S., Cunningham, K., and Rao, R.
(2003). Antifungal activity of amiodarone is mediated by disruption of cal-
cium homeostasis. J. Biol. Chem. 278, 28831–28839.
Guscetti, F., Nath, N., and Denko, N. (2005). Functional characterization of
human proapoptotic molecules in yeast S. cerevisiae. FASEB J. 19, 464–466.
Harris, M. H., Vander Heiden, M. G., Kron, S. J., and Thompson, C. B. (2000).
Role of oxidative phosphorylation in Bax toxicity. Mol. Cell. Biol. 20, 3590–
Heiman, M. G., and Walter, P. (2000). Prm1p, a pheromone-regulated multi-
spanning membrane protein, facilitates plasma membrane fusion during
yeast mating. J. Cell Biol. 151, 719–730.
Herker, E., Jungwirth, H., Lehmann, K. A., Maldener, C., Frohlich, K. U.,
Wissing, S., Buttner, S., Fehr, M., Sigrist, S., and Madeo, F. (2004). Chrono-
logical aging leads to apoptosis in yeast. J. Cell Biol. 164, 501–507.
Ihrie, R. A., Reczek, E., Horner, J. S., Khachatrian, L., Sage, J., Jacks, T., and
Attardi, L. D. (2003). Perp is a mediator of p53-dependent apoptosis in diverse
cell types. Curr. Biol. 13, 1985–1990.
Iida, H., Nakamura, H., Ono, T., Okumura, M. S., and Anraku, Y. (1994).
MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane
protein, is required for Ca2?influx and mating. Mol. Cell. Biol. 14, 8259–8271.
Iida, H., Yagawa, Y., and Anraku, Y. (1990). Essential role for induced Ca2?
influx followed by [Ca2?]i rise in maintaining viability of yeast cells late in the
mating pheromone response pathway. A study of [Ca2?]i in single Saccharo-
myces cerevisiae cells with imaging of fura-2. J. Biol. Chem. 265, 13391–13399.
Ivanovska, I., and Hardwick, J. M. (2005). Viruses activate a genetically
conserved cell death pathway in a unicellular organism. J. Cell Biol. 170,
Jin, H., Carlile, C., Nolan, S., and Grote, E. (2004). Prm1 prevents contact-
dependent lysis of yeast mating pairs. Eukaryot. Cell 3, 1664–1673.
Kaur, R., Castano, I., and Cormack, B. P. (2004). Functional genomic analysis
of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of
calcium signaling and mitochondria. Antimicrob. Agents Chemother. 48,
Koonin, E. V., and Aravind, L. (2002). Origin and evolution of eukaryotic
apoptosis: the bacterial connection. Cell Death Differ. 9, 394–404.
Kraus, P. R., Fox, D. S., Cox, G. M., and Heitman, J. (2003). The Cryptococcus
neoformans MAP kinase Mpk1 regulates cell integrity in response to antifungal
drugs and loss of calcineurin function. Mol. Microbiol. 48, 1377–1387.
Letts, V. A., Felix, R., Biddlecome, G. H., Arikkath, J., Mahaffey, C. L.,
Valenzuela, A., Bartlett, F. S., 2nd, Mori, Y., Campbell, K. P., and Frankel,
W. N. (1998). The mouse stargazer gene encodes a neuronal Ca2?-channel
gamma subunit. Nat. Genet. 19, 340–347.
Levin, D. E. (2005). Cell wall integrity signaling in Saccharomyces cerevisiae.
Microbiol. Mol. Biol. Rev. 69, 262–291.
Longo, V. D., Mitteldorf, J., and Skulachev, V. P. (2005). Programmed and
altruistic ageing. Nat. Rev. Genet. 6, 866–872.
Madeo, F., Engelhardt, S., Herker, E., Lehmann, N., Maldener, C., Proksch, A.,
Wissing, S., and Frohlich, K. U. (2002). Apoptosis in yeast: a new model
system with applications in cell biology and medicine. Curr. Genet. 41,
Madeo, F., Frohlich, E., Ligr, M., Grey, M., Sigrist, S. J., Wolf, D. H., and
Frohlich, K. U. (1999). Oxygen stress: a regulator of apoptosis in yeast. J. Cell
Biol. 145, 757–767.
Madeo, F., Herker, E., Wissing, S., Jungwirth, H., Eisenberg, T., and Frohlich,
K.-U. (2004). Apoptosis in yeast. Curr. Opin. Microbiol. 7, 655–660.
Marchetti, O., Moreillon, P., Glauser, M. P., Bille, J., and Sanglard, D. (2000).
Potent synergism of the combination of fluconazole and cyclosporine in
Candida albicans. Antimicrob. Agents Chemother. 44, 2373–2381.
Moser, M. J., Geiser, J. R., and Davis, T. N. (1996). Ca2?-calmodulin promotes
survival of pheromone-induced growth arrest by activation of calcineurin and
Ca2?-calmodulin-dependent protein kinase. Mol. Cell. Biol. 16, 4824–4831.
Mozdy, A. D., McCaffery, J. M., and Shaw, J. M. (2000). Dnm1p GTPase-
mediated mitochondrial fission is a multi-step process requiring the novel
integral membrane component Fis1p. J. Cell Biol. 151, 367–380.
Muller, E. M., Locke, E. G., and Cunningham, K. W. (2001). Differential
regulation of two Ca2?influx systems by pheromone signaling in Saccharo-
myces cerevisiae. Genetics 159, 1527–1538.
Muller, E. M., Mackin, N. A., Erdman, S. E., and Cunningham, K. W. (2003).
Fig1p facilitates Ca2?influx and cell fusion during mating of Saccharomyces
cerevisiae. J. Biol. Chem. 278, 38461–38469.
Noma, S., Iida, K., and Iida, H. (2005). Polarized morphogenesis regulator
Spa2 is required for the function of putative stretch-activated Ca2?-permeable
channel component Mid1 in Saccharomyces cerevisiae. Eukaryot. Cell 4, 1353–
Okamoto, K., and Shaw, J. M. (2005). Mitochondrial morphology and dynam-
ics in yeast and multicellular eukaryotes. Annu. Rev. Genet. 39, 503–536.
Ono, T., Suzuki, T., Anraku, Y., and Iida, H. (1994). The MID2 gene encodes
a putative integral membrane protein with a Ca2?-binding domain and shows
mating pheromone-stimulated expression in Saccharomyces cerevisiae. Gene
Onyewu, C., Blankenship, J. R., Del Poeta, M., and Heitman, J. (2003). Ergos-
terol biosynthesis inhibitors become fungicidal when combined with cal-
cineurin inhibitors against Candida albicans, Candida glabrata, and Candida
krusei. Antimicrob. Agents Chemother. 47, 956–964.
Paidhungat, M., and Garrett, S. (1997). A homolog of mammalian, voltage-
gated calcium channels mediates yeast pheromone-stimulated Ca2?uptake
and exacerbates the cdc1(Ts) growth defect. Mol. Cell. Biol. 17, 6339–6347.
Pozniakovsky, A. I., Knorre, D. A., Markova, O. V., Hyman, A. A., Skulachev,
V. P., and Severin, F. F. (2005). Role of mitochondria in the pheromone- and
amiodarone-induced programmed death of yeast. J. Cell Biol. 168, 257–269.
Priault, M., Camougrand, N., Kinnally, K. W., Vallette, F. M., and Manon, S.
(2003). Yeast as a tool to study Bax/mitochondrial interactions in cell death.
FEMS Yeast Res. 4, 15–27.
Qi, M., and Elion, E. A. (2005). Formin-induced actin cables are required for
polarized recruitment of the Ste5 scaffold and high level activation of MAPK
Fus3. J. Cell Sci. 118, 2837–2848.
Reinoso-Martin, C., Schuller, C., Schuetzer-Muehlbauer, M., and Kuchler, K.
(2003). The yeast protein kinase C (PKC) cell integrity pathway mediates
tolerance to the antifungal drug caspofungin through activation of Slt2p
mitogen-activated protein kinase signaling. Eukaryot Cell 2, 1200–1210.
Mating and Cell Death in Yeast
Vol. 17, August 20063421
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning. A
Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Sanglard, D., Ischer, F., Marchetti, O., Entenza, J., and Bille, J. (2003). Cal-
cineurin A of Candida albicans: involvement in antifungal tolerance, cell mor-
phogenesis and virulence. Mol. Microbiol. 48, 959–976.
Severin, F. F., and Hyman, A. A. (2002). Pheromone induces programmed cell
death in S. cerevisiae. Curr. Biol. 12, R233–R235.
Sherman, F., Hicks, J B., and Fink, G. R. (1986). Methods in Yeast Genetics,
Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sprague, G. F., Jr., and Herskowitz, I. (1981). Control of yeast cell type by the
mating type locus. I. Identification and control of expression of the a-specific
gene BAR1. J. Mol. Biol. 153, 305–321.
Sprague, G. F., Jr., and Thorner, J. W. (1992). Pheromone response and signal
transduction during the mating process of Saccharomyces cerevisiae. In: The
Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression,
ed. E. W. Jones, J. R. Pringle, and J. R. Broach, Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory Press, 657–744.
Steinbach, W. J., Schell, W. A., Blankenship, J. R., Onyewu, C., Heitman, J.,
and Perfect, J. R. (2004). In vitro interactions between antifungals and immu-
nosuppressants against Aspergillus fumigatus. Antimicrob. Agents Chemother.
Valtz, N., and Herskowitz, I. (1996). Pea2 protein of yeast is localized to sites
of polarized growth and is required for efficient mating and bipolar budding.
J. Cell Biol. 135, 725–739.
Van Itallie, C. M., and Anderson, J. M. (2006). Claudins and epithelial para-
cellular transport. Annu. Rev. Physiol. 68, 403–429.
Wallis, J. W., Chrebet, G., Brodsky, G., Rolfe, M., and Rothstein, R. (1989). A
hyper-recombination mutation in S. cerevisiae identifies a novel eukaryotic
topoisomerase. Cell 58, 409–419.
Wang, H. G., Pathan, N., Ethell, I. M., Krajewski, S., Yamaguchi, Y., Shibasaki,
F., McKeon, F., Bobo, T., Franke, T. F., and Reed, J. C. (1999). Ca2?-induced
apoptosis through calcineurin dephosphorylation of BAD. Science 284, 339–
Wilson, H. L., Wilson, S. A., Surprenant, A., and North, R. A. (2002). Epithelial
membrane proteins induce membrane blebbing and interact with the P2X7
receptor C terminus. J. Biol. Chem. 277, 34017–34023.
Withee, J. L., Mulholland, J., Jeng, R., and Cyert, M. S. (1997). An essential role
of the yeast pheromone-induced Ca2?signal is to activate calcineurin. Mol.
Biol. Cell 8, 263–277.
Zhao, C., Jung, U. S., Garrett-Engele, P., Roe, T., Cyert, M. S., and Levin, D. E.
(1998). Temperature-induced expression of yeast FKS2 is under the dual
control of PKC and calcineurin. Mol. Cell. Biol. 18, 1013–1022.
N.-N. Zhang et al.
Molecular Biology of the Cell3422