Department of Biochemistry and Biophysics, Successful zygote formation during yeast
mating requires cell fusion of the two haploid mating
partners. To ensure that cells do not lyse as they remodel their cell wall, the fusion event is both temporally and spatially regulated: the cell wall is degraded only after cell-cell contact and only in the region of
cell-cell contact. To understand how cell fusion is regulated, we identified mutants defective in cell fusion
based upon their defect in mating to a fus1 fus2 strain
(Chenevert, J., N. Valtz, and I. Herskowitz. 1994. Genetics 136:1287-1297). Two of these cell fusion mutants are defective in the FPS1 gene, which codes for a glycerol facilitator (Luyten, K., J. Albertyn, W.F. Skibbe,
B.A. Prior, J. Ramos, J.M. Thevelein, and S. Hohmann.
1995. EMBO [Eur. Mol. Biol. Organ.] J. 14:1360-1371).
To determine whether inability to maintain osmotic balance accounts for the defect in cell fusion in these
mutants, we analyzed the behavior of an fps1
THE joining of two cells occurs during certain specialized cell-cell interactions such as sperm-egg fusion
during fertilization, myoblast fusion during myotube formation, and gamete fusion during yeast mating.
Intercellular fusion requires successful completion of a
number of different events, the molecular details of which are poorly understood. The interacting cells must first recognize and adhere to each other. Extracellular material
separating the interacting cells must then be removed. The
zona pellucida surrounding the egg, extracellular matrix
components separating myoblasts, and cell wall material
separating haploid yeast cells must be removed to place
the plasma membranes of the interacting cells into apposition. Finally, the plasma membranes of the two cells fuse,
forming a single heterokaryon which can then undergo fusion of intracellular organelles.
The mating pathway of Saccharomyces cerevisiae culminates in the fusion of two haploid cells of opposite mating
type (a and Although pheromones activate cells for fusion, cell wall
degradation does not begin until the mating partners contact each other. Initially, cell surface agglutinins mediate
attachment of the mating partners (Lipke and Kurjan,
1992 Products of the FUS1-FUS3, FUS5-FUS8, and CEF1
genes are required for cell fusion (Trueheart et al., 1987 Cell fusion requires that cell contact be sensed and that
the cell surface be remodeled in response to this contact.
To prevent cell lysis and to maintain cell integrity, cell wall
degradation must be highly regulated, occurring only after
cell-cell contact and only in the region of cell-cell contact.
We show that the glycerol facilitator, Fps1p, is required
for cell fusion. Our studies on Fps1p provide evidence that
the osmotic state of the cell regulates cell fusion. We present
additional studies suggesting that protein kinase C, previously recognized for its role in osmotic regulation (Davenport et al., 1995 Yeast Strains and Media
Yeast strains are described in Table I. Standard yeast growth conditions
and genetic manipulations are described in Rose et al. (1990) Table I.
Yeast Strains and Plasmids Used in This Study
Yeast Plasmids and Transformations
YEpGPD1 is a 2 µ URA3 plasmid (derived from YEplac195) containing
the GPD1 gene, as described in Albertyn et al. (1994 Strain Construction
The GPD1 gene was deleted from strains using pUCgpd1
Mating Assays
Quantitative mating was as described in Valtz and Herskowitz (1996) Cloning of FPS1
FPS1 was cloned by complementation of the mating defect of the M8 mutant (Chenevert et al., 1994
Analysis of fps1 Segregants obtained by crossing an fps1 Assaying of Intracellular Glycerol
Intracellular glycerol was assayed enzymatically using a glycerol determination kit (Boehringer Mannheim Biochemicals, Indianapolis, IN) essentially as described (Albertyn et al., 1994a Identification of Mutants Defective in Cell Fusion
Mutants defective in cell fusion were identified based
upon their defect in mating to a fus1 fus2 mutant (Chenevert et al., 1994 To identify additional cell fusion mutants, mutants exhibiting a mating defect but normal shmoo morphology
(class 4 mutants in Chenevert et al., 1994 M8 and M11 exhibit normal pheromone signaling and
events leading up to cell-cell contact. They produce and
respond to pheromone normally, as assayed by cell cycle
arrest and shmoo formation (Chenevert et al., 1994 M8 and M11 Are Defective in the FPS1 Gene
To clone the gene defective in the M8 mutant, the strain
was transformed with a high copy YEp24-derived library,
and 22,000 transformants were screened for ability to mate
with a fus1 fus2 strain. Five plasmids were identified that
restored mating. All contained a common 6.5-kb overlapping DNA segment. This segment was subcloned into
pRS316, a CEN-ARS vector, and further subcloning was
performed to identify the minimal fragment capable of
complementation (Fig. 2 c). A 2.2-kb XhoI-HindIII fragment (pJP27) restored mating to the M8 and M11 mutants.
This fragment contains the FPS1 ORF, which fully restored mating and cell fusion to M8 and M11 when integrated at its genomic locus (Fig. 3 and data not shown).
The mutation responsible for the mating defect of M8 and
M11 was demonstrated to be allelic to FPS1 by following
the segregation of M8 and M11 in crosses where the FPS1
locus was marked (see Materials and Methods). The plasmid complementation and segregation analyses indicate
that the mating defect of M8 and M11 strains is due to mutations in FPS1, which are designated fps1-1 and fps1-2,
respectively.
Mating Defect of fps1 Analysis of strains deleted for FPS1 confirmed that Fps1p
is required for cell fusion. FPS1 was deleted from the genome by replacing the FPS1 coding sequence with either
LEU2 or URA3 (Fig. 2 b; see Materials and Methods).
fps1
Quantitative mating assays demonstrated that, like fus1
fus2 strains, fps1 Table II.
Quantitative Mating Defect of fps1
Abstract
Materials and Methods
Results
Discussion
Footnotes
Acknowledgements
Abbreviations used in this paper
References
Abstract
mutant
with reduced intracellular glycerol levels because of a
defect in the glycerol-3-phosphate dehydrogenase
(GPD1) gene (Albertyn, J., S. Hohmann, J.M. Thevelein, and B.A. Prior. 1994. Mol. Cell. Biol. 14:4135-
4144): deletion of GPD1 partially suppressed the cell
fusion defect of fps1 mutants. In contrast, overexpression of GPD1 exacerbated the defect. The fusion defect
could also be partially suppressed by 1 M sorbitol. These observations indicate that the fusion defect of
fps1 mutants results from inability to regulate osmotic
balance and provide evidence that the osmotic state of
the cell can regulate fusion. We have also observed that
mutants expressing hyperactive protein kinase C exhibit a cell fusion defect similar to that of fps1 mutants.
We propose that Pkc1p regulates cell fusion in response to osmotic disequilibrium. Unlike fps1 mutants, fus1
and fus2 mutants are not influenced by expression of
GPD1 or by 1 M sorbitol. Their fusion defect is thus
unlikely to result from altered osmotic balance.
) into an a/
diploid zygote. The events leading up to cell-cell contact are well characterized. Haploid
cells secrete peptide pheromones (a-factor by a cells and
-factor by
cells) that are important for intercellular recognition and for preparing cells for fusion. These pheromones activate a G protein-coupled receptor on the surface of the opposite mating partner, which in turn activates
a mitogen-activated protein (MAP)1 kinase cascade, inducing a morphological response (shmoo formation), cell
cycle arrest, and transcriptional induction (for reviews see
Kurjan, 1992
; Sprague and Thorner, 1992
; Bardwell et al., 1994
; Herskowitz, 1995
). The mating pheromones prepare
cells to fuse by inducing expression and localization of fusion components. In particular, synthesis of Fus1p and
Fus2p, proteins required for cell fusion, is induced by
pheromone (Trueheart et al., 1987
; McCaffrey et al., 1987
;
Elion et al., 1995
). These proteins are localized to the region of future cell contact (Trueheart et al., 1987
; Elion et
al., 1995
). Cells polarize the actin cytoskeleton and secretory apparatus toward their selected mating partner by detecting a pheromone gradient (Jackson and Hartwell,
1990
; Madden and Snyder, 1992
; Segall, 1993
). As a result,
new membrane and cell wall material is deposited at the
site of future cell contact (Field and Schekman, 1980
; Adams and Pringle, 1984
; Novick and Botstein, 1985
; Hasek
et al., 1987
; Read et al., 1992
), which may be important for
localized cell wall modifications (Lipke et al., 1976
; Tkacz
and MacKay, 1979
; Schekman and Brawley, 1979
; Baba et
al., 1989
) and targeting of the fusion machinery.
), which is reversible by sonication. The cell walls then
become irreversibly attached. Once cell-cell contact occurs, a thinning of the cell wall is observed that begins in
the center of the region of cell contact and proceeds toward the edges (Osumi et al., 1974
). Cell wall degradation
and remodeling normally occur quickly, so that few cells in
a population of mating cells are adhered but not fused (Trueheart et al., 1987
). In mutants defective in cell fusion, zygote formation is blocked after the cells have adhered
but before the intervening wall has been degraded, producing a dumbbell-shaped structure called a prezygote.
The persistence of the cell wall in these mutants creates a
physical barrier between mating partners, preventing cytoplasmic mixing and nuclear fusion (Trueheart et al., 1987
;
McCaffrey et al., 1987
).
;
McCaffrey et al., 1987
; Elion et al., 1990
, 1995
; Kurihara et
al., 1994
; Elia and Marsh, 1996
). Fus1p is a transmembrane
protein with an intracellular SH3 domain (Trueheart et al.,
1987
; Trueheart and Fink, 1989
). Fus2p has no similarity to
known proteins (Elion et al., 1995
). Despite their lack of
homology, Fus1p and Fus2p have overlapping functions,
as overexpression of one can partially suppress loss of the
other. Absence of both proteins results in a synthetic fusion defect. In addition, the fusion defect is greatly enhanced when both mating partners are mutant, suggesting
that at least some activities required for cell fusion can be
provided by either partner (Trueheart et al., 1987
). Unlike
FUS1 and FUS2, which are specifically required for cell fusion, the other FUS genes have additional functions during
mating. For instance, FUS3, whose role in cell fusion is unknown, encodes a MAP kinase that functions in the pheromone response pathway (Elion et al., 1990
). FUS5, FUS8,
and CEF1 genes correspond to AXL1, RAM1, and STE6,
respectively. These genes were previously identified for
their role in a-factor production (Adames et al., 1995
;
Powers et al., 1986
; Kuchler et al., 1989
), suggesting that
high levels of pheromone may play a role in cell fusion
(Elia and Marsh, 1996
; Brizzio et al., 1996
). It is also possible that these proteins (e.g., Ste6p, the a-factor transporter) are required for cell fusion independently of their role in pheromone production or secretion (Elia and Marsh,
1996
). Whether the products of these genes play direct
roles in cell fusion or are involved in regulating fusion is
unclear.
), negatively regulates cell fusion, further
linking osmosensing pathways to regulation of cell fusion.
Materials and Methods
. Cells were
grown at 30°C in yeast extract/peptone/dextrose medium unless otherwise
noted. DNA manipulations were performed as described in Sambrook et
al. (1989)
.
b) (kindly provided
by S. Hohmann, Katholieke University, Leuven, Belgium). pJP67 (YCp50-DS1) is a YCp50-derived plasmid containing the PKC1-R398P allele as
described in Nonaka et al. (1995)
(kindly provided by Y. Takai, Osaka
University, Osaka, Japan). The 4.3-kb SphI fragment containing PKC1-R398P from pJP67 was cloned into BamHI and SalI sites of pRS306 to
generate pJP72. This plasmid was used to integrate PKC1-R398P at its genomic locus, generating strain JP317. Plasmids containing PKC1 under
control of the GAL1 promoter are pDL242 (pGAL1[PKC1-R398A]),
pDL293 (pGAL1[PKC1::HA]), and pDL295 (pGAL1[PKC1-K853R:: HA]) as described in Watanabe et al. (1994)
. They were kindly provided
by D. Levin (Johns Hopkins University, Baltimore, MD). pJW192 codes
for a RAS2-green fluorescent protein (GFP) fusion protein under control
of the GPD promoter on a 2µ TRP1 marked plasmid (kindly provided by
J. Whistler, University of California, Berkeley). Yeast transformations
were performed by the lithium acetate method (Ito et al., 1983
).
::TRP1, a construct designed to replace GPD1 with TRP1 as described in Albertyn et
al. (1994
b) (kindly provided by S. Hohmann). Strains were confirmed to
be gpd1
by their sensitivity to high osmolarity media and by PCR analysis. fus1
strains were constructed using pJP2, which contains a substitution of the FUS1 open reading frame (ORF) by TRP1. This plasmid was
generated by cloning the 1.9-kb PstI-KpnI fragment containing FUS1 into
the PstI-KpnI sites of pBluescript KS+. The FUS1 ORF was removed by
cloning a ClaI-HincII fragment containing TRP1 into the AccI-HincII
sites of FUS1. fus2
strains were generated using pKOFUS2, a plasmid in
which the 1.6-kb HindIII fragment containing FUS2 was replaced by the
1.1-kb HindIII fragment containing URA3. fus1
and fus2
strains were
confirmed by their defective mating and by PCR analysis. fps1
deletion strains were generated using either pJP31 or pJP52. pJP31 replaces FPS1
with LEU2; pJP52 replaces FPS1 with URA3 (see Fig. 1 b). The BglII-
HindIII fragment containing FPS1 was cloned into the BamHI-HindIII
sites of pUC18. To generate pJP31, LEU2 was removed from pUC18-LEU2 (Herskowitz collection), in which LEU2 is cloned into the SalI site
of pUC18. A PstI-XbaI fragment, containing LEU2, was cloned into the
NsiI-AccI site of FPS1. pJP52 was constructed from a plasmid in which a
111-nucleotide fragment was inserted at the stop codon of FPS1, generating a BamHI site 70 nucleotides 3
to the stop codon. This plasmid was cut
with BamHI and NsiI and a BamHI-NsiI fragment containing URA3 was
inserted. The URA3 fragment was obtained from pSM32 (Herskowitz collection), a pUC18 plasmid containing URA3. fps1
strains were confirmed by defective mating and by PCR analysis.
Fig. 1.
Morphological phenotype of cell fusion mutants. (a) Wild-type (IH3160), (b) M8 (IH3179), (c) M11 (IH3182), (d) fps1
(JP147), and (e) PKC1-R398P (JP317) strains were mated on filters to wild-type strain IH3186 (two left columns) or to wild-type strain
JP333 carrying the RAS2-GPF fusion plasmid (two right columns) as described in Materials and Methods. Nuclei were visualized by
DAPI staining (second column). RAS2-GFP was visualized by fluorescence microscopy (fourth column).
[View Larger Version of this Image (79K GIF file)]
except that 6 × 106 cells of each mating partner were mixed. Mating assays
scored microscopically were performed by mixing equal numbers of log phase a and
cells (6 × 106), collecting cells on 0.45-µm filters (Millipore
Corp., Bedford, MA), and incubating on YEPD plates for ~4 h at 30°C.
Cells were resuspended in 5 ml 70% ethanol by vortexing, washed, and resuspended in 50% glycerol + 1 µg/ml 4
,6-diamidino-2-phenylindole
(DAPI; Sigma Chemical Co., St. Louis, MO). Samples were sonicated and viewed with a microscope at 100× (Axioskop; Carl Zeiss, Inc., Thornwood, NY). Percentage of prezygotes was defined as prezygotes/(prezygotes + zygotes). At least 100 partnered cells (zygotes + prezygotes) were
counted per sample. Numbers represent the average of at least three experiments unless otherwise noted. Assays in which one partner contained
the RAS2-GFP plasmid were performed in essentially the same manner,
except that strains were grown in SD-TRP (Rose et al., 1990
) to select for
the plasmid. In this case, cells were resuspended from the filter into 5 ml
YEPD by vortexing, sonicated, and viewed with a microscope at 100×
(BX50; Olympus Corp., Lake Success, NY). Mating assays scored on
plates were performed by spreading a lawn of 9 × 106 log phase fus1 fus2
mating tester cells on a YEPD plate. Patches of cells grown on YEPD (or
on minimal media to select for plasmids) were replica plated to the lawn.
After incubation at 30°C for 3.5-4.5 h, plates were replica plated to media
selective for growth of diploids. For cells expressing PKC1 alleles under
control of the GAL1 promoter (pDL242, pDL293, pDL295), patches were
grown on raffinose-URA plates to select for the plasmid but maintain the
GAL1 promoter inactive. They were replica plated to a lawn of an MAT
fus1 fus2 strain at 30°C, on either YEPD or YEPGalactose. Cells were allowed to mate for 3.75 h and then replica plated to select for diploids in
the presence of glucose.
). Five plasmids that rescued the mating defect
were isolated from a 2µ YEp24-derived library (Carlson and Botstein,
1982
) from 22,000 transformants screened. All contained a common 6.5-kb overlapping DNA segment. An EcoRI-SalI fragment was subcloned
into pRS316, a CEN-ARS vector, and deletion analysis was performed to
identify the complementing ORF (see Fig. 2 c). A plasmid containing a
2.2-kb XhoI-HindIII fragment (pJP27) fully restored mating to the M8
and M11 mutants. The 2.4-kb BglII-HindIII segment containing FPS1
was cloned into pRS306, a URA3-marked, integrating vector to form
pJP30, which was able to restore mating and fusion when integrated at the
FPS1 genomic locus. A wild-type strain was transformed with the integrating plasmid, marking the FPS1 locus with URA3 (JP150), and then
crossed to M8 and M11 mutants, which were ura3. 12 of 13 complete tetrads from the M8 cross showed 2:2 segregation of Ura3+:Ura3
and
Mating+:Mating
phenotypes. One tetrad showed 1:3 segregation of
Ura3+:Ura3
and 2:2 segregation of Mating+:Mating
, presumably because of gene conversion at URA3. 10 complete tetrads from the M11
cross showed 2:2 segregation of Ura3 and mating phenotypes. In all
spores, the Ura3+ phenotype cosegregated with mating proficiency, indicating that the mutations responsible for the mating defects of M8 and
M11 are linked to FPS1.
Fig. 2.
Restriction map (a), disruption constructs (b), and deletion analysis (c) of FPS1 region. The ability of each plasmid to
complement the mating defect of the M8 and M11 mutants is indicated on the right: +, complementation; , no complementation. pJP are plasmids carrying the indicated segments. Restriction enzymes: S, SalI; B, BamHI; Bg, BglII; Xh, XhoI; N, NsiI; K,
KpnI; H, HindIII; X, XbaI; E, EcoRI. Additional information is
given in Materials and Methods and in the text.
[View Larger Version of this Image (13K GIF file)]
mpk1
Double Mutants
strain with an mpk1
strain
were allowed to germinate on YEPD containing 1 M sorbitol at 25°C.
Cells were passaged on YEPD (except the mpk1
fps1
double mutant,
which was grown on YEPD + 1 M sorbitol), streaked for single colonies
on either YEPD or YEPD + 1 M sorbitol, and grown for 2 d at 25°C.
). Cells were grown to mid-log
phase in YEPD unless selecting for plasmids, in which case they were
grown in SD-URA (Rose et al., 1990
). From each culture, three 10-ml aliquots were separately filtered onto 0.45-µm filters (Millipore Corp.),
washed quickly with 5 ml ice-cold YEPD, and resuspended in 2 ml 0.5 M
Tris-HCl, pH 7.5, by vortexing. Samples were heated to 95°C for 10 min,
and cell debris was removed by centrifugation. Aliquots were pooled, and
glycerol determinations were performed as per manufacturer's specifications. Protein concentrations were determined using the Bio-Rad protein
assay (Bio Rad Laboratories, Hercules, CA). Glycerol concentrations
were normalized to total protein. Fold increase in glycerol concentrations
was determined by normalizing to the level of intracellular glycerol in
wild-type cells. Data represent the average of at least three experiments.
Results
). fus1 fus2 double mutants are mildly
compromised in mating to a wild-type strain but are severely defective in mating to fus1 or fus2 strains (Trueheart et al., 1987
; Elion et al., 1995
). Hence, a screen to
find mutants defective in mating to a fus1 fus2 strain
should identify mutants defective in FUS1, FUS2, and other genes required for fusion. To determine if any of the
mutants were defective in FUS1 or FUS2, plasmids containing these genes were tested for their ability to restore
mating to the mutants, which identified one mutant as defective in FUS2 (Chenevert et al., 1994
).
) were examined
microscopically to ascertain if prezygotes accumulated
when the mutants were mated to a wild-type strain. Prezygotes were scored as structures in which the nuclei of mating partners remained unfused, as evidenced by two distinct DAPI staining structures, and in which a septum was
visible between adherent mating partners. 9 of 13 mutants
exhibited increased frequency of prezygotes, indicative of
a fusion defect (data not shown). The mutants were of two
classes: five were a cell specific and four were non-cell
type specific, exhibiting mating defects as both a and
cells. Two mutants of the latter class, designated M8 and
M11 (B6 and J10, respectively, in Chenevert et al., 1994
),
were further characterized and are described here.
; Philips, J., unpublished observations). We observed that, in
matings between M8 or M11 mutants and a wild-type partner, the percentage of partnered cells ([prezgotes + zygotes]/total cells) was normal, suggesting that the defect in
mating is not due to inability of the partners to find or adhere to each other. Fig. 1 illustrates the aberrant morphological structures that accumulated in mating mixes in
which one partner is M8 or M11. More than 30% of the
partnered cells were prezygotes (Fig. 1, b and c). In contrast, in mating reactions between wild-type partners (Fig.
1 a), <1% of the partnered cells were prezygotes. Further evidence that cell fusion was blocked before plasma membrane fusion was obtained by mating cells to a partner that
produces a RAS2-GFP fusion protein (kindly provided by
J. Whistler), which localizes green fluorescence around the
periphery of the cell (Whistler, J., and J. Rine, personal
communication). In mating reactions containing wild-type
cells, zygotes showed the green fluorescent signal throughout the entire zygote (Fig. 1 a). In contrast, when mutants
were mated to the wild-type partner containing RAS2-
GFP, prezygotes were found in which the green fluorescent signal remained restricted to one cell, indicating a
failure of plasma membrane fusion and cytoplasmic mixing (Fig. 1, b and c). We conclude that M8 and M11 mutants are defective in cell fusion but are normal for earlier
events of mating.
Fig. 3.
The FPS1 gene complements the mating defect of M8
and M11. (Right) WT (JP150), M8 (JP154), and M11 (JP158), all
carrying FPS1 plasmid pJP30 at the FPS1 genomic locus; (left)
WT (JP153), M8 (JP157), and M11 (JP161), all carrying the vector alone (pRS306). Strains were mated to a MAT fus1 fus2
strain (IH2351) as described in Materials and Methods.
[View Larger Version of this Image (123K GIF file)]
Mutants
strains, like fps1-1 and fps1-2 mutants, were defective in mating to fus1 fus2 strains (Fig. 4) and accumulated
prezygotes that were morphologically indistinguishable
from those observed in mating reactions with fps1-1 and
fps1-2 mutants (Fig. 1 d). In mating mixes of fps1
or fps1-2 mutants to a wild-type partner, 30-45% of partnered cells were prezygotes. To determine if the fps1-1 allele was
quantitatively similar to the fps1
allele, FPS1 was deleted
in two different fps1-1 strains. These fps1
deletion strains
behaved like the fps1-1 parent strains with respect to the
percentage of prezygotes accumulated (data not shown).
Thus, fps1-1 and fps1-2 appear to be null alleles of FPS1.
FPS1 deletion mutants in the EG123 strain background
have a similar defect in cell fusion (data not shown).
Fig. 4.
Suppression of
mating defect of fps1 by
gpd1
and 1 M sorbitol. (a)
Patches of MATa strains,
WT (IH3160), fps1
(JP147), fus1
(JP52) (left) and gpd1
(JP168), fps1
gpd1
(JP165),
and fus1
gpd1
(JP285)
(right), were mated to a MAT
fus1 fus2 strain (IH2351) as
described in Materials and
Methods. (b) Patches of MAT
strains, fps1
(JP226)
and fus2
(JP257) (left) and
fps1
gpd1
(JP233) and
fus2
gpd1
(JP287) (right),
were mated to a MATa fus1
fus2 strain (IH2353) as described in Materials and
Methods. (c) Patches of
MATa strains, WT (IH3196), and fps1
(JP147), were
mated on YEPD (left) or
YEPD containing 1 M sorbitol (right) to a MAT
fus1
fus2 strain (IH2351).
[View Larger Version of this Image (73K GIF file)]
strains were mildly defective in mating,
exhibiting a two- to threefold decrease in diploid formation (Table II). Unlike fus1 fus2 mutants, which exhibit an
enhanced defect when both mating partners are mutant
(Trueheart et al., 1987
; McCaffrey et al., 1987
; Elion et al.,
1995
), fps1
mutants did not mate significantly worse or
accumulate more prezygotes when mated to an fps1
partner than to a wild-type partner. In fact, when fps1
mutants are mated to wild-type cells, they yielded 61 ± 17%
or 32 ± 5% prezygotes, depending upon the mating type
of the mutant, whereas matings between two fps1
mutants yield 18 ± 9% prezygotes. Thus, unlike fus1 and fus2
mutants, matings between fps1
mutants may exhibit
some suppression of the fusion defect. In contrast, fps1
mutants mated to fus1 fus2 mutants at a much lower efficiency compared with wild type, a decrease of ~25-fold
(Table II).
Mutants
Glycerol Accumulation in fps1 Mutants Is Correlated
with Defective Cell Fusion
Fps1p is a member of the major intrinsic protein family, an
evolutionarily conserved family of channel proteins that
transport small molecules (for review see Reizer et al.,
1993). It is most similar to the Escherichia coli glycerol facilitator, GlpF, and has been proposed to function as a
glycerol transporter in yeast (Luyten et al., 1995
). Consistent with its role as a glycerol transporter, fps1
mutants
contain approximately twofold more intracellular glycerol
than wild-type strains, suggesting that they are defective in
glycerol efflux (Luyten et al., 1995
). We observed a similar
increase in intracellular glycerol in our fps1
mutant (Table III).
Table III.
Intracellular Glycerol Concentrations in fps1 |
To ascertain whether the defect in cell fusion of fps1
strains results from elevated levels of intracellular glycerol, we determined whether reducing intracellular glycerol levels restored cell fusion. We reduced intracellular
glycerol levels by deleting the GPD1 gene, which encodes
the NADH-dependent glycerol-3-phosphate dehydrogenase (GPDH) required for the first step of glycerol biosynthesis (Gancedo et al., 1968
). Although yeast cells have a
second NADH-dependent GPDH (Gpd2p), Gpd1p is responsible for ~95% of the NADH-dependent GPDH activity in the cell (Albertyn et al., 1994
b). We found that the
level of intracellular glycerol in the fps1
GPD1 mutant
was 2.1-fold higher than in the wild-type strain (FPS1
GPD1) and was only 1.1-fold higher than wild-type strains
in the fps1
gpd1
mutant (Table III).
We next determined whether this decrease in intracellular glycerol level in fps1 gpd1
mutants suppressed the
mating defect. We found that deletion of GPD1 suppressed the mating defect of fps1
strains, whereas deletion of GPD1 in FPS1 strains had no effect on mating (Fig.
4). To determine if gpd1
suppressed the cell fusion defect
of fps1
strains, we microscopically assayed the double
mutant for accumulation of prezygotes: deletion of GPD1
in an fps1
mutant decreased the number of prezygotes
from 42% to 14% (Table IV). Again, we saw little effect of
the GPD1 deletion in an FPS1 strain. Thus, deletion of
GPD1 partially restored both intracellular glycerol levels
and mating to an fps1
mutant.
Table IV.
Effect of Altering GPD1 Expression on the Defect in
Cell Fusion of fps1 |
If increased intracellular glycerol is responsible for the
cell fusion defect of fps1 mutants, we hypothesized that
this defect would be exacerbated by further increasing intracellular glycerol levels. Overexpression of GPD1 in
fps1
strains raises intracellular glycerol levels (Luyten et
al., 1995
). We observed that overexpression of GPD1 in
fps1
mutant strain JP200 led to a 1.8-fold increase in glycerol levels in comparison with the fps1
strain carrying a
control plasmid (JP401) (Table III, seventh and eighth
lines). We observed that the glycerol-overproducing strain
JP200 exhibited an enhanced defect in cell fusion, producing 84% prezygotes in comparison with strain JP401,
which yielded 20% prezygotes when mated to a wild-type
strain (Table IV). No significant increase in prezygotes
was observed in the FPS1 strain carrying the GPD1 plasmid. We also examined the ability of these strains to mate
with partners that overexpress GPD1. Once again, we observed that mating with JP200 yielded nearly fivefold
more prezygotes than JP401: 33% vs 7% (Table IV, second column, seventh and eight lines). Overexpression of
GPD1 increased the levels of intracellular glycerol and exacerbated the defect in cell fusion of fps1
mutants. We
conclude that the defect in cell fusion in fps1
mutants
correlates with increased levels of intracellular glycerol.
In contrast with what was observed when GPD1 was
overexpressed in the fps1 mutant, we found that when
GPD1 was overexpressed in the mating partner there was
a decrease in prezygotes formed, from 20 to 7% for the
fps1
strain carrying the control plasmid (Table IV, seventh line) and from 84 to 33% for the fps1
strain carrying
the GPD1 plasmid (Table IV, eighth line). One explanation for this improvement in fusion is that overexpression of
GPD1 in the mating partner led to increased extracellular
glycerol, which restored osmotic balance to the fps1
mutant. Consistent with the idea that glycerol produced by
the mating partner may osmotically stabilize the fps1
mutant, we found that deletion of GPD1 in the partner
somewhat exacerbated the defect of the fps1
mutant.
fps1
mutants mated to GPD1 strains produced 42% prezygotes, which increased to 55% when mated to a
gpd1
partner (Table IV, third line). Additionally, fps1
gpd1
mutants mated to GPD1 strains produced 14%
prezygotes, which increased to 26% when mated to a
gpd1
partner (Table IV, fourth line).
High Osmolarity Partially Suppresses the Cell Fusion
Defect of fps1 Mutants
The correlation between increased intracellular glycerol
levels and a defect in cell fusion suggests at least two possible causes of the cell fusion defect. Intracellular glycerol itself may inhibit cell fusion. Another possibility is that an
imbalance between intracellular and extracellular solute
levels inhibits fusion. To distinguish between these possibilities, we determined whether restoration of osmotic balance by 1 M sorbitol could suppress the cell fusion defect
of fps1 mutants. If elevated intracellular glycerol per se
inhibits cell fusion, then 1 M sorbitol should either have no
effect or may exacerbate the cell fusion defect of fps1
mutants, since 1 M sorbitol induces GPD1 expression
(Hirayama et al., 1995
). If the mating defect is due to osmotic imbalance, then mating in the presence of 1 M sorbitol may suppress the defect. We observed the latter: the
mating defect of fps1
mutants was partially alleviated by
1 M sorbitol (Fig. 4 c). Because this mating assay involves
mating to a fus1 fus2 strain, it was possible that the improvement in mating resulted from an effect of the 1 M
sorbitol on the fus1 fus2 partner rather than on the fps1
partner. To address this issue, we examined the effect of
1 M sorbitol on fps1
mutants mated to a wild-type strain.
We found that the fusion defect was suppressed in the
presence of 1 M sorbitol; the number of prezygotes declined from 37 to 10% (Table V). Moreover, the presence
of 1 M sorbitol did not improve mating by fus1 fus2 mutants (data not shown) or result in decreased levels of
prezygotes (Table V). We conclude that the mating defect
of fps1
mutants can be suppressed by restoring osmotic
balance to these cells.
Table V.
1 M Sorbitol Partially Suppresses the Cell Fusion
Defect of fps1 |
Additional evidence that the cell fusion defect of fps1
mutants is not due to an absolute increase in glycerol but
rather to a difference between intracellular and extracellular glycerol comes from comparing the behavior of wild-type cells overexpressing GPD1 and fps1
mutants. Both
of these strains contained approximately twofold more intracellular glycerol than wild-type strains containing vector alone (Table III, sixth and seventh lines), yet the fps1
mutant exhibited a more severe cell fusion defect (Table
IV, sixth and seventh lines). One explanation for this difference is that the wild-type cells, which contain a higher
level of glycerol as a result of overexpression of GPD1,
can efficiently release this glycerol, so that extracellular
glycerol levels also increase. The fps1
mutant, which releases glycerol inefficiently, has higher intracellular glycerol and decreased extracellular glycerol (Luyten et al.,
1995
). These data suggest that osmotic imbalance, rather than the absolute level of intracellular glycerol, accounts
for the cell fusion defect of fps1
mutants.
Activated Alleles of PKC1 Inhibit Cell Fusion
The protein kinase C pathway is induced by conditions in
which intracellular solute is higher than extracellular solute (Davenport et al., 1995). Because fps1
mutants have
higher intracellular glycerol concentrations than wild-type
cells, we wondered whether the cell fusion defect of fps1
mutants was influenced or mediated by the PKC1 pathway. If activation of the PKC1 pathway is responsible for
the defect in cell fusion of fps1
mutants, then activation of Pkc1p in an otherwise wild-type background should
give a similar defect. We therefore examined the effect of
expressing an activated allele of PKC1 on mating and cell
fusion.
A CEN-ARS plasmid containing such an allele of PKC1
(PKC1-R398P) was introduced into a wild-type strain
(IH3196) to generate strain JP300. PKC1-R398P alters the
pseudosubstrate binding site of Pkc1p, creating a dominant, activated allele (Nonaka et al., 1995). Expression of
this allele under control of its own promoter had no detectable effect on cell viability (data not shown). The activated allele did, however, result in a mating defect similar to that of the fps1
mutant (Fig. 5 a). Assays of cell cycle
arrest and shmoo formation indicated that JP300 responded
normally to pheromone (data not shown). Furthermore,
JP300 produced pheromone normally, as assayed by halo
formation. When JP300 was mated to a wild-type strain,
prezygotes accumulated. JP300 exhibited normal partnership, indicating that its defect did not result from inability to respond or adhere to its partner, but rather to a defect
in cell fusion (Table VI, first and second lines; Fig. 1 e). We
conclude that activation of the PKC1 pathway is sufficient
to cause a defect in cell fusion. We were able to activate
the PKC1 pathway at the time of mating by expressing an
activated allele of PKC1 (PKC1-R398A) under control of
the GAL1 promoter (Watanabe et al., 1994
). Expression of this activated allele when cells were mating resulted in a mating defect, whereas no significant mating defect was
seen when cells expressed wild-type PKC1 or PKC1-K853R,
an allele mutated in the kinase domain (Fig. 5 b). Hence, a
constitutively active allele of Pkc1p expressed during mating causes a mating defect.
Table VI. The PKC1-R398P Allele Causes a Defect in Cell Fusion |
Pkc1p Does Not Act through Fps1p to Inhibit Cell Fusion
Activated alleles of Pkc1p and loss of function of FPS1
caused quantitatively very similar cell fusion defects (Table VI), raising the possibility that Pkc1p negatively regulates cell fusion by inhibiting Fps1p. If the only target of
Pkc1p for inhibiting cell fusion were Fps1p, then the activated allele of Pkc1p should not exacerbate the defect in
fusion of an fps1 strain. However, we found that the
PKC1-R398P allele increased the defect in cell fusion of
the fps1
strain (Table VI, third and fourth lines). In addition, if Pkc1p negatively regulates Fps1p, then deletion of
GPD1 should suppress the PKC1-R398P allele, as it partially suppressed an fps1
mutant (Fig. 4 a; Table IV). Deletion of GPD1 did not, however, suppress the PKC1-R398P allele, suggesting that Pkc1p acts downstream or
parallel to glycerol accumulation (Table VI, second and
sixth lines; Figs. 5 and 7).
fps1 mpk1
Double Mutants Require Osmotic
Support for Viability
One explanation for the defect in fps1 mutants is that increased activity of the PKC1 pathway accounts, at least in
part, for the defect in cell fusion. Additional evidence that
this pathway is active came from analysis of a mutant defective in both FPS1 and MPK1, which codes for the MAP
kinase regulated by Pkc1p (Lee et al., 1993
; Torres et al.,
1991
). Although both fps1
and mpk1
mutants grow normally at 25°C (Van Aelst et al., 1994; Lee et al., 1993
), we
were unable to obtain double mutant segregants when
spores were germinated at 25°C on YEPD in eight tetrads
analyzed. The fps1
mpk1
double mutant was obtained,
however, when spores were germinated in the presence of
1 M sorbitol: 14 double mutants were obtained from 18 tetrads; all were unable to grow on YEPD lacking sorbitol.
The remaining 58 spores, none of which were double mutants, grew normally on unsupplemented YEPD at 25°C
(Fig. 6). The inviability of the fps1
mpk1
strain demonstrates that the fps1
mutant, in the absence of osmotic
stabilizing agents, requires MPK1 for viability, and thus
that Mpk1p is active in fps1
mutants.
Mutations in FUS1 and FUS2 Are Not Suppressed by Altering Osmotic Conditions
We have carried out a variety of analyses to determine
whether the cell fusion defect of fus1 and fus2 mutants has
a similar basis to that of fps1 mutants. First, we determined whether deletion of GPD1 restored mating and cell
fusion to fus1 or fus2 mutants. Although deletion of GPD1
improved mating in fps1 mutants, it did not improve matings of fus1 and fus2 mutants (Fig. 4, a and b). Similarly,
microscopic examination of mating mixes did not reveal a
significant change in prezygote accumulation in fus1 gpd1
or fus2 gpd1 double mutants compared with fus1 GPD1 or
fus2 GPD1 strains. Moreover, overexpression of GPD1
did not exacerbate the cell fusion defect of fus1 mutants
(data not shown). We conclude that the cell fusion defect
of fus1 and fus2 strains is unlikely to be due to an accumulation of intracellular glycerol. Additional evidence that
the defect in cell fusion in fus1 and fus2 mutants differs
from that in fps1
mutants came from analyzing the ability of mutants to mate in the presence of 1 M sorbitol. Accumulation of prezygotes was not detectably altered for
fus1
mutants by mating in the presence of 1 M sorbitol,
whereas fus2
and fus1 fus2 strains showed an exacerbation of the fusion defect, a result opposite to that seen with
fps1
mutants (Table VI). Thus, fus1 and fus2 mutants differ from fps1
mutants in their genetic interactions with
GPD1 and in their response to 1 M sorbitol. We conclude
that it is unlikely that the cell fusion defect associated with
mutations in FUS1 and FUS2 is due to an inability to
maintain osmotic stability.
During conjugation, haploid cells of opposite mating type reorganize their cell walls to allow cell fusion. The signals that control cell wall degradation and membrane fusion and the machinery that mediates these processes are not known. We have found that the FPS1 gene, which codes for a glycerol transporter, is essential for efficient cell fusion. We identified fps1 mutants based on their defect in mating to an enfeebled (fus1 fus2) mating partner and showed that they are specifically defective in cell fusion: they accumulated prezygotes during mating but were normal for pheromone signaling. The fusion defect of fps1 mutants correlates with their increased level of intracellular glycerol relative to wild-type cells: the defect was partially suppressed by reducing intracellular glycerol and exacerbated by further increasing intracellular glycerol levels. The defect appears to result from osmotic imbalance rather than a high level of glycerol per se since extracellular 1 M sorbitol partially relieved the fusion defect. We propose that, during mating, cells monitor their osmotic state before committing to breaking down and remodeling their cell wall. In particular, under conditions of osmotic imbalance, such as in strains lacking Fps1p, cells interrupt cell wall breakdown. The fps1 mutant thus reveals a checkpoint for cell wall breakdown during mating.
Because fps1 mutants accumulate a high level of intracellular glycerol, we reasoned that they experience a situation analogous to that of wild-type cells exposed to hypotonic conditions, which would lead to activation of the
PKC1 pathway (Davenport et al., 1995; Kamada et al.,
1995
). We therefore anticipated that constitutive activation of the PKC1 pathway, e.g., due to alteration of Pkc1p itself, would cause a fusion defect similar to that of fps1
mutants. This prediction was borne out. Additional analyses of the constitutively activated PKC1 mutant indicate
that Pkc1p functions downstream of Fps1p and glycerol
accumulation. We suggest that activation of Pkc1p couples
sensation of hypoosmotic conditions to inhibition of cell
fusion. Other genes required for cell fusion, in particular FUS1 and FUS2, do not appear to participate in this osmolarity checkpoint, as mutants defective in these genes are
not influenced by osmolarity.
Osmotic Balance Governs Cell Fusion
During vegetative growth, fps1 mutants accumulate approximately twofold more intracellular glycerol than do
wild-type cells, which does not cause any apparent growth
defect (Van Aelst et al., 1991; Philips, J., unpublished observations). This increase in intracellular glycerol causes a
defect in cell fusion during mating. In principle, Fps1p,
which contains six putative membrane-spanning domains
(Van Aelst et al., 1991
), might play a role in mating that is
distinct from its role in transport. Our analyses, however, indicate that its role in mating is a consequence of its function in glycerol transport and that, in its absence, the process of cell wall breakdown is inhibited. Our observations
indicate that glycerol functions as an osmolyte during the
mating process rather than being directly involved, e.g., in
signaling between the mating partners. This conclusion
comes from our observations that glycerol per se is not required during mating since the defect of fps1 mutants can
be suppressed by extracellular sorbitol or by deleting GPD1, which reduces intracellular glycerol levels. We
thus favor the hypothesis that mating cells of yeast are exceptionally sensitive to osmotic disequilibrium before undergoing the potentially lethal morphogenetic changes required for cell fusion. Cells lacking FPS1 sense that they
are not osmotically balanced and respond by blocking cell
wall breakdown. In other words, cells possess a checkpoint
that ensures that they do not degrade their cell wall under
hypoosmotic conditions.
It is unclear whether the osmotic imbalance perceived
by the fps1 mutant results from a difference between the
mutant and its environment or a disparity between mating
partners. If the osmolarity between mating partners were
monitored, we might expect that an a fps1 and fps1 mutant mated to each other would form zygotes at normal
frequency. Although the number of prezygotes does not
return to wild-type levels when fps1
mutants are mated with fps1
mutants, the reduction in the number of prezygotes compared with fps1
mutants mated to wild type
may be significant (Table II), indicating that a difference
in osmolarity between mating partners may be monitored.
The PKC1 Pathway May Mediate the Cell Fusion Checkpoint
Support for the hypothesis that osmosensing pathways
regulate cell fusion was obtained by analyses of the PKC1
pathway. This pathway is required for maintenance of cell
integrity and cell wall construction (for review see Errede
and Levin, 1993) and is induced when cells are subjected
to conditions, such as hypoosmotic shock, that threaten
cell integrity (Davenport et al., 1995
; Kamada et al., 1995
).
We found that activation of Pkc1p using a constitutively activated allele of PKC1 led to a fusion defect essentially
identical to that of fps1 mutants: PKC1-R398P and fps1
strains accumulated prezygotes that were morphologically
indistinguishable from each other.
One possibility is that Pkc1p blocks cell fusion by inhibiting FPS1. Two experiments suggest that this is not the
case. First, the PKC1-R398P mutation has a more severe
phenotype in an fps1 mutant than in a wild-type strain.
Additionally, cell fusion is not restored to the PKC1-R398P mutant by reducing intracellular glycerol levels due
to deletion of GPD1. These data, in conjunction with the
previously demonstrated role of Pkc1p in responding to hypoosmotic shock, lead us to propose that Pkc1p lies
downstream of Fps1p and Gpd1p and functions as a part
of a checkpoint to monitor osmotic balance during mating
(Fig. 7).
If activation of the PKC1 pathway is responsible for the
defect in cell fusion of fps1 mutants, then Pkc1p should
be active in fps1
mutants and deletion of pkc1 should
suppress the mating defect. Because 1 M sorbitol suppresses the mating defect of fps1
mutants, and pkc1
mutants require 1 M sorbitol for viability (Levin and Bartlett-Heubusch, 1992
; Paravicini et al., 1992
), it is impossible to ask whether pkc1
suppresses the mating defect. However, evidence that the PKC1 pathway is active in
fps1 mutants comes from the analysis of mutants defective
in both Fps1p and Mpk1p, the MAP kinase in the PKC1
pathway (Lee et al., 1993
; Torres et al., 1991
). We have observed that fps1
mpk1
mutants are inviable unless supported osmotically, indicating that Mpk1p is active in the
fps1 mutants. The FPS1 mpk1 strains are inviable only at high temperatures (37°C) (Lee et al., 1993
), whereas the
fps1 mpk1 strains are also inviable at low temperatures
(25°C). One explanation for the inviability of the fps1
mpk1 double mutant is that fps1 mutants depend upon
Mpk1 activity to respond to the osmotic imbalance caused
by their inefficient release of glycerol.
At least two different models could explain the role of PKC1 in cell fusion. According to one view, Pkc1p negatively regulates cell fusion as part of a checkpoint to ensure that cells are not osmotically vulnerable before fusion (Fig. 7 a). In this case, Pkc1p is used only under conditions, such as hypoosmotic shock, that make cell fusion particularly dangerous to cell integrity. According to a second view, Pkc1p constitutively inhibits cell fusion, so that cell fusion occurs only when Pkc1p is antagonized (Fig. 7 b). According to this model, Pkc1p is a key regulator of cell fusion, inhibiting fusion until a signal turns it off. In this case, the cell fusion defect of fps1 mutants results from activation of the osmotic sensing pathway, which prevents the Pkc1p-dependent inhibition from being relieved during cell fusion.
Previous work has shown that Mpk1p is activated in response to mating pheromone (Errede et al., 1995; Zarzov
et al., 1996
), presumably because of activation of Pkc1p,
and is necessary for viability of yeast cells under these conditions (Errede et al., 1995
). Our observations indicate, in
contrast, that activation of Pkc1p inhibits a late step in
mating. To explain the apparently paradoxical actions of
the PKC1 pathway in mating, we suggest, as in the second
model above, that Pkc1p is a negative regulator of cell fusion (Fig. 7 b) that, during normal mating, is activated and
then subsequently inhibited. We propose that Pkc1p is
first activated in response to pheromone and thereby inhibits cell wall degradation during initial stages of pheromone response and projection formation. When mating
partners come in contact with each other, they generate a
mechanical force as the cell walls become irreversibly adhered to each other, which generates a signal to turn off
the PKC1 pathway. We suggest that the signal to turn off
the PKC1 pathway is a mechanical signal. It has been previously suggested that the PKC1 pathway responds to
membrane stretch due to various stimuli, such as low external osmolarity, high temperature, and drug-induced
membrane stretch (Kamada et al., 1995
).
Proteins Required for Cell Fusion: Regulators and Machinery
Genes required for cell fusion have been identified by a
number of different strategies (Trueheart et al., 1987; McCaffrey et al., 1987
; Elion et al., 1990
, 1995
; Kurihara et al.,
1994
; Elia and Marsh, 1996
). The studies presented here
allow us to make a distinction between proteins that regulate the process of fusion (such as Pkc1p or, more indirectly, Fps1p) and potential participants in fusion itself
(such as Fus1p and Fus2p). This distinction comes from
the following observations. Fps1p is not directly required
for cell fusion: its requirement can be relieved by deleting
GPD1 or by mating in the presence of high osmolarity. Rather, Fps1p is required to maintain osmotic balance and
thereby avoid tripping a checkpoint that inhibits cell fusion. We have shown here that Pkc1p also governs fusion:
cell expressing activated forms are defective in fusion presumably because they activate the cell fusion checkpoint.
It is not clear whether Fus1p and Fus2p are regulators of
fusion or play more direct roles in this process, or whether
they are targets for the inhibition mediated by the PKC1
pathway. The fusion defect of fus1 and fus2 mutants is not
influenced by deletion of GPD1 or osmotic stabilizers, and
thus it is unlikely that these mutants exhibit a fusion defect by evoking the osmotic checkpoint.
Our findings on fusion during yeast mating may have
implications for other examples of cell fusion. Maintenance
of cell integrity is essential for all cell fusion processes. We
therefore expect that processes such as sperm-egg and
myoblast fusion may also be regulated by osmolarity. It
would be striking if protein kinase C were involved in
monitoring osmotic balance during sperm-egg or myoblast fusion. Eyster and McFarland (1995) have, in fact, reported that endogenous modulators of protein kinase C
can regulate myogenesis. Perhaps activated versions of
protein kinase C could block sperm-egg or myoblast fusion as they block fusion between mating partners in yeast.
The targets of the inhibition triggered by the osmolarity-induced checkpoint in yeast remain to be determined. We
are seeking to identify such targets by genetic strategies. These proteins might be directly involved in cell fusion
during mating and illuminate mechanisms relevant to other
cell fusion events.
Received for publication 21 March 1997 and in revised form 17 June 1997.
Please address all correspondence to Ira Herskowitz, Department of Biochemistry and Biophysics, Programs in Genetics and Cell Biology, University of California, San Francisco, San Francisco, CA 94143-0448. Tel.: (415) 476-4985. Fax: (415) 502-5145. e-mail: philips{at}socrates.ucsf.eduWe thank members of our laboratory and S. Doberstein for valuable discussion, S. Hohmann, D. Levin, Y. Takai, J. Chenevert, and J. Whistler for providing plasmids; and S. Hohmann for communicating results before publication. We also thank R. Tabtiang, L. Huang, and F. Banuett for helpful comments on the manuscript, as well as C. Boone for suggesting the RAS2-GFP plasmid.
This work was supported by Research and Program Project Grants from the National Institutes of Health (NIH) to I. Herskowitz. J. Philips was supported by a Medical Scientist Training Program grant from the NIH, supplemented by the Sussman Fund, the Markey Program in Biological Sciences, and the Herbert W. Boyer Fund.
DAPI, 4,6-diamidino-2-phenylindole;
GFP, green fluorescent protein;
MAP, mitogen-activated protein;
ORF, open reading frame;
YEPD, yeast extract/peptone/dextrose.
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