Molecular Biology of the Cell
Vol. 20, 2909–2919, June 15, 2009
The Class V Myosin Myo2p Is Required for Fus2p
Transport and Actin Polarization during the Yeast Mating
Jason M. Sheltzer* and Mark D. Rose
Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1014
Submitted September 10, 2008; Revised March 30, 2009; Accepted April 20, 2009
Monitoring Editor: David G. Drubin
Mating yeast cells remove their cell walls and fuse their plasma membranes in a spatially restricted cell contact region.
Cell wall removal is dependent on Fus2p, an amphiphysin-associated Rho-GEF homolog. As mating cells polarize,
Fus2p-GFP localizes to the tip of the mating projection, where cell fusion will occur, and to cytoplasmic puncta, which
show rapid movement toward the tip. Movement requires polymerized actin, whereas tip localization is dependent on
both actin and a membrane protein, Fus1p. Here, we show that Fus2p-GFP movement is specifically dependent on Myo2p,
a type V myosin, and not on Myo4p, another type V myosin, or Myo3p and Myo5p, type I myosins. Fus2p-GFP tip
localization and actin polarization in shmoos are also dependent on Myo2p. A temperature-sensitive tropomyosin
mutation and Myo2p alleles that specifically disrupt vesicle binding caused rapid loss of actin patch organization,
indicating that transport is required to maintain actin polarity. Mutant shmoos lost actin polarity more rapidly than
mitotic cells, suggesting that the maintenance of cell polarity in shmoos is more sensitive to perturbation. The different
velocities, differential sensitivity to mutation and lack of colocalization suggest that Fus2p and Sec4p, another Myo2p
cargo associated with exocytotic vesicles, reside predominantly on different cellular organelles.
How cells create and maintain asymmetry is a fundamental
problem in cell biology. In yeast cells undergoing mitotic
growth, polarized secretion is directed toward the develop-
ing bud (Pruyne and Bretscher, 2000). The septin ring, which
forms at the mother-daughter neck, acts as a diffusion bar-
rier to limit the flow of asymmetrically localized proteins out
of the bud (Barral et al., 2000). Mating yeast must also
localize proteins to specific sites on the plasma membrane,
though they lack an analogous structure to prevent retro-
grade diffusion. How mating yeast selectively polarize cer-
tain proteins remains a largely unexplored question.
The budding yeast, Saccharomyces cerevisiae, has two hap-
loid mating types, MATa and MAT?, which can conjugate to
form a diploid cell (reviewed in Marsh and Rose, 1997).
Haploid yeast cells secrete a peptide pheromone (a-factor or
?-factor) that triggers a MAP kinase signaling cascade in
cells of the opposite mating type (Bardwell, 2005). In re-
sponse to pheromone, haploid cells arrest their cell cycle at
G1, begin apical growth in the direction of the pheromone
gradient (commonly called “shmooing”), and up-regulate
the transcription of mating-specific genes (Wilkinson and
Pringle, 1974; Fields et al., 1988; Segall, 1993). When mating
partners make contact, the intervening cell wall is degraded,
and the plasma membranes fuse to form a continuous
surface (Gammie et al., 1998). Finally, apposing nuclei are
pulled together by cytoplasmic microtubules, allowing
karyogamy to occur (Meluh and Rose, 1990; Molk et al.,
2006). After conjugation is complete, mating-specific pro-
cesses are down-regulated, and the cell is able to resume
Efficient mating requires intracellular and cell surface po-
larization toward a mating partner (Chenevert et al., 1994;
Bagnat and Simons, 2002). Pheromone signaling activates
the formin Bni1p, which triggers the reorganization of the
actin cytoskeleton and directs new cell wall growth toward
the formation of a mating projection (Evangelista et al., 1997;
Matheos et al., 2004). Additionally, proteins required for cell
wall degradation localize to the tip of the mating projection,
where cell fusion will occur. Fus1p and Fus2p have been
identified as key mediators of cell fusion: both proteins
localize to the shmoo tip, and crosses between fus1 or fus2
pairs result in profound mating defects (Trueheart et al.,
1987; Elion et al., 1995). Fus1p is an O-glycosylated mem-
brane-spanning protein that regulates fusion pore formation
(Nolan et al., 2006), whereas Fus2p is a cytoplasmic protein
that contains a putative guanine-nucleotide exchange factor
(GEF) domain and may be involved in transduction of the
fusion signal (Paterson et al., 2008). Fus2p acts in conjunction
with Rvs161p, a BAR domain–containing, amphiphysin-like
protein that has various roles in endocytosis and vesicular
trafficking (Crouzet et al., 1991; Sivadon et al., 1995; Brizzio et
al., 1998; Friesen et al., 2006; Paterson et al., 2008). Because
BAR domain proteins typically form dimers that interact
with the curved surfaces of membranes (Peter et al., 2004),
Fus2p-Rvs161p heterodimers may localize to the surface of
vesicles that have been observed to cluster at the junction
between mating cells (Gammie et al., 1998; Paterson et al.,
In a recent study, Paterson et al. (2008) tagged Fus2p with
green fluorescent protein (GFP) to observe Fus2p dynamics
This article was published online ahead of print in MBC in Press
on April 29, 2009.
* Present address: Department of Biology, Massachusetts Institute of
Technology, Cambridge, MA 02139.
Address correspondence to: Mark D. Rose (email@example.com).
© 2009 by The American Society for Cell Biology2909
in vivo. In shmoos, Fus2p-GFP was found to localize pri-
marily to the tip of the mating projection. Surprisingly,
Fus2p-GFP also appeared as cytoplasmic puncta that were
observed moving rapidly toward the shmoo tip. Puncta
movement was dependent on polymerized actin, whereas
Fus2p-GFP’s localization to the shmoo tip was dependent on
both actin and Fus1p, which may also function as a cortical
In S. cerevisiae, actin-dependent motility occurs via one or
more members of the myosin family of molecular motors, or
via Arp2/3-mediated actin polymerization (Brown, 1997;
Bretscher, 2003). The classical type II myosin, Myo1p, is
required for cytokinesis. The type I myosins, Myo3p and
Myo5p, function during the internalization step of endocy-
tosis, and the type V myosins, Myo2p and Myo4p, transport
various cargoes along actin cables. Myo2p is implicated in
vesicular and organelle transport, whereas Myo4p is known
to transport mRNA and elements of the cortical endoplasmic
reticulum (ER). The actin-nucleating ability of Arp2/3 may
drive the movement of mitochondria and endosomes (Boldogh
et al., 2001; Chang et al., 2003), although this function re-
mains controversial (Itoh et al., 2002; Altmann et al., 2008).
In this study, we sought to identify the protein(s) respon-
sible for Fus2p’s actin-dependent movement. We find that
Fus2p is transported by Myo2p along actin cables to the
shmoo tip, where it becomes anchored to the plasma mem-
brane by Fus1p. Fus2p transport was distinct from the trans-
port of Sec4p, suggesting that the majority of these proteins
reside on distinct cellular structures. Surprisingly, actin or-
ganization was highly dependent on both Myo2p and tro-
pomyosin in shmoos but not in mitotic cells, suggesting that
the maintenance of cytoskeletal polarity differs between
these growth regimes.
MATERIALS AND METHODS
General Yeast Techniques
Yeast manipulations and general techniques were performed as previously
described (Rose et al., 1990). Temperature-sensitive strains were grown at
23°C; all other strains were grown at 30°C. For induction with mating pher-
omone, cultures were grown to early log phase and then treated with syn-
thetic alpha-factor (Department of Molecular Biology Syn/Seq Facility,
Princeton University) to a final concentration of 10 ?g/ml. Temperature-
sensitive strains were incubated in pheromone for 120 min at 23°C; all other
strains were incubated in pheromone for 90 min at 30°C.
Strain and Plasmid Construction
Yeast strains and plasmids used in this study are listed in Tables 1 and 2.
FUS1 disruptions were performed using one-step gene replacement. The
Fus2p-mCherryFP construct (pMR5821) was created by in vivo recombination
(Oldenburg et al., 1997). Three fragments were generated by PCR to form the
construct. A PCR fragment containing homology to the vector along with 500
base pairs of the 5? untranslated region (UTR) plus the first 360 base pairs of
FUS2 was amplified with primers JP21 (vector sequence is in uppercase
letters, FUS2 sequence is in lowercase letters: TAGGGCGAATTGGGTAC-
CGGGCCCCCCCTCGAGGTCGACGGTATCGATgtcccacctgcttggtgg) and RC
ATTCAGTCTCACGAC) using genomic DNA as a template. A second PCR
fragment containing the mCherry fluorescent protein coding sequence with
flanking FUS2 homology was amplified from pMR5598 using primers Fus2-
AGGGCGAGGAGG) and mChFP-Fus2106 (ATAAAATTTGCATCCCTCGT-
GAGGAGAATTCTTGTACAGCTCGTCCATGCCG). A third fragment containing
the C-terminal portion of FUS2 and homology to the vector was amplified using
primers RC mChFP-Fus2106 (CGGCATGGACGAGCTGTACAAGAATTCTC-
CTCACGAGGGATGCAAATTTTAT) and JP22 (vector sequence is in uppercase
letters, FUS2 sequence is in lowercase letters: GCTGGAGCTCCACCGCGGTG-
GCGGCCGCTCTAGAACTAGTGGATCCCCctgctccagcgcagtagt) with genomic
DNA as the template. The three PCR products were transformed along with the
pRS415 vector cut with BamHI into a fus2? strain. Plasmid DNA was extracted
from the transformants and the FUS2-mCherryFP fusion construct was confirmed
by restriction digestion. The functionality was verified by complementation of
the fus2 mating defect and by localization of Fus2p-mCherryFP in response to
Live Cell Microscopy
To visualize GFP fluorescence, early log phase cells were incubated with
pheromone then placed on an agar pad containing the appropriate selective
media supplemented with 10 ?g/ml alpha factor. Cells were visualized using
a DeltaVision deconvolution microscope (Applied Precision, Issaquah, WA),
based on a Nikon TE200 (Melville, NY), using a 100? objective, a 50 W Hg
lamp, and a Cool Snap ER CCD camera (Roper Scientific, Trenton, NJ). For
short time courses, images were acquired every 0.36 or 0.46 s using 0.2-s
exposures and 2 ? 2 binning. For Z sections, 24 images were acquired at a
spacing of 0.2 ?m, using 0.4-s exposures, 2 ? 2 binning, and a 93% neutral
density filter. GFP fluorescence was visualized using a FITC filter set (Chroma,
Temperature-shift assays were conducted using the Delta T4 Culture Dish
System (Bioptechs, Butler, PA), and 0.17-mm culture dishes (Bioptechs) were
coated with 20 ?l of concanavalin-A (0.1 mg/ml in 20 mM NaOAc, pH 5.8) for
5 min and then washed three times with phosphate-buffered saline (PBS).
Early log phase cells that had been pretreated with pheromone were then
transferred onto the dish and allowed to bind for 5 min. After binding, excess
medium was removed, and fresh medium supplemented with 10 ?g/ml
alpha factor was added. Dishes were placed on a Delta T4 stage Adapter
(Bioptechs) and visualized as described above.
To visualize actin, cells were fixed for 10 min in 4% formaldehyde and then
washed twice with PBS. Subsequently, cells were resuspended in 50 ?l of PBS
and treated with 25 ?l of Texas red-phalloidin (Molecular Probes, Eugene,
OR) for 1 h in the dark. Cells were washed three times with PBS and then
placed on agarose pads for microscopy. Texas red-phalloidin staining was
visualized using a rhodamine filter set.
Indirect immunofluorescence of Sec4p was performed as previously de-
scribed (Walch-Solimena et al., 1997). Cells were treated with pheromone for
1.5 h at 23°C and shifted to 37°C for varying times before fixation. mAb C123
against Sec4p, a generous gift of P. Novick (UC San Diego, CA), was used
undiluted. To detect the anti-Sec4p, Alexafluor 64–conjugated goat anti-
mouse secondary antibody (Molecular Probes) was used at 1:500, and cells
were visualized using a Cy5 filter set.
Images were deconvolved and collapsed for analysis using the SoftWorx
Imaging software (Applied Precision). Velocity measurements were made on
and contrast were linearly increased, and where needed, pixel density was
resampled using a bicubic algorithm in Photoshop CS3 (Adobe, San Jose, CA).
Myo2p Is Required for the Localization of Fus2p
Paterson et al. (2008) previously demonstrated that Fus2p is
transported via an actin-dependent process to the tip of the
mating projection, where it is anchored to the plasma mem-
brane by Fus1p. To identify the protein(s) responsible for
Fus2p movement, we examined the distribution of Fus2p in
strains containing deletions or conditional mutations in the
various yeast myosins. The type II myosin encoded by
MYO1 was excluded from our initial survey of the yeast
myosin family, because it has no known function in yeast
outside of cytokinesis and its expression is down-regulated
in the presence of pheromone (Brown, 1997; Roberts et al.,
2000). Additionally, because MYO3 and MYO5 are partially
redundant, we utilized a myo3? myo5? strain in which
growth was rescued by a plasmid carrying a temperature-
sensitive allele of MYO5 (myo5-1; Geli and Riezman, 1996).
Fus2p-GFP localized to the shmoo tip normally in both
myo4? FUS1 and myo4? fus1? strains (Figure 1). Consistent
with previous observations, Fus2p-GFP was visible as a
dense spot at the tip of the mating projection in pheromone-
treated FUS1 cells, but assumed a broader distribution in
fus1? strains. Fus2p localization was also unaffected in
pheromone-treated myo3? myo5? [myo5-1] FUS1 and
J. M. Sheltzer and M. D. Rose
Molecular Biology of the Cell2910
Segall, J. E. (1993). Polarization of yeast cells in spatial gradients of alpha
mating factor. Proc. Natl. Acad. Sci. USA 90, 8332–8336.
Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B.N.G., Palmer,
A. E., and Tsien, R. Y. (2004). Improved monomeric red, orange and yellow
fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat.
Biotech. 22, 1567–1572.
Sivadon, P., Bauer, F., Aigle, M., and Crouzet, M. (1995). Actin cytoskeleton
and budding pattern are altered in the yeast rvs161 mutant: the Rvs161
protein shares common domains with the brain protein amphiphysin. Mol.
Gen. Genet. 246, 485–495.
Trueheart, J., Boeke, J. D., and Fink, G. R. (1987). Two genes required for cell
fusion during yeast conjugation: evidence for a pheromone-induced surface
protein. Mol. Cell. Biol. 7, 2316–2328.
Uyeda, T. Q., Abramson, P. D., and Spudich, J. A. (1996). The neck region of
the myosin motor domain acts as a lever arm to generate movement. Proc.
Natl. Acad. Sci. USA 93, 4459–4464.
Valdez-Taubas, J., and Pelham, H.R.B. (2003). Slow diffusion of proteins in the
yeast plasma membrane allows polarity to be maintained by endocytic cy-
cling. Curr. Biol. 13, 1636–1640.
Walch-Solimena, C., Collins, R. N., and Novick, P. J. (1997). Sec2p mediates
nucleotide exchange on Sec4p and is involved in polarized delivery of post-
Golgi vesicles. J. Cell Biol. 137, 1495–1509.
Wedlich-Soldner, R., Altschuler, S., Wu, L., and Li, R. (2003). Spontaneous cell
polarization through actomyosin-based delivery of the Cdc42 GTPase. Science
Whyte, J.R.C., and Munro, S. (2002). Vesicle tethering complexes in membrane
traffic. J. Cell Sci. 115, 2627–2637.
Wilkinson, L. E., and Pringle, J. R. (1974). Transient G1 arrest of S. cerevisiae
cells of mating type alpha by a factor produced by cells of mating type a. Exp.
Cell Res. 89, 175–187.
Myosin V Required for Fus2p Transport
Vol. 20, June 15, 20092919