1208 | C. A. Ydenberg et al. Molecular Biology of the Cell
MBoC | ARTICLE
Cdc42p and Fus2p act together late in yeast cell
Casey A. Ydenberg*, Richard A. Stein, and Mark D. Rose
Department of Molecular Biology, Princeton University, Princeton, NJ 08544
ABSTRACT Cell fusion is the key event of fertilization that gives rise to the diploid zygote
and is a nearly universal aspect of eukaryotic biology. In the yeast Saccharomyces cerevisiae,
several mutants have been identified that are defective for cell fusion, and yet the molecular
mechanism of this process remains obscure. One obstacle has been that genetic screens have
mainly focused on mating-specific factors, whereas the process likely involves housekeeping
proteins as well. Here we implicate Cdc42p, an essential protein with roles in multiple aspects
of morphogenesis, as a core component of the yeast cell fusion pathway. We identify a point
mutant in the Rho-insert domain of CDC42, called cdc42-138, which is specifically defective
in cell fusion. The cell fusion defect is not a secondary consequence of ineffective signaling or
polarization. Genetic and morphological data show that Cdc42p acts at a late stage in cell
fusion in concert with a key cell fusion regulator, Fus2p, which contains a Dbl-homology do-
main. We find that Fus2p binds specifically with activated Cdc42p, and binding is blocked by
the cdc42-138 mutation. Thus, in addition to signaling and morphogenetic roles in mating,
Cdc42p plays a role late in cell fusion via activation of Fus2p.
Cell fusion is widespread in the eukaryotic kingdom. In addition to
the familiar fertilization events necessary to restore the diploid state
after meiosis (Primakoff and Myles, 2002), fusion occurs throughout
the development of many multicellular organisms, including the for-
mation of muscle, bone, and the mammalian placenta (Vignery,
2000; Potgens et al., 2002; Horsley and Pavlath, 2004; Chen and
Olson, 2005). In contrast to virus–cell fusion and fusion between in-
tracellular membranes, the molecular mechanism of cell fusion is
not well understood.
Cell fusion in budding yeast occurs during mating events between
haploid cells (Marsh and Rose, 1997; Ydenberg and Rose, 2008).
Yeast haploid cells of the a and α mating types each secrete phero-
mones sensed by a receptor expressed by the opposite cell type.
This induces characteristic gene expression and developmental
changes resulting in polarized growth or “shmooing” in the direction
of the mating partner, followed by cell fusion and nuclear fusion. Al-
though numerous mutants have been identified that block fusion,
many of them are now known to have prior defects in pheromone
signaling and polarization, suggesting that the cell fusion defect is a
secondary consequence of inefficient completion of earlier mating
events (Brizzio et al., 1996; Dorer et al., 1997; Paterson et al., 2008).
In contrast, four proteins have been identified that appear to
play a direct role in yeast cell fusion without having detectable de-
fects in earlier steps of mating. Mutations in FUS1, FUS2, and
RVS161 block the removal of cell wall material between mating pr-
ezygotes (Trueheart et al., 1987; Trueheart and Fink, 1989; Brizzio
et al., 1998), whereas PRM1 mutants block fusion after cell wall re-
moval but before plasma membrane fusion (Heiman and Walter,
2000). The molecular function of these proteins is not known, but it
may involve the trafficking and/or fusion of mating-specific vesicles
that are visualized at the cell fusion zone (Gammie et al., 1998).
These vesicles are delocalized in fus1 mutants, as well as in polariza-
tion mutants (e.g., bni1 and spa2). The vesicles are properly local-
ized in fus2 and rvs161 mutants, suggesting that these mutations
affect later stages of cell fusion than fus1. The behavior of the vesi-
cles in prm1 mutant zygotes has not been reported.
Fus2p and Rvs161p form a complex that is transported to the
tips of fusing cells (Brizzio et al., 1998; Paterson et al., 2008; Sheltzer
Daniel J. Lew
Received: Aug 26, 2011
Revised: Jan 27, 2012
Accepted: Jan 31, 2012
This article was published online ahead of print in MBoC in Press (http://www
.molbiolcell.org/cgi/doi/10.1091/mbc.E11-08-0723) on February 9, 2012.
*Present address: Rosenstiel Center for Biomedical Research, Brandeis University,
Waltham, MA 02454.
Address correspondence to: Mark D. Rose (firstname.lastname@example.org).
Abbreviations used: BAR, Bin1, amphiphysin, Rvs; GAP, GTPase activating
protein; GEF, GTP exchange factor.
© 2012 Ydenberg et al. This article is distributed by The American Society for Cell
Biology under license from the author(s). Two months after publication it is avail-
able to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported
Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
“ASCB®,“ “The American Society for Cell Biology®,” and “Molecular Biology of
the Cell®” are registered trademarks of The American Society of Cell Biology.
Volume 23 April 1, 2012 Cdc42 and Fus2p in yeast cell fusion | 1209
between the mating partners remains intact) and partially unfused
zygotes (in which there is a gap in the septum that fails to open
completely). Both types of unfused zygotes are indicative of the
defect in Fus2p function. The fus2ΔDbl mutant produced a large
number of unfused and partially unfused zygotes (Figure 1E). Again,
the phenotype is not as strong as a null fus2Δ strain, in which the
majority of zygotes are fully unfused (compare to Figure 2B). These
results indicate that the fus2ΔDbl allele is hypomorphic and that this
domain is critically important but not absolutely required for Fus2p’s
cell fusion activity.
Mutations in the Rho-insert domain are defective
in cell fusion
The interaction between activated Cdc42p and Fus2p suggested
that Cdc42p may play a direct, specific role in cell fusion. Cdc42p
has at least two defined roles in the mating pathway already: it is
required for response to pheromone by activation of the PAK Ste20p
(Moskow et al., 2000) and for defining the site of polarization of the
shmoo (Nern and Arkowitz, 2000). One mutant allele of cdc42 that
exhibits a defect in cell fusion has been previously characterized,
cdc42-V36M (Barale et al., 2006). However, given the many poten-
tial roles for Cdc42p in mating, the specific defect of cdc43-V36M
remained unclear. To look for additional alleles of CDC42 that might
be specifically defective for Fus2p-mediated cell fusion, we used an
and Rose, 2009). Rvs161p is a BAR domain–
containing protein that plays a separate role
in endocytosis (Crouzet et al., 1991) by sta-
bilizing membrane curvature (Peter et al.,
2004). Fus2p contains a Dbl-homology
domain, suggesting that it interacts with a
Rho-type G protein. In support of this, a
large-scale two-hybrid screen identified an
interaction between Fus2p and Cdc42p in
the GTP-bound form (Nelson et al., 2004).
Yeast Cdc42p plays multiple essential
roles during growth and morphogenesis
(Johnson, 1999; Richman et al., 1999;
Kozminski et al., 2000; Adamo et al., 2001),
including during mating (Simon et al., 1995;
Zhao et al., 1995). In late G1, Cdc42p estab-
lishes polarized growth toward a specific
area of the cell surface to grow a new bud.
During mating, the budding axis is overrid-
den by cues that direct Cdc42p activity
along the axis of the pheromone gradient
(Nern and Arkowitz, 1998, 1999). Cdc42p
has been implicated in cell fusion (Barale
et al., 2006), but the stage at which it acts
and its molecular functions are not clear.
Here we present evidence that Cdc42 has
multiple roles in cell fusion, that Fus2p is a
direct effector of Cdc42p signaling, and that
Cdc42p and Fus2p act together late in the
cell fusion pathway. Our results have impli-
cations for the molecular mechanism of
yeast cell fusion, the function of an unchar-
acterized domain in Cdc42p, and conserva-
tion between fungal and metazoan cell fu-
The Fus2p Dbl-homology domain
promotes cell fusion
Fus2p contains a Dbl-homology domain (Paterson et al., 2008), sug-
gesting that it may act as a guanine-nucleotide exchange factor for
one of the six yeast Rho proteins. Conversely, a two-hybrid study
suggested that Fus2p interacted with Cdc42p-Q61L, a GTP-locked
allele (Nelson et al., 2004). These results suggested that Fus2p may
be an effector for Cdc42p, since it interacted exclusively with the
active form of the GTPase.
To assess the importance of the Dbl-homology domain in vivo,
we examined the phenotype caused by a deletion of this domain.
Fus2p-ΔDbl–green fluorescent protein (GFP) localized to the nucleus
in mitotic cells and at the shmoo tip in pheromone-arrested cells,
similar to wild-type Fus2p-GFP (Patterson et al., 2008). Therefore
the Dbl-homology domain is required for neither localization in mi-
totic and pheromone-arrested cells nor the regulated change in lo-
calization (Figure 1A). Both proteins were detected at similar levels
(Figure 1B). When this deletion was integrated into the genome to
replace the endogenous FUS2 locus, the resulting strain was signifi-
cantly defective for the formation of diploids, although not quite as
defective as a complete deletion mutant (fus2Δ; Figure 1C). To ex-
amine the morphology of the zygotes formed by this strain, wild-
type and fus2-ΔDbl strains were mated to a fus2Δ tester strain and
stained with FM4-64 to visualize the plasma membrane (Figure 1D).
We then counted the fully unfused zygotes (in which the septum
FIGURE 1: The Dbl-homology domain is required for cell fusion. (A) Fus2p-ΔDbl-GFP localizes
to the shmoo tip. Cells (MY9181) transformed with a plasmid expressing either Fus2p-GFP
(pMR5469) or Fus2p-ΔDbl-GFP (pMR5883) were treated with pheromone as indicated for 2 h
and examined. (B) Fus2p-ΔDbl-GFP is expressed at similar levels as the wild-type Fus2p-GFP
protein. The same strains as in A were analyzed by Western blot using anti-GFP. (C) Diploid
formation is defective in fus2-ΔDbl. Wild type (DDY1300), fus2Δ (MY10016), and fus2-ΔDbl
(MY10933) were mated to fus1 fus2 (MY10798) for 4 h at 30°C. (D, E) Cell fusion is defective in
fus2-ΔDbl. The same strains as in C were mated to fus2 (MY10797) for 2 h, washed into azide,
and stained with FM4-64 to label the plasma membrane. (D) Examples of fusion-defective
zygotes. Wild-type zygote (DDY1300xMY10797), partial Fus− zygote (MY10933xMY10797), and
full Fus− zygote (MY10933xMY10797). (E) Percentage of zygotes of each morphology produced
in matings with the indicated genotype. Gray bars, partial Fus− zygotes; black bars, full Fus−
zygotes. n ≥ 133 zygotes imaged in two independent experiments.
1210 | C. A. Ydenberg et al. Molecular Biology of the Cell
alanine-scanning collection created previously (Kozminski et al.,
2000). Twenty mutant alleles, annotated as wild type for growth,
were mated to a wild-type strain or a fusion-defective tester strain
and examined for the ability to form diploids by a plate mating as-
say (Figure 2A and Supplemental Figure S1). Because the fus2Δ
single mutant exhibits an obvious defect when mated against a
compromised partner (Figure 2A) but not when mated to the wild-
type strain, we reasoned that this approach would allow us to iden-
tify alleles that were specifically defective in cell fusion. One allele,
cdc42-127, did not mate with either the fus1 fus2 or the wild-type
partner, indicating that it has a sterile (Ste−) phenotype (Figure 2A).
This mutation, V33A, lies within the Switch I region and was not ana-
lyzed further. Two alleles, cdc42-137 and -138, exhibited significant
mating defects with the fus1 fus2 tester strain but not with the wild-
type strain, suggesting that they represent cell fusion–specific (Fus−)
alleles (Figure 2B). The two mutations, D121A and D122A, respec-
tively, lie within the Rho-insert domain (Figure 2C) and alter con-
secutive negatively charged residues.
To confirm that the mating defect against the compromised
strain was due to reduced cell fusion efficiency, we quantified zy-
gote morphology in these mutants. For these experiments, we used
a bilateral mating configuration in which both partners contain the
same mutation of interest. Under these conditions, wild-type zy-
gotes typically have no visible septum between the mating partners,
whereas cdc42-137 and 138 zygotes frequently had all or part of the
septum intact, indicating that they had not fully fused (Figure 2B;
44% of cdc42-137 zygotes and 75% of cdc42-138 zygotes were fu-
sion defective compared with 8% of wild-type zygotes).
Given the location of these alleles within the Rho-insert domain,
we examined two other mutations constructed in the alanine-scan-
ning collection that altered residues in this region but that had not
exhibited a defect in the plate mating screen. Both mutants showed
a significant cell fusion defect; cdc42-105 (E127A, K128A) produced
20% fusion defective zygotes, and cdc42-107 (R131A, R133A,
R135A) produced 28% fusion defective zygotes (Figure 1C). These
defects were not as strong as those for cdc42-137 and cdc42-138,
explaining why they were missed in the less sensitive plate mating
assay. Together the data indicate that the Rho-insert domain of
Cdc42p, which had not previously been shown to have a specific
function, plays a significant and unexpected role in cell fusion. To
further characterize the defect in cell fusion, we focused on the more
potent cdc42-138 allele.
The fusion defect in cdc42-138 is not due to defective
Cdc42p plays a known role in the pheromone-signaling pathway
(Simon et al., 1995; Zhao et al., 1995). One possibility was that the
defect in cell fusion is simply due to partly compromised signaling
(Brizzio et al., 1996). To isolate Cdc42p from any effects on the
pheromone pathway, we used a STE20-ΔCRIB mutation (Lamson
et al., 2002), which removes the Cdc42p-binding domain in Ste20p,
thereby rendering the protein and the pheromone response
FIGURE 2: Mutants in the Cdc42p Rho-insert domain are defective
for cell fusion. (A, B) A screen for cell fusion–specific alleles of Cdc42p.
Wild type (WT; DDY1300), fus2 (MY10016), and 20 alleles from a
Cdc42p alanine-scanning set were mated to an anti-WT strain (JY431;
A) or an anti– fus1 fus2 strain (JY429; B). Shown are the alleles
discussed in the text. Note that the matings against JY431 are more
dilute than against JY429, so that colony formation against the two
testers cannot be compared with one another. Images are
representative of two independent experiments. (B) Microscopic
examination of zygotes. Bilateral matings of the indicated genotype
were mated for 2 h at 30°C, washed into azide, and stained with
FM4-64. Shown is the percentage of zygotes of each morphology in
the indicated genotypes (WT, DDY1300 × DDY1301; fus2, MY10016 ×
MY10303; cdc42-138, DDY1354 × DDY1355; cdc42-V36M, MY10547 ×
MY10554; see Kozminski et al., 2000, for other strain numbers). Gray
bars, partial Fus− zygotes; black bars, full Fus− zygotes. n ≥ 122
zygotes imaged in at least two independent experiments.
(C) Structure of Cdc42p indicating mutations affecting cell fusion.
Mutations are mapped on a space-filling model of human Cdc42p
(Brookhaven Protein Database, accession number 1AN0). The
Rho-insert domain is shown in yellow, with mutations indicated in
shades of red, correlated with the severity of the defect. The Switch I
domain is shown in green, with the V36M mutation in dark blue. The
Switch II domain is in light blue. Pseudo–wild-type mutations with no
effect on cell fusion are not shown.
Volume 23 April 1, 2012 Cdc42 and Fus2p in yeast cell fusion | 1211
pathway insensitive to signaling from Cdc42p. In the STE20-ΔCRIB
background, cdc42-138 had no effect on pheromone signaling
(Figure 3A). In contrast, mating between STE20-ΔCRIB cdc42-138
double mutants was significantly more defective than matings be-
tween the STE20-ΔCRIB controls (Figure 3B). Therefore cdc42-138
is defective in an aspect of mating that is independent of signaling
to Ste20p. We used cytoplasmic transfer assays to confirm that the
mating defect was due to a cell fusion defect (Figure 3, C and D).
When a fus2Δ tester strain expressing cytoplasmic GFP was mated
to a STE20-ΔCRIB strain, the GFP was transferred to the partner in
95% of the zygotes (corresponding to Fus+ and partial Fus− zygotes).
When the STE20-ΔCRIB strain also contained the cdc42-138 muta-
tion, cytoplasmic transfer was observed in only 49% of zygotes, in-
dicating that cdc42-138 had a full cell fusion defect in more than half
of the zygotes in this strain (n ≥ 469). We conclude that CDC42 plays
a role in cell fusion that is independent of its role in signal
A second possible explanation for the cell fusion defect in
cdc42-138 is that the mutants produce reduced levels of a-factor or
α-factor, leading to a fusion defect due to reduced signaling in the
partner (Brizzio et al., 1996). Because the genes required for pro-
duction and secretion of a-factor and α-factor are different, cell fu-
sion mutations that affect pheromone levels are mating-type spe-
cific (Brizzio et al., 1996). In crosses between cdc42-138 strains and
wild-type (WT) strains, the efficiency of cell fusion was independent
of whether the a-cell or the α-cell contained the mutation (Figure
3E; a WT × α cdc42-138 produced 32% fusion-defective zygotes,
compared with 41% for a cdc42-138 × α WT). Moreover, the defect
was significantly stronger when both strains contained the
cdc42-138 mutation, showing that Cdc42p function plays a role in
cell fusion in both parents. These data indicate that the fusion de-
fect in cdc42-138 is not due to defective pheromone signaling.
CDC42 plays independent roles in cell polarization
Previously a point mutation within the Switch I domain of Cdc42p
(cdc42-V36M) was reported to cause a cell fusion–defective pheno-
type (Barale et al., 2006). To ensure that any differences observed
between cdc42-V36M and cdc42-138 would not be due to strain
background, we re-created this allele in our strain background. We
confirmed that cdc42-V36M caused a cell fusion defect, producing
a significant number of unfused zygotes (Figure 2B; 53% had fusion
FIGURE 3: The cell fusion defect in cdc42-138 is not due to defective
pheromone signaling. (A) cdc42-138 has no defect in signaling in the
context of STE20-ΔCRIB. STE20Δ−CRIB (MY10925; black bars) and
STE20-ΔCRIB cdc42-138 (MY10926; gray bars), transformed with
fus1-LacZ (pSB231), were treated with the indicated concentrations of
α-factor for 90 min and their response measured via β-galactosidase
assay. Error bars represent SD of three independent experiments.
(B–D) cdc42-138 still exhibits a cell fusion defect in the STE20-ΔCRIB
background. (B) STE20-ΔCRIB (MY10925) and STE20-ΔCRIB cdc42-
138 (MY10926) were mated to fus2 (MY10797) for 4 h at 30°C, and
diploids were selected by replica plating to selective media (C, D)
Cytoplasmic transfer assays were performed using the same strains as
in B, in which one partner expresses cytoplasmic GFP. (C) Examples of
zygotes showing transfer (top) or in which cell fusion was blocked
(bottom). The latter phenotype corresponds to the “Full” Fus− cell
fusion defect. (D) Quantification of the data in C. More than 460
zygotes were imaged in two independent experiments. (E) The defect
in cdc42-138 is not mating-type specific. WT and cdc42-138 cells were
mated in the orientations indicated. Gray bars, partial Fus− zygotes;
black bars, full Fus− zygotes. n ≥ 176 zygotes imaged in two
1212 | C. A. Ydenberg et al. Molecular Biology of the Cell
Together, these data indicate that the cdc42-138 mutant re-
sponds to pheromone and polarizes normally. Therefore, the block
in cell fusion is not likely to be a secondary consequence of defects
in earlier events in mating. In contrast, the cdc42-V36M mutant po-
larizes poorly in response to pheromone, suggesting that the fusion
defect may be largely due to indirect effects on the cytoskeleton
during cell polarization.
Cdc42-138 acts late in cell fusion
Several classes of cell fusion mutants are now known that can be
distinguished by their morphological characteristics. Mutations in
SPA2, FUS1, FUS2, and RVS161 all block prezygotes at the stage of
cell wall removal (Gammie et al., 1998). As discussed earlier, SPA2
and BNI1 primarily affect polarization, and mutants form broad ad-
hesion zones between the fusing cells. The respective phenotypes
can be distinguished at the level of electron microscopy; fus2 and
rvs161 mutants have vesicles tightly clustered at the zone of cell fu-
sion, similar to wild-type zygotes prior to fusion. In contrast, fus1
and spa2 mutants contain broadly dispersed vesicles that do not
localize to a particular point. Double-mutant and suppression analy-
sis showed that fus1 and fus2 work in overlapping but distinct path-
ways, whereas fus2 and rvs161 work together (Trueheart et al., 1987;
Brizzio et al., 1998; Gammie et al., 1998).
Electron microscopy of cdc42-138 bilateral zygotes revealed that
they contain intact cell walls between the mating partners (Figure 5).
Vesicles were observed to be tightly clustered in a single region at
the zone of cell fusion (Figure 5, B and C; 35% of sections through
cdc42-138 prezygotes had vesicles, n = 48; of these, 89% had clus-
tered vesicles, n = 19). This phenotype is very similar to that of fus2
Mutations that affect the polarization of cells in response to pher-
omone (e.g., spa2, bni1, pea2) exhibit corresponding defects in cell
fusion (Dorer et al., 1997; Gammie et al., 1998). Because Cdc42p is
critically important for polarity during both mating and mitotic
growth (Chant, 1999; Pruyne and Bretscher, 2000; Casamayor and
Snyder, 2002), we next examined the morphological responses of
cdc42-138 and cdc42-V36M to pheromone.
When wild-type or cdc42-138 cells were treated with phero-
mone, they formed characteristic pointed mating projections
(Figure 4A). The actin cytoskeleton became polarized, with actin
patches concentrated in the projection and cables oriented toward
the shmoo tip (Figure 4A; 85% of wild-type cells and 81% of cdc42-
138 cells had polarized actin). In contrast, cdc42-V36M cells were
round or oblong, without an obvious projection of any kind (Figure
4A). Actin patches were not tightly localized at any particular point
but instead were dispersed throughout the cell (Figure 4A; only 18%
had polarized actin).
We also examined the localization of Fus2p. Fus2p-GFP localizes
tightly to the shmoo tip in wild-type cells but is usually dispersed or
nuclear in polarization mutants such as bni1 and spa2 (Paterson
et al., 2008). Wild-type and cdc42-138 shmoos contained predomi-
nantly tip-localized Fus2p-GFP (Figure 4B; Fus2p-GFP was predomi-
nantly polarized in 98 and 80% of cells, respectively). The small but
significant (p = 0.004, Fisher’s exact test) decrease in Fus2-GFP lo-
calization is consistent with an interaction with Cdc42p. In contrast,
in the majority of cdc42-V36M cells (56%), Fus2p-GFP was dispersed
or partially nuclear. In only 44% of cdc42-V36M cells was Fus2p-GFP
localized to a point on the cortex (usually at one of the ends of the
oval-shaped cells). However, even in these cells, localization was not
wild type; time-lapse imaging of Fus2p-GFP in cdc42-V36M showed
that the cortical spots were unstable, moving rapidly away from the
cortex and showing significant nuclear localization (Supplemental
Movie S2). Similar unstable localization was observed in spa2 and
bni1 mutants (Paterson et al., 2008).
FIGURE 4: cdc42-138 is not defective in cell polarization in response
to pheromone. (A) Actin localization in cdc42-138 and cdc42-V36M.
WT (DDY1300), cdc42-138 (DDY1354), and cdc42-V36M (MY10547)
were treated with α-factor for 90 min, fixed, and stained with Texas
red–phalloidin. Representative images are shown. n ≥ 241 cells in
three independent experiments. (B) Fus2p-GFP localization in
cdc42-138 and V36M. The same strains were transformed with
FUS2::GFP104 (pMR5482) and imaged after treatment with α-factor
for 90 min. Representative images are shown. n ≥ 55 cells in two
FIGURE 5: EM of cdc42-138 and fus2 zygotes. cdc42-138 bilateral
zygotes (DDY1354 × DDY1355) were examined by electron
microscopy. (A) A zygote, showing the unfused nuclei and septum,
containing cell wall material. Image is representative of 91 zygotes.
(B) The waist regions of the same zygotes, showing vesicles clustered
at the zone of cell fusion. Images are representative of 32 zygotes in
which vesicles were observed. The vesicles were clustered in 91% of
this class. (C) Dark plaques and a membrane invagination at the cell
fusion zone. These phenotypes were observed in 21 and 10% of all
zygotes examined, respectively. (D) A fus2 zygote, for comparison.
Scale bar, 0.5 μm.
Volume 23 April 1, 2012 Cdc42 and Fus2p in yeast cell fusion | 1213
p = 0.04, chi-square) but significantly different from the multiplicative
prediction for independent pathways (36%, p = 0.004, chi-square).
Because of the significant difference from the multiplicative model,
we conclude that the cdc42-138 and fus2 mutations largely affect the
same pathway for cell fusion. However, the smaller difference from
the phenotype of the single mutant suggests that one or both pro-
teins may also contribute an independent function to cell fusion.
Fus2p Interacts with GTP-bound Cdc42p
Our genetic data indicate that Fus2p and Cdc42p work in the same
pathway; they do not distinguish which protein acts upstream. Be-
cause Fus2p has a Dbl-homology domain required for cell fusion,
one possibility is that Fus2p is a guanine nucleotide exchange factor
(GEF) for Cdc42p. However, numerous attempts to demonstrate
GEF activity have been unsuccessful, even under conditions
where Cdc24p (the GEF for Cdc42p) showed strong activity
(Ydenberg, Stein, Andrianantoandro, and Rose, unpublished data).
We next considered the possibility that Fus2p is a Cdc42p effector,
interacting specifically with activated, GTP-bound Cdc42p.
We began by confirming the Fus2p-Cdc42p interaction in an in
vitro system. Wild-type Cdc42p, Cdc42p-Q61L, the GTP-locked
mutant, and Cdc42p-T17N, the GDP-locked mutant, were ex-
pressed as GST fusions in Escherichia coli. Equal amounts of puri-
fied GST-Cdc42p were added to yeast extracts containing full-length
Fus2p, which was internally FLAG-epitope tagged at residue 104.
This protein was shown previously to support wild-type levels of cell
fusion, in vivo (Paterson et al., 2008). All three forms of Cdc42p
coimmunoprecipitated with Fus2p-FLAG; however roughly 20-times-
higher levels of Cdc42p-Q61L (GTP bound) were bound compared
with either Cdc42p-T17N (GDP bound) or wild type (Figure 7A).
These results confirm that Cdc42p can bind to Fus2p and suggest
that Fus2p is not a GEF for Cdc42p. Indeed, because the interaction
was strongest with activated Cdc42p, we conclude that that Fus2p
is more likely to be a effector or to play a role in localizing activated
Cdc42p to the fusion site.
To identify the region required for binding to Cdc42p, various de-
letions of Fus2p were constructed and used in the coimmunoprecipi-
tation assay, using Cdc42p-Q61L (Figure 7, B and C). The most effi-
cient binding was observed with full-length protein and the deletion
lacking residues 580–677. Of interest, although the Dbl-homology
domain was sufficient to see some binding to Fus2p, it was not
(Figure 5D) and rvs161 but distinct from that
of spa2 and fus1 (Gammie et al., 1998). We
also observed dark-staining plaques and
membrane invaginations in cdc42-138 zy-
gotes (Figure 5C; 31% had plaques and 5%
had invaginations, n = 48). These pheno-
types were previously shown to be unique to
fus2 and rvs161 mutants and were not ob-
served in fus1 and spa2 mutants (Gammie
et al., 1998). Furthermore, the formation of
these structures was dependent on FUS1
and SPA2 function because they were not
observed in fus1 fus2 or spa2 fus2 double
mutants. The fact that cdc42-138 formed
these structures provides evidence that the
SPA2 and FUS1 pathways remain functional.
To further characterize the morphology
of cdc42-138, we measured the breadth of
the zygote cell fusion zones (referred as the
zygote waistlines). In wild-type matings, un-
fused prezygotes have an average waistline
of 1.0 ± 0.3 μm, which expands to 1.6 ± 0.2 μm soon after nuclear
fusion (Gammie et al., 1998). When only unfused prezygotes were
considered, cdc42-138 bilateral zygotes had waistlines measuring
1.4 ± 0.3 μm (n = 48). It is surprising that this was significantly nar-
rower than for fus2 prezygotes (1.7 ± 0.5 μm, n = 19, p = 0.008,
Student’s t test). We reasoned that because the cdc42-138 mutant
has a weaker phenotype than the fus2 mutant, a significant portion
of the zygotes with broad zones may have fused, leading to a shorter
average waistline. In support of this, when the entire population of
fused and unfused zygotes was considered, there was no difference
between the average waistline of the cdc42-138 and fus2 mutant
zygotes (1.6 ± 0.4 μm, n = 91, vs. 1.7 ± 0.5 μm, n = 20, respectively;
p = 0.22). Most important, the data provide additional evidence that
the cdc42-138 mutant is morphologically dissimilar from polariza-
tion mutants, which have significantly wider waistlines than the fus2
mutant (Gammie et al., 1998). Together the data show that the
cdc42-138 mutant is morphologically most similar to fus2 and rvs161
mutants. Furthermore, they show that CDC42 plays a role in the very
latest stages of cell fusion.
FUS2 and CDC42 work together
To test whether FUS2 and CDC42 act in the same pathway for cell
fusion, we constructed a fus2Δ cdc42-138 double mutant and com-
pared its phenotype to that of the two single mutants. We reasoned
that if cdc42-138 specifically compromised Fus2p function in cell fu-
sion (or vice versa), then the double mutants should show no more
severe a defect than the most defective single mutant. Such behavior
is observed between rvs161 and fus2 mutations, whereas double
mutants between either of these mutations and fus1 or spa2 muta-
tions are significantly more severe (Brizzio et al., 1996). For this ex-
periment, we used a cytoplasmic transfer assay (Figure 6A). In this
assay, only full Fus− zygotes with no plasma membrane fusion are
scored as being defective. The fus2 donor strain was able to transfer
cytoplasmic GFP to 92% of wild-type cells (n = 764) but was signifi-
cantly defective for transfer to fus2 (46% transfer, n = 778, p = 0.001,
chi-square) and cdc42-138 (72% transfer, n = 710, p = 0.001, chi-
square; Figure 6B). If the two proteins act in two independent path-
ways, then we predict that the defect of the double mutant should be
multiplicative, normalized for the frequency observed for wild type
(i.e., [0.72 × 0.46]/0.92). Transfer to the cdc42-138 fus2 double mu-
tant was somewhat more defective than to fus2 alone (42%, n = 554,
FIGURE 6: Fus2p and Cdc42p work in the same pathway. (A) Cytoplasmic transfer assay. Strains
of different genotypes were mated to MY10797 (fus2 PGPD1-GFP) for 2 h at 30°C. An example of
a zygote displaying cytoplasmic transfer (top) and one not displaying transfer (bottom) are
shown. (B) WT (DDY1300), cdc42-138 (DDY1354), fus2 (MY10016), and fus2 cdc42-138
(MY10310) were mated to MY10797, and the percentage of zygotes showing transfer were
counted. The total number of zygotes counted in three independent experiments are shown.
1214 | C. A. Ydenberg et al. Molecular Biology of the Cell
required for binding; binding was also ob-
served for protein fragments containing resi-
dues 415–677. These results suggest that
more than one region of Fus2p interacts with
Cdc42p. To analyze this further, we examined
the nucleotide specificity of binding to the
two regions of Fus2p. Binding to the DBH
domain showed strong specificity for binding
to the GTP-bound Cdc42p-Q61L but not to
the other forms of Cdc42p (Figure 7D). The
415–677 fragment showed lower specific
binding, although it still bound preferentially
to Cdc42p-Q61L (Figure 7E). We conclude
that the region of Fus2p that binds Cdc42
extends beyond the DBH domain.
As a final test, we examined whether the
cdc42 mutations affected the interaction
with Fus2p. The cdc42-V36M and cdc42-
138 alleles were constructed in the plasmid
expressing the GTP-locked Q61L mutation
and the purified proteins tested for binding
to Fus2p-FLAG (Figure 7F). Both proteins
showed a significant decrease in binding
relative to wild-type Cdc42p (cdc42-V36M
and cdc42-138 exhibited 7 and 4% binding,
respectively). These results corroborate the
FIGURE 7: Fus2p binds to Cdc42p in vitro. (A) Fus2p binds most strongly to the activated
GTP-bound form of Cdc42p. Wild-type, Cdc42p-Q61L (GTP-locked), and Cdc42p-T17N
(GDP-bound) proteins were expressed as GST fusions in E. coli and purified on glutathione
Sepharose. The Cdc42p-GST fusions were incubated with yeast cell extracts expressing FLAG
epitope–tagged Fus2p for 1 h at 4°C, and interacting proteins were precipitated with anti-FLAG
Sepharose. The Cdc42p-GST fusions were detected by Western blot using anti-GST and
chemiluminescent imaging. Top, bound Cdc42 proteins. Bottom, input Cdc42 proteins. Numbers
indicate the relative level of binding of each Cdc42p species normalized to Cdc42p-Q61L. In the
rightmost “no Fus2p” lane, Cdc42p-Q61L was used. (B) Map of Fus2p deletion mutants. The
Dbl-homology domain is indicated (Dbl H). The extents of the deletions, in amino acid residues,
are indicated. Note that the FLAG epitope is inserted at residue 104. (C) Cdc42p binds to more
than one domain of Fus2p. Yeast strains expressing the deletion constructs shown in B were
used in binding experiments with GST-
Cdc42p-Q61L. The “no Fus2p” control used
yeast extracts in which Fus2p was not
epitope tagged. FLAG-tagged Fus3-KD
(kinase dead) was used as an unrelated
protein control. Top, relative levels of bound
GST-Cdc42p-Q61L proteins are indicated.
Middle, input GST-Cdc42p-Q61L. Bottom,
the levels of the input FLAG-tagged Fus2p
deletion proteins. Proteins bound to the
anti-FLAG Sepharose were eluted and
detected by Western blotting with anti-FLAG
antibody. The deletion proteins are indicated
by a red dot and the Fus3-KD protein with a
blue dot, and the heavy and light
immunoglobulin chains are indicated (H and
L, respectively). The levels of the proteins
relative to full-length Fus2p are indicated at
the bottom. (D) Nucleotide specificity of
binding to the DBH domain. The indicated
Cdc42p proteins were incubated with
Fus2p105-415. Top, bound Cdc42p proteins;
relative levels of binding are indicated.
Bottom, input Fus2p105-415. In this experiment
some degradation of the Fus2p deletion
fragment was observed. In the rightmost
three lanes, Cdc42p-Q61L was used.
(E) Nucleotide specificity of binding to
415–677. As in D, except that the Cdc42p
proteins were incubated with Fus2p415-677.
Top, bound Cdc42p proteins; relative levels
of binding are indicated. Bottom, input
Fus2p415-677. (F) Cdc42p mutations diminished
Fus2p binding. The two mutations affecting
cell fusion were introduced into the Cdc42p-
Q61L construct and used for binding to
Volume 23 April 1, 2012 Cdc42 and Fus2p in yeast cell fusion | 1215
Fus2p/Rvs161p may play a role in facilitating the exocytosis of se-
creted proteins required for cell wall dissolution.
A role for the Rho-insert domain
The Rho-insert domain is a ∼20-residue loop containing a 13-resi-
due α-helix unique to the Rho subfamily of small G proteins (Feltham
et al., 1997). The residues of Cdc42p required for cell fusion (D121
and D122) are within four charged amino acids at one end of the
Rho-insert domain. Previous work in yeast was not informative as to
possible function; individual mutations to alanine did not affect
growth; mutation of all four was lethal (Kozminski et al., 2000). A role
in cell fusion represents the first specific function identified for this
region of the protein in yeast.
Although little is known about Cdc42p’s Rho-insert domain in
yeast, hints may be gleaned from human Cdc42 and Rac1, where it is
involved in the interactions with a variety of proteins, including Rho
GDP dissociation inhibitors (Wu et al., 1997; Richman et al., 2004),
formins (Richman et al., 2004; Lammers et al., 2008), and IQGAPs
(McCallum et al., 1996; Li et al., 1999; Owen et al., 2008). It is possible
that in yeast other protein interactions may be affected in addition to
Fus2p. Although the yeast formin Bni1p is required for cell fusion, it
also is required for cell polarization (Evangelista et al., 1997; Matheos
et al., 2004; Paterson et al., 2008), likely acting prior to the function
identified by cdc42-138. Of interest, the yeast IQGAP, Iqg1p, regu-
lates exocytosis by localizing Sec3p to polarized growth sites (Osman
et al., 2002), and Sec3p interacts with activated Cdc42p (Zhang et al.,
2001, 2008). Continuous secretion has been shown to be required for
the late stages of cell fusion (Grote, 2010). However, a specific require-
ment for either Iqpg1p or Sec3p in cell fusion has not been tested.
Cdc42 plays roles at multiple stages of mating
and cell fusion
Analyzing the many roles of GTPases has required extensive use of
point mutations to specifically interfere with individual functions
(Mosch et al., 1999; Kozminski et al., 2000; Adamo et al., 2001; Saka
et al., 2001; Barale et al., 2006; Heinrich et al., 2007). For example,
this approach demonstrated that cdc42-V36M affects cell fusion but
not mitotic growth (Barale et al., 2006). We found that cdc42-V36M
did not polarize in response to pheromone and showed unstable
localization of Fus2p-GFP (Figure 3), closely resembling spa2 and
bni1. Fus1p-GFP also failed to localize in cdc42-V36M (Barale et al.,
2006). We conclude that cdc42-V36M is primarily defective for po-
larization and may affect cell fusion indirectly, possibly through
Bni1p. The cdc42-V36M mutant may also have a specific defect in
cell fusion, which is masked by the earlier polarization defect.
In contrast, cdc42-138 mutant cells polarized normally and were
specifically defective in cell fusion. Epistasis analysis showed that
Spa2p acts upstream of Fus1p, which in turn acts upstream of Fus2p
(Gammie et al., 1998). The morphology of cdc42-138 most closely
resembled fus2, arguing that this allele disrupts a function down-
stream of Fus1p and Spa2p. In confirmation, double-mutant analy-
sis showed that fus2 and cdc42-138 act at the same step.
We conclude that Cdc42p plays multiple roles during cell fusion.
Roles in pheromone signaling, via Ste20p (Simon et al., 1995; Zhao
et al., 1995), and polarization, via Bni1p (Evangelista et al., 1997), are
well established. We propose a separate function in cell fusion medi-
ated by the Rho-insert domain in association with Fus2p/Rvs161p.
A conserved role of Cdc42 homologues in eukaryotic
Among metazoan cell fusion events, Drosophila myoblast fusion
may be the best understood (Chen and Olson, 2004). Ultrastructural
genetic data and suggest that cdc42-138, as well as cdc42-V36M,
may interfere with cell fusion by disrupting the interaction between
Cdc42GTPp and Fus2p.
Several lines of evidence previously suggested an interaction be-
tween Cdc42p and Fus2p. First, they interact by two-hybrid assay
(Nelson et al., 2004). Second, BEM1, encoding a scaffold for Cdc42p
and its effectors, is a high-copy suppressor of fus2 (Fitch et al., 2004).
Third, high-copy FUS2 suppresses the mating defects of bem1 mu-
tations (Leberer et al., 1996). Finally, Fus2p contains a Dbl-homology
domain (Paterson et al., 2008), which is shared with activators of
Rho-GTPases. However, Cdc42p has numerous functions and inter-
action partners and so could potentially have multiple direct and
indirect roles during mating.
To implicate CDC42 specifically in cell fusion, we sought mutant
alleles defective in cell fusion but not other steps of mating. One
mutation, cdc42-138, caused a strong defect in cell fusion but not in
mitotic growth, which was not due to impaired pheromone signaling
or cell polarization. The mutant phenotype closely resembled fus2
mutations and analysis of double mutants showed that cdc42-138
and fus2 mutations conferred defects in the same step in cell fusion.
The Dbl-homology domain in Fus2p is required for as much as
90% of Fus2p’s function in cell fusion. The presence of residual func-
tion suggests that Fus2p may have other roles in cell fusion or more
than one interaction with Cdc42p. Indeed, we found that Cdc42p
interacts with the Dbl-homology domain and additional sequences
Although Fus2p contains a Dbl-homology domain found in other
Cdc42p GEFs, we did not detect GEF activity (unpublished data).
Instead, we found that Fus2p interacted more strongly with Cdc42-
Q61L, the GTP-bound form of Cdc42p, in vitro, consistent with the
two-hybrid data (Nelson et al., 2004). Because GEFs are expected
to bind more strongly to the GDP-bound G-protein, these findings
suggest that Fus2p is not a GEF but may instead serve either as a
mating-specific effector for Cdc42p or to localize the activated
Cdc42p to the cell fusion zone. It is notable that another putative
GEF, Lte1p, requires its GEF domain for cortical localization and not
for activation of Tem1p (Geymonat et al., 2009).
If Fus2p is an effector, what might be the role of the Fus2p–
Cdc42p interaction in promoting cell fusion? Fus2p binds the BAR
domain–containing protein Rvs161p, and the interaction is essential
for cell fusion (Brizzio et al., 1998). BAR-domain proteins preferen-
tially bind to curved membranes (Peter et al., 2004), and Rvs161p,
together with Rvs167p, binds to membranes and promotes curva-
ture in vitro (Friesen et al., 2006; Youn et al., 2009). Mutations in
Rvs161p that inhibit membrane binding also inhibit mating (Youn
et al., 2009), suggesting that Fus2p/Rvs161p’s role in mating re-
quires membrane interaction.
Fus2p localizes near clustered vesicles, and both Fus2p and the
vesicles are partly delocalized in fus1 mutants (Gammie et al., 1998;
Paterson et al., 2008). Deletion of neither Fus2p’s Dbl-homology
domain nor cdc42-138 affected Fus2p localization. Moreover, vesi-
cles clustered normally in cdc42-138. These data suggest that the
Cdc42p interaction might regulate an activity associated with Fus2p/
Rvs161p rather than its localization. Recent work showed that mem-
brane bending by the vesicle protein synaptotagmin is required for
vesicle fusion (Hui et al., 2009). Perhaps Cdc42p regulates the de-
gree of curvature induced by Fus2p/Rvs161p, deforming vesicle
and/or target membranes in aid of membrane fusion. Alternatively,
Fus2p/Rvs161p might interact with the curved outer surface of the
vesicle fusion pore, stabilizing it to promote fusion. In either case,
1216 | C. A. Ydenberg et al. Molecular Biology of the Cell
used to create pMR5917, which is equivalent but in the integration
vector pRS406. pMR5917 was cut with HindIII and fus2Δ105-414::GFP
integrated in DDY1300 by the loop-in/loop-out procedure. A loop-
out containing GFP fluorescence was denoted MY10933.
To create pMR6115 and pMR6116, mutations were introduced
into pB2082 by site-directed PCR mutagenesis (Phusion; Thermo
Fisher Scientific, Waltham, MA). Deletion mutants in FUS2 were
generated by site-directed PCR mutagenesis using pMR5480
(Paterson et al., 2008) as a template. Deletions were confirmed by
sequencing, and pheromone-induced protein expression was con-
firmed by trichloroacetic acid (TCA) precipitation–Western blot.
β-Galactosidase assays were performed essentially as described
(Adams et al., 1997), using cell extracts prepared from ∼1.2 ×
108 cells. Actin staining was performed as described with minor
modifications (Adams et al., 1997), using cells treated with 6 μM
α-factor for 90 min.
GST-Cdc42p and derivatives were expressed and purified from E. coli
as described (Gladfelter et al., 2002) with minor modifications. For
expression of Fus2p-FLAG in yeast, MY9181, transformed with
pMR5480 (or derivative), was grown to early exponential phase
(OD600 = 0.2) and treated with 10 μg/ml synthetic mating phero-
mone (Syn/Seq Facility, Department of Molecular Biology, Princeton
University, Princeton, NJ) for 2 h. Cell extracts were prepared as in
Brizzio et al. (1998). The cleared lysate was divided into three frac-
tions, and each fraction was incubated with 5 μg of bacterially
expressed GST-Cdc42 or derivative as indicated. Binding proceeded
for 1 h at 4°C, with gentle rotation. After incubation, 30 μl of equili-
brated anti-FLAG Sepharose (Sigma-Aldrich, St. Louis, MO)
was added to each reaction and the incubation continued at 4°C for
1 h. The anti-FLAG Sepharose was washed five times and finally
studies revealed clustered, electron-dense vesicles across the cell
fusion zone (Doberstein et al., 1997), similar to yeast cell fusion and
vertebrate myoblast fusion (Lipton and Konigsberg, 1972). Recent
work showed that, as in yeast, fusion occurs at a single site, coinci-
dent with accumulation of actin (Sens et al., 2010). Cdc42p homo-
logues Drac1 and Drac2 are also required for myoblast fusion
(Hakeda-Suzuki et al., 2002) and most likely activated by an uncon-
ventional GEF (Erickson et al., 1997; Brugnera et al., 2002). More-
over, Cdc42p is required for mouse myoblast fusion (Vasyutina et al.,
2009). The conserved requirement for Cdc42p-related proteins in
cell fusion suggests that yeast cell fusion will yield important insights
into the mechanism in higher cells.
MATERIALS AND METHODS
General yeast methods, strains, and plasmid construction
Yeast media, general methods, and transformations were performed
as described previously (Adams et al., 1997) with minor modifica-
tions. Strains and plasmids are listed in Tables 1 and 2, respectively.
To create MY10016, the fus2Δ::HIS3 deletion was amplified
from MY9181 and transformed into DDY1300. Correct integration
was confirmed by PCR across the FUS2 locus. MY10547 and
MY10554 were created as described (Kozminski et al., 2000). To
create MY10925 and MY10926, pPP1043 (Lamson et al., 2002)
was cut with BamHI and integrated in DDY1300 and DDY1354,
respectively, by the loop-in/loop-out procedure. Correct loop-
outs were confirmed by PCR across the STE20 locus. To create
MY10797 and MY10798, pEG746 (Jin et al., 2004) was cut with
BglII and transformed into JY428 and JY429, respectively. The
integration was confirmed by examining the resulting strains for
pMR5883 was created by a multistep cloning procedure in the
vector pRS416 (Sikorski and Hieter, 1989). A similar procedure was
α fus2Δ3 his4-34 trp1Δ1 ura3-52
JY428 G. Fink (Whitehead Institute,
Kozminski et al. (2000)
Kozminski et al. (2000)
Kozminski et al. (2000)
Kozminski et al. (2000)
Paterson et al. (2008)
α fus1Δ1 fus2Δ3 trp1Δ1 ura3-52
α his4-34 trp1Δ1 ura3-52
a ura3-52 leu2-3112 his3Δ200 lys2-801 CDC42::LEU2
α ura3-52 leu2-3112 his3Δ200 lys2-801 CDC42::LEU2
a ura3-52 leu2-3112 his3Δ200 lys2-801 cdc42-138::LEU2
α ura3-52 leu2-3112 his3Δ200 lys2-801 cdc42-138::LEU2
a ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 fus2Δ::HIS3
a ura3-52 leu2-3112 his3Δ200 lys2-801 CDC42::LEU2 fus2Δ::HIS3
α ura3-52 leu2-3112 his3Δ200 ade2-1 fus2Δ::HIS3
a ura3-52 leu2-3112 his3Δ200 lys2-801 cdc42-138::LEU2 fus2Δ::HIS3
a ura3-52 leu2-3112 his3Δ200 lys2-801 cdc42-V36M::LEU2
α ura3-52 leu2-3112 his3Δ200 ade2-1 cdc42-V36M::LEU2
α fus2Δ3 his4-34 trp1Δ1 ura3-52 SSO1::PGPD1-GFP::URA3
α fus1Δ1 fus2Δ3 trp1Δ1 ura3-52 SSO1::PGPD1-GFP::URA3
a ura3-52 leu2-3112 his3Δ200 lys2-801 CDC42::LEU2 STE20ΔCRIB
a ura3-52 leu2-3112 his3Δ200 lys2-801 cdc42-138::LEU2 STE20ΔCRIB
a ura3-52 leu2-3112 his3Δ200 lys2-801 CDC42::LEU2 fus2ΔDbl::GFP
a ura3-52 leu2-3112 his3Δ200 pep4::HIS3 bar1::kanMX fus2::natMX
aStrains containing cdc42-105, cdc42-107, and cdc42-137 are isogenic to DDY1300 (or DDY1301). See Kozminski et al. (2000).
TABLE 1: Strains used this study.
Volume 23 April 1, 2012 Cdc42 and Fus2p in yeast cell fusion | 1217
by SDS–PAGE, and transferred to a nitrocellulose filter. GFP-tagged
Fus2p was detected using mouse anti-GFP (Roche, Indianapolis, IN)
at 1:1000. FLAG-tagged Fus2p was detected with anti-FLAG (Sigma-
Aldrich) at 1:5000. Rabbit anti-Kar2 (our laboratory) was used at
1:50,000. Glutathione S-transferase (GST)–tagged Cdc42p was de-
tected with anti-GST (Cell Signaling Technologies, Beverly, MA) at
resuspended in SDS loading buffer. Experiments using the Fus2p
deletion mutants were performed similarly, except that constructs
were transformed into MY12402, which harbored deletions of the
fus2, pep4, and bar1 genes.
Mating and cell fusion assays
Plate mating assays were performed as described (Gammie and
Rose, 2002) by replica plating strains together on yeast extract/pep-
tone/dextrose (YEPD) and selecting diploids by replica plating to
minimal media or drop-in media. Microscopic examination of zy-
gotes and cytoplasmic transfer assays were performed as described
(Grote, 2008), using ∼1.0 × 106 cells of each mating type. Zygotes
were processed for electron microscopy as described (Gammie
et al., 1998), using ∼1.2 × 108 cells of each mating type, separated
over five filters and mated on YEPD for 2 h.
Images were acquired using the DeltaVision Microscopy System
(Applied Precision, Issaquah, WA) using a TE200 inverted micro-
scope (Nikon, Melville, NY) and a Coolsnap HQ CCD camera (Pho-
tometrics, Tucson, AZ) and a 100× objective with a numerical aper-
ture of 1.4. Fus2p-GFP images were acquired as previously described
(Ydenberg and Rose, 2009). Figures were prepared for publication
using Adobe Photoshop and Adobe Illustrator (Adobe, San Jose,
CA). For presentation purposes, pixel density was increased using
bicubic resampling, where necessary.
For Western analysis of Fus2p-GFP, protein was prepared from
∼6.0 × 106 exponential phase cells by TCA precipitation, separated
We thank Danny Lew, Keith Kozminski, Peter Pryciak, Eric Grote,
and Robert Arkowitz for providing strains and reagents and Satoshi
Yoshida and Bruce Goode for critical comments on the manuscript.
This work was supported by National Institute of General Medical
Sciences Grant GM37739 to M.D.R.
pSB231 CEN URA3 fus1-LacZTrueheart
et al. (1987)
et al. (2008)
et al. (2008)
pMR5469 CEN URA3 PGAL1-FUS2::GFP104
pMR5482 CEN URA3 FUS2::GFP104
CEN URA3 PGAL1-fus2Δ105-414::GFP
pMR5480 FUS2::FLAG104 URA3 CENPaterson
et al. 2008
FUS21-580::FLAG104 URA3 CEN
FUS2105-677::FLAG104 URA3 CEN
FUS2105-580::FLAG104 URA3 CEN
FUS2415-677::FLAG104 URA3 CEN
FUS2105-415::FLAG104 URA3 CEN
GST-CDC42 Q61L D122A
et al. (2002)
et al. (2002)
et al. (2002)
TABLE 2: Plasmids used in this study.
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