Cell Motility and the Cytoskeleton 53:251–266 (2002)
The Alpha-Factor Receptor C-terminus Is
Important for Mating Projection Formation
and Orientation in Saccharomyces cerevisiae
Laura G. Vallier,1 Jeffrey E. Segall,2 and Michael Snyder1*
1
Department of Molecular, Cellular, and Developmental Biology, Yale University,
New Haven, Connecticut
2
Department of Anatomy and Cell Biology, Albert Einstein College of Medicine,
New York, New York
Successful mating of MATa Saccharomyces cerevisiae cells is dependent on Ste2p,
the ␣-factor receptor. Besides receiving the pheromone signal and transducing it
through the G-protein coupled MAP kinase pathway, Ste2p is active in the establishment and orientation of the mating projection. We investigated the role of the carboxyl
terminus of the receptor in mating projection formation and orientation using a spatial
gradient assay. Cells carrying the ste2-T326 mutation, truncating 105 of the 135 amino
acids in the receptor tail including a motif necessary for its ligand-mediated internalization, display slow onset of projection formation, abnormal shmoo morphology, and
reduced ability to orient the mating projection toward a pheromone source. This
reduction was due to the increased loss of mating projection orientation in a pheromone gradient. Cells with a mutated endocytosis motif were defective in reorientation
in a pheromone gradient. ste2-⌬296 cells, which carry a complete truncation of the
Ste2p tail, exhibit a severe defect in projection formation, and those projections that
do form are unable to orient in a pheromone gradient. These results suggest a complex
role for the Ste2p carboxy-terminal tail in the formation, orientation, and directional
adjustment of the mating projection, and that endocytosis of the receptor is important
for this process. In addition, mutations in RSR1/BUD1 and SPA2, genes necessary for
budding polarity, exhibited little or no defect in formation or orientation of mating
projections. We conclude that mating projection orientation depends upon the carboxyl terminus of the pheromone receptor and not the directional machinery used in
budding. Cell Motil. Cytoskeleton 53:251–266, 2002. © 2002 Wiley-Liss, Inc.
Key words: polarized cell growth; endocytosis; mating; pheromone receptor; pheromone gradient; yeast
INTRODUCTION
The ability of cells to respond to external cues and
generate dynamic changes in cell architecture, cell morphology, and cellular function has been well documented. Examples include axon formation and guidance
in neurons, slime mold aggregation, and the response to
mating pheromone in yeast mating partners. How sites of
polarized cell growth are selected and how subsequent
polarized cell growth is directed and maintained in response to external cues is not well understood.
Yeast undergoes polarized cell growth during three
distinct developmental stages of its life cycle [Roemer et
al., 1996]. Vegetative cells form new buds in a precise
pattern: haploid yeast cells bud adjacent to the previous
site of cytokinesis, whereas diploids bud from either
pole. When starved for nitrogen, yeast cells form
pseudohyphae in which cells elongate and invade their
© 2002 Wiley-Liss, Inc.
environment to forage for sources of nutrients [Gimeno
et al., 1992].
Laura G. Vallier’s present address is Department of Biochemistry,
Columbia University, 701 W. 168th St. HHSC720, New York City,
NY 10032.
Grant sponsor: NIH National Institute of General Medical Sciences
National Research Service Award; Grant number: GM15937-01;
Grant sponsor: National Science Foundation; Grant number: MCB
9304992; Grant sponsor: National Institute of Health; Grant number:
GM36494.
*Correspondence to: Michael Snyder, Department of Molecular, Cellular, and Developmental Biology, PO Box 208103, Yale University,
New Haven, CT 06520-8103. E-mail: michael.snyder@yale.edu
Received 4 February 2002; Accepted 15 May 2002
Published online 11 October 2002 in Wiley InterScience (www.
interscience.wiley.com). DOI: 10.1002/cm.10073
252
Vallier et al.
During mating in yeast, cells differentiate and form
projections to become specialized cells called shmoos
[Levi, 1956]. Mating is initiated when partners respond
to cell-type specific mating pheromones produced by
cells of the opposite mating type; MAT␣ cells produce
␣-factor and MATa cells produce a-factor. The pheromone binds a seven-transmembrane receptor specific for
the cell type (Ste2p, the ␣-factor receptor, is produced by
MATa cells and Ste3p, the a-factor receptor, by MAT␣
cells), which triggers a common G-protein coupled pheromone-responsive MAP kinase cascade that effects
many cellular responses. Cells exposed to mating pheromone arrest in G1; the actin cytoskeleton is transiently
depolarized and repolarized towards the mating partner
resulting in the formation of a mating projection [Gehrung and Snyder 1990; Hasek et al., 1987; Madden et
al., 1992; Read et al., 1992].
Projections are directed toward mating partners by
means of a pheromone gradient emanating from each
partner. Several lines of evidence indicate that mating
yeast cells track pheromone gradients to direct projection
growth. First, wild-type cells pick partners secreting the
highest level of pheromone [Jackson and Hartwell 1990].
Second, addition of exogenous pheromone to a mating
mixture drastically reduces mating efficiently [Dorer et
al., 1995]. Third, cells orient their projections in a spatial
gradient of pheromone [Segall, 1993]. The mechanisms
of how cells establish and maintain polarized growth
toward their partner once they have initiated projection
formation have not been explored.
Thus far, a limited number of genes important for
shmoo formation have been identified in yeast. Certain
mutations in FAR1 lead to projection formation at axial
sites and the failure to orient toward mating partners
[Chang and Herskowitz 1990; Valtz and Peter 1997;
Valtz et al., 1995]. Mutants that have a disrupted actin
cytoskeleton are defective in mating projection formation. These include mutations in the gene encoding actin,
ACT1, the Rho1p GTPase and its exchange factor Rom2p
and the GTPase activating protein, Bem2p [Read et al.,
1992; Drgonova et al., 1999; Manning et al., 1997; Wang
and Bretscher 1995]. Other mutants that affect cell polarity such as the Cdc42p GTPase [Johnson 1999], its
exchange factor Cdc24p, [Simon et al., 1995; Toenjes et
al., 1999; Zhao et al., 1995], spa2 [Gehrung and Snyder,
1990], bem1 [Chenevert et al., 1992], ste2-T326
[Konopka et al., 1988], slk1 [Costigan et al., 1992], afr1
[Konopka 1993; Konopka et al., 1995], pea2 [Chenevert
et al., 1994; Sheu et al., 1998], and the fig mutants
[Erdman et al., 1998] also exhibit mating projection
defects.
Many of these proteins localize to the shmoo tip
where they might be expected to participate in mating
projection orientation. These include Spa2p [Snyder
1989; Snyder et al., 1991] Sph1p [Roemer et al., 1998],
Cdc24p [Toenjes et al., 1999], Bud6p and Bni1p [Evangelista et al., 1997].
Due to the limitations of mating and morphological
assays, it has been impossible to evaluate the roles of the
majority of these mutants in both projection formation
and the orientation of the projection toward their mating
partners under physiological conditions. Standard shmoo
assays evaluate the ability of yeast cells to form projections in a vast excess of pheromone. We used a visual
technique, the spatial gradient assay, to directly investigate steps and gene products necessary to form the mating projection and to orient the mating projection during
the initiation and process of polarized growth. We find
that the carboxyl terminus of the pheromone receptor is
required for proper mating projection formation and orientation in yeast. We also provide evidence that endocytosis of the receptor is critical for cells to maintain and
improve growth toward pheromone sources.
MATERIALS AND METHODS
Yeast Strains
Yeast strains are listed in Table I. Strains constructed for this study were created using standard methods [Sambrook et al., 1989; Sherman et al., 1986].
Strains Y1200 and Y971 were constructed by integrating
plasmid sequences linearized with StuI carrying either
the STE2 allele (pLV17) or the ste2–⌬296 allele (pLV15)
at the ura3-52 locus in a strain lacking the STE2 gene
(KBY16) (gift of K. Blumer). The presence of the allele
was monitored by stable uracil prototrophy. Plasmid
pLV17 was constructed by ligating a EagI - EcoRI STE2
fragment from pLV16 into YIp5; pLV16 is the STE2
genomic sequences from pYJE111 (gift of J. Thorner) on
a BamHI fragment ligated into Bluescript SK at the
BamHI site; pLV15 carries the ste2–⌬296 allele and was
constructed from a two kilobase EcoRI - SalI fragment of
pYJE106⌬296 (gift of J. Thorner) inserted into YIp5
linearized with EcoRI and SalI. The resulting strains
were maintained on SC-Ura medium [Sherman et al.,
1986] prior to being assayed in the spatial gradient assay
[Segall, 1993].
Spatial Gradient Assay
The spatial gradient assay (SGA) was performed
essentially as outlined in Segall [1993]. Briefly, MATa
cells were grown to early log phase and 1 ⫻ 108 cells
were collected and resuspended in 0.5 ml YPD broth
[Sherman et al., 1986]. A monolayer of cells was immobilized in low melt agarose on a YPD plate (1% yeast
extract, 2% peptone, 2% dextose, 1% agar), then overlaid
with YPD broth. A needle discharging ␣-factor (Sigma
Ste2p C-Terminus Role in Mating Polarity
253
TABLE I. Strains Used in This Study
Strain
ABY355
KBY16
RH1722
RH1860
RH1906
Y602
Y604
Y971
Y1200
7413–3–3
7440-1
Genotype
Source
MATa rsr1-⌬::URA3 ura3 leu2 ade3 trp1
MATa mf␣1::LYS2 mf␣2::LEU2 ste2-⌬::HIS3 sst1-⌬5 ura3-52 trp1-903 ade2-101
his3-⌬200 leu2–3, 112 lys2-801
MATa leu2 his4 bar1-1 ste2⌬::LEU2 ura3::STE2-URA3
MATa leu2 his4 bar1-1 ste2⌬::LEU2 ura3::ste2–345Stop-URA3
MATa leu2 his4 bar1-1 ste2⌬::LEU2 ura3::ste2–337A,345Stop-URA3
MATa ura3-52 lys2-801 ade2-101 trp1⌬ spa2⌬1::URA3 his3-⌬200 trp1⌬
MATa ura3-52 lys2-802 ade2-101 trp1⌬ his3-⌬200
MATa ura3-52::pLV15-ste2-⌬296-URA3 trp1–903 ade2-101 his3⌬200
leu2–3,112 lys2–801 mf␣1::LYS2 mf␣2::LEU2 ste2⌬::HIS3 sst1⌬5
MATa ura3-52::pLV17-STE2-URA3 mf␣1::LYS2mf␣2::LEU2
trp1-903 ade2-101 his3-⌬200 leu2–3, 112 lys2-801 ste2-⌬::HIS3 sst1-⌬5
MATa cry1 SUP4-3ts ade2-1o his4-580a lys2o trp1a leu2-3, 112 ura3-52 ste2-T326
MATa cry1 SUP4-3ts ade2-1o his4-580a lys2o trp1a leu2-3, 112 ura3-52
[Bender and Pringle, 1989]
[Blumer et al., 1988; Reneke et al., 1988]
T6901) at the indicated concentration was placed 10 ␮
above the surface, the dish was overlaid with light mineral oil (Sigma M5904) to prevent evaporation, and the
responses of the cells to the resulting diffusion gradient
were captured using time lapse video-microscopy. Responses of the cells in the field were analyzed using NIH
Image and Microsoft Excel software. Average values are
expressed as ⫾ the standard error of the mean unless
otherwise stated. Ideal or optimal concentration of pheromone is defined as the minimal amount of pheromone
necessary for cells to respond: wild-type cells require
65–70 nM pheromone in the pipet for response to occur
whereas hypersensitive strains such as bar1 and certain
STE2 alleles require less pheromone to elicit response
[Segall, 1993]. Strains carrying both bar1 and a hypersensitive STE2 allele show increased sensitivity over
those carrying either single mutation [Segall, 1993].
Strains RH1860, RH1906, Y971, and Y1200 were grown
to early log in SC-Ura, harvested, and then resuspended
in YPD, processed, and assayed as above. Strains Y971,
Y1200, RH1860, RH1722, and RH1906 were analyzed at
25°C; all others were analyzed at 30°C. In the reorientation assay, the subset of cells that initiated mating
projection formation on the cell surface at an angle
greater than 45° away from the pheromone source were
analyzed for their ability to redirect their projections
toward the pheromone source.
Isotropic Pheromone Assay
Pheromone assays were performed essentially as in
Roemer et al. [1998]. Briefly, logarithmically growing
MATa cells were harvested and washed once in SC–Ura.
Washed cells were incubated at room temperature in 5
␮g/ml ␣-factor; after 1 h an additional 5 ␮g/ml ␣-factor
was added to the cells and incubated for a second hour.
Cells were then washed, fixed, and photographed.
[Rohrer et al., 1993]
[Rohrer et al., 1993]
[Rohrer et al., 1993]
Lab strain
Lab strain
This study (see Materials and Methods)
This study (see Materials and
(Methods)
[Konopka et al., 1988]
[Konopka et al., 1988]
RESULTS
To examine the ability of different yeast strains to
orient mating projection growth, we used a spatial gradient assay (SGA) in which a pipet filled with ␣-factor
mating pheromone is positioned near a group of MATa
cells. In this assay, cells arrest growth and initiate mating
projection formation toward the pheromone source [Segall, 1993; Valtz et al., 1995; Nern and Arkowitz, 1998].
We first examined mating projection orientation more
extensively using wild-type strains. A pipet filled with
␣-factor was positioned near a group of MATa cells and
cell cycle arrest, cell morphology and mating projection
orientation were monitored as a function of time. To
quantify the ability to orient the mating projection, the
angle that occurs between a line from the tip of the
projection to the pipet tip and the line along the actual
direction of projection growth was measured and converted to a cosine value for each cell. Measurements
were taken at the onset of projection formation and 4 h
later. Thus, for optimal orientation (angle equals 0°), the
cosine equals one and for random orientation (angle
equals ⫾ 90°), the cosine equals zero with a large standard deviation. We found that at optimal pheromone
concentrations (67 nM), wild-type cells grow toward the
mating source with an orientation cosine at 4 h of 0.84 ⫾
0.03 (average cosine ⫾ standard error of the mean)
consistent with that reported previously (see Table IIA)
[Segall, 1993].
This high degree of orientation might be due to the
fact that the projections initiate in a favorable direction
relative to the pheromone source or that they continually
track the pheromone gradient and improve their orientation. Consistent with the latter possibility, we note that
projection growth at the start of the experiment is less
well oriented than the same cells 4 h later (0.54 vs. 0.84).
To examine this possibility more directly, we analyzed
254
Vallier et al.
TABLE II. Components Required for Vegetative Polarity Are Not Required for Mating Polarized Cell Growth*
A. Spatial gradient assay
Relevant genotype
rsr1⌬::URA3
spa2⌬::TRP1
WT
Time required to
initiate projection
(min)
Direction of
projection formation
at 0 h
Direction of
projection formation
at 4 h
N
222 ⫾ 11
222 ⫾ 14
253 ⫾ 10
0.59 ⫾ 0.08
0.40 ⫾ 0.08
0.54 ⫾ 0.06
0.85 ⫾ 0.04
0.69 ⫾ 0.06
0.84 ⫾ 0.03
44
47
65
B. Reorientation assay
Relevant genotype
rsr1⌬
spa2⌬
WT
Direction of projection
formation at 0 h
Direction of projection
formation at 4 h
N
0.05 ⫾ 0.12
0.01 ⫾ 0.10
0.14 ⫾ 0.09
0.80 ⫾ 0.06
0.66 ⫾ 0.08
0.77 ⫾ 0.07
16
26
26
* All strains were examined at 67 nM pheromone in the pipet. Data are the average time in minutes of projection initiation (0 h) or of the cosine
of the angle formed between the tip of the pipet to the tip of the mating projection and the line formed by the actual direction of projection growth
(average ⫾ standard error of the mean). For perfect orientation, cosine equals one; random orientation, cosine equals zero. Zero hours is the time
at which the projection is first visible as a slight rounding on the cell surface and 4 h is 4 h after projection initiation. A: Spatial gradient assay.
B: Reorientation assay. See text and Materials and Methods for details. rsr1⌬, ABY355; spa2⌬, Y602; WT, 7440-1 (see Table I for complete
genotypes). N, number of cells analyzed.
those cells that initiated their projection growth greater
than ⫾ 45° away from the pheromone source (26 of 65
cells) for their ability to improve directional growth
toward the pheromone source (a process that we call
reorientation). We found that after 4 h, 92% of this subset
of wild-type cells reoriented toward the pheromone
source with an orientation cosine of 0.77 ⫾ 0.07 (see
Table IIB). Therefore, wild-type cells are able to reorient
efficiently toward the pheromone source.
Bud Site Selection Genes Are Dispensable For
Mating Projection Orientation in an ␣-Factor
Gradient
Many genes have been identified that are necessary
for the successful and efficient mating of two yeast
partners. However, the role of each identified gene product is just now beginning to be understood. We first
examined whether proteins that had been implicated in
various aspects of vegetative polarity or mating were
necessary for the production and orientation of the mating projections using the SGA. Strains carrying representative mutations in bud site selection genes, structural
element genes, signal transduction genes, the ␣-factor
receptor gene STE2, and those mutants that have poor or
altered projection formation were analyzed.
We analyzed two representative mutants defective
in aspects of vegetative polarized cell growth for their
ability to orient projections in the SGA. Mutants carrying
a spa2⌬ mutation display a bud site selection defect in
older diploid mothers [Madden and Snyder, 1992; Snyder, 1989], a defect in low density mating [Gehrung and
Snyder, 1990] and are defective in the default mating
pathway [Dorer et al., 1995]. Mutants carrying a rsr1⌬
(bud1⌬) mutation are defective in haploid and diploid
bud site selection, resulting in a random budding pattern
[Bender and Pringle 1989; Chant and Herskowitz 1991].
We found that both MATa rsr1⌬/bud1⌬ and MATa
spa2⌬ mutant cells are able to orient mating projections
along an ␣-factor pheromone gradient in the SGA (Table
IIA). rsr1⌬/bud1⌬ and spa2⌬ cells initiate projection
formation at 222 ⫾ 11 min and 221 ⫾ 14 min, respectively, which is similar to wild-type cells (253 ⫾ 10 min).
After 4 h, rsr1⌬ mutant cells form and orient projections
as well as wild type (average cosine of projection orientation after 4 h ⫽ 0.85 ⫾ 0.04 and 0.84 ⫾ 0.03, respectively). spa2⌬ cells are also able to orient a mating
projection toward a mating partner (average cosine at 4 h
⫽ 0.69 ⫾ 0.06), although slightly less well than either
rsr1⌬ or wild-type cells. Thus, the mating defect seen in
spa2⌬ mutants is unlikely to be due to problems in
mating projection orientation. In summary, Rsr1p and
Spa2p do not play critical roles in mating projection
orientation, and the mechanism used in directing projection growth during mating appears to require different or
additional components or mechanisms than those controlling budding patterns.
We also tested whether these cells were able to
reorient toward the pheromone source. From the set of
cells subjected to the spatial gradient assay, we analyzed
the subset that initiated mating projection formation on
the cell surface at an angle greater than 45° away from
the pheromone source for their ability to orient their
projections toward the source. We found that MATa
rsr1⌬ and MATa spa2⌬ mutant cells are able to effi-
Ste2p C-Terminus Role in Mating Polarity
ciently reorient mating projections along an ␣-factor
pheromone gradient as well as wild-type MATa cells
(average cosine of projection orientation after 4 h ⫽
0.80 ⫾ 0.06 for rsr1⌬, 0.66 ⫾ 0.08 spa2⌬, and 0.77 ⫾
0.07 for WT; see Table IIB); as before, the values for
spa2⌬ mutants are slightly lower than wild type (0.66 vs.
0.77) (Table IIB).
Spa2p shares 30% sequence identity with Sph1p
over each of three 100 –amino acid domains [Arkowitz
and Lowe, 1997; Roemer et al., 1998]. Mating morphology defects for sph1⌬ mutants are similar but less severe
than those observed for spa2⌬ mutants; sph1⌬ spa2⌬
double mutants exhibit a more severe shmoo morphology
defect than either single mutant. Therefore, we determined if the nearly wild-type levels of orientation in
spa2⌬ mutants might be due to the redundant function of
Sph1p by examining a spa2⌬ sph1⌬ mutant using the
SGA. We found that these double mutants do not display
more severe orientation defects than spa2⌬ mutants
alone (data not shown). In addition, the sph1⌬ mutant
orients as well as wild type (data not shown), consistent
with previous data showing little mating or morphology
defect in this strain [Roemer et al., 1998]. Therefore the
slight defect observed in spa2⌬ mutants is not due to the
redundant function of SPH1⌬.
We also examined two other mutants involved in
the mating response, kss1⌬ and afr1⌬ [Elion et al., 1991;
Konopka et al., 1995]. Kss1p is a protein involved in the
G-protein coupled MAP-kinase cascade that is required
for mating response induction. The AFR1 gene encodes a
protein that is associated with the Ste2p ␣-factor receptor
and shows localization to the shmoo neck. Neither mutant displayed defects in projection orientation (data not
shown).
The Carboxyl Terminus of STE2p Is Required for
Rapid Onset of Shmoo Formation and Projection
Orientation
The STE2 gene encodes the ␣-factor receptor, a
431–amino acid protein that is a member of the seventransmembrane superfamily of G-protein coupled receptors; this family includes rhodopsin and the ␤-adrenergic
receptor [Bockaert and Pin, 1999; Burkholder and Hartwell, 1985]. Various truncations of the cytoplasmic tail
of Ste2p cause increased hypersensitivity to ␣-factor and
poor or altered projection formation [Blumer et al., 1988;
Konopka and Jenness 1991; Konopka et al., 1988]. To
test whether the cytoplasmic carboxyl terminus of the
Ste2p receptor participated in orientation, we examined a
MATa ste2–T326 mutant (truncated at residue 326, which
removes all but 30 amino acids of the carboxy terminal
cytosolic domain) and a MATa STE2 isogenic wild-type
strain in the SGA. We found that MATa ste2–T326 cells
255
were defective in projection initiation, morphology, and
orientation (Fig. 1).
The time required to initiate projections and the
ability to orient projections in the SGA was quantified for
both mutant and wild-type cells. MATa ste2-T326 cells
require almost twice as long for projection initiation as an
isogenic wild-type strain (Table III) and the projections
were much larger than wild type. Wild-type cells take
253 ⫾ 10 min to initiate projection formation whereas
ste2-T326 cells take 425 ⫾ 16 min. At the ideal pheromone concentration for wild type (67 nM in the pipet)
described by Segall [1993] (see Materials and Methods),
ste2-T326 cells are able to establish the initial direction
of projection growth similar in accuracy to wild-type
cells: the cosine at 0 h is 0.47 ⫾ 0.07 compared to 0.54 ⫾
0.06, respectively (standard deviation ⫽ 0.52 for both
wild-type and ste2-T326 cells). However, after 4 h,
ste2-T326 cells exhibit a reduced ability compared to
wild-type cells to improve the orientation of their projections along the pheromone gradient: the cosine at 4 h
is 0.45 ⫾ 0.06 for ste2-T326 compared to 0.84 ⫾ 0.03 for
wild type (standard deviation is 0.51 for ste2-T326 compared to 0.26 for wild type). The fact that cells carrying
ste2-T326 do not exhibit improved orientation at 4 h is
not due to the tenfold hypersensitivity of ste2-T326 mutants to pheromone [Konopka et al., 1988], which would
cause a perceived saturation of receptors on the cell
surface. When ste2-T326 mutant cells were tested in the
SGA using a tenfold lower pheromone concentration (6.7
nM), orientation at 4 h (cosine at 4 h ⫽ 0.57 ⫾ 0.08) was
not significantly improved. Wild–type cells are unable to
respond to this pheromone concentration. Thus, the cytoplasmic carboxyl terminus is dispensable for initial
orientation but is necessary for continued improvement
of direction toward the mating partner.
When the concentration of pheromone in the gradient is increased, we found that the ability of wild-type
cells to orient toward a pheromone source was inversely
proportional to the concentration of the pheromone. One
possibility is that receptor occupation by pheromone
becomes more uniform over the surface of the cell as the
pheromone concentration in the gradient increases and
thus affects the ability of the cell to detect the pheromone
source. This is consistent with the possibility that a
difference between receptor occupancy on the stimulus
side of the cell and the opposite side of the cell is
necessary for that cell to orient toward the stimulus
[Segall, 1993]. At the highest concentration tested (333
nM), the orientation of wild-type cells at 4 h in the SGA
bears a striking similarity to that seen for the ste2-T326
cells (compare wild type at 333 nM pheromone: cosine at
4 h ⫽ 0.50 ⫾ 0.09 (standard deviation equals 0.60) to
ste2-T326 at 6.7 nM: cosine at 4 h ⫽ 0.57 ⫾ 0.08
(standard deviation ⫽ 0.55).
256
Vallier et al.
Fig. 1. ste2-T326 mutants are defective in mating projection formation and orientation. Wild-type (top) and ste2-T326 (bottom) MATa
cells were photographed at 0 h (left) and at 5 or 8 h, respectively
(right), after being in a diffusion gradient of pheromone emanating
from a micropipet (out-of-focus object at the right in all panels) at
30°C. In wild-type cells, mating projections grow and orient along the
pheromone source; ste2-T326 cells take longer to initiate projection
formation and the responding cells have a greater volume with projections
frequently seen growing away from the gradient (direction of growth for
representative cells is marked with arrow). Wild-type strain is 7440-1 and
ste2-T326 strain is 7413-3-3. Concentration of pheromone in the pipet is
67 nM for both strains. Scale bar ⫽ 10 ␮.
The reduction in the ability of ste2-T326 cells to
orient may be due to the fact that cells that initiate growth
in an unfavorable direction are unable to improve their
orientation. We, therefore, examined those cells that initiate their projections not directly in line with the pheromone source for their ability to improve directional
growth toward the pheromone source. Cells that initiated
their projections greater than ⫾ 45° away from the pheromone source were examined for projection reorientation. We found that the subset of ste2-T326 cells that
initiated their projections at an angle greater than ⫾ 45°
from the pheromone source (i.e., cosine at 0 h is less than
0.71) were slightly more impaired in their overall ability
to orient in the gradient (compare Table IIIA and B) as
compared to wild-type cells. At the optimal pheromone
concentration for ste2-T326 (6.7 nM), the improvement
over the 0-h time point is somewhat enhanced but not to
the extent of wild-type cells at their ideal pheromone
concentration (67 nM) (ste2-T326 cosine at 4 h ⫽ 0.48 ⫾
0.10 compared to wild-type cosine at 4 h, which equals
0.77 ⫾ 0.07). Thus, ste2-T326 cells that are not initially
oriented toward the pheromone also are partially impaired in reorienting toward the pheromone gradient.
ste2-T326 Cells Frequently Lose Orientation
Toward the Pheromone Source
The modest defect observed in the average cosine
values for orientation of the ste2-T326 cells (see Table
Ste2p C-Terminus Role in Mating Polarity
257
TABLE III. ste2-T326 Mutants Are Defective in the Onset of Projection Formation and the Improvement of Projection
Orientation*
␣-factor
(nM)
6.7
67
134
333
A. Ability to orient in an ␣-factor gradient
WT
Cosine at 0 h
Cosine at 4 h
Time to
initiate
projection
nd
0.54 ⫾ 0.06
0.36 ⫾ 0.08
0.33 ⫾ 0.10
nd
0.84 ⫾ 0.03
0.67 ⫾ 0.07
0.50 ⫾ 0.09
nd
253 ⫾ 10
225 ⫾ 13
231 ⫾ 16
␣-factor
(nM)
6.7
67
134
333
ste2-T326
N
Cosine at 0 h
65
47
46
0.52 ⫾ 0.07
0.47 ⫾ 0.07
0.40 ⫾ 0.08
0.45 ⫾ 0.08
Cosine at 4 h
Time to
initiate
projection
N
0.57 ⫾ 0.08
0.45 ⫾ 0.06
0.46 ⫾ 0.09
0.36 ⫾ 0.08
464 ⫾ 18
425 ⫾ 16
445 ⫾ 23
408 ⫾ 19
49
63
47
56
N
26
34
32
27
B. Ability to reorient in an ␣-factor gradient
WT
ste2-T326
Cosine at 0 h
Cosine at 4 h
Time to
initiate
projection
nd
0.14 ⫾ 0.09
0.07 ⫾ 0.09
⫺0.19 ⫾ 0.10
nd
0.77 ⫾ 0.07
0.66 ⫾ 0.10
0.41 ⫾ 0.10
nd
251 ⫾ 15
232 ⫾ 16
251 ⫾ 25
N
Cosine at 0 h
Cosine at 4 h
Time to
initiate
projection
26
28
25
0.15 ⫾ 0.09
0.09 ⫾ 0.08
0.15 ⫾ 0.09
⫺0.05 ⫾ 0.10
0.48 ⫾ 0.10
0.28 ⫾ 0.10
0.26 ⫾ 0.12
0.24 ⫾ 0.12
473 ⫾ 22
434 ⫾ 22
425 ⫾ 25
370 ⫾ 24
* All strains were examined at the concentration of pheromone indicated in the first column. Data are the average ⫾ the standard error of the
mean. Cosines are determined from the angle formed between the tip of the pipet to the tip of the mating projection and the line formed by the
actual direction of projection growth. Time is the minutes after exposure to pheromone for projection formation to occur (0 h). Zero hours is
the time at which the projection is first visible as a slight rounding on the cell surface and 4 h is 4 h after projection initiation. A: Spatial gradient
assay. B: Reorientation assay. See Material and Methods and text for further details. WT, 7440-1; ste2-T326, 7413-3-3 (see Table I for complete
genotypes). nd, not determined; N, number of cells analyzed.
III) do not seem to reflect the defect seen in the visual
record (see Fig. 1). We hypothesized that the ste2-T326
average cosine values might not be representative of the
visual record because some of the ste2-T326 cells lose
orientation at various times during the assay. Consistent
with this interpretation, at a concentration of 6.7 nM
pheromone in the pipet, the standard deviation for
ste2-T326 cells at 4 h was almost twice as high as that
observed for wild type (see Table IIIA and B). To determine if this large standard deviation was due to the loss
of orientation in mutant cells, we measured how many
cells analyzed in Table IIIB failed to maintain or improve
orientation by comparing values at 4 h to those at 0 h
(Fig. 2). We found that 15% of STE2 wild-type cells lose
orientation, whereas ste2-T326 cells lose orientation almost three times more frequently than wild-type cells at
their respective ideal pheromone concentration. Wildtype cells lose orientation with greater frequency as
the ␣-factor concentration is increased. In contrast,
ste2-T326 cells consistently exhibit a high rate of loss of
orientation at all pheromone concentrations. Reorienting
cells display similar characteristics for both wild type
and ste2-T326 (data not shown). Thus, MATa cells carrying the ste2-T326 allele appear to be defective in maintaining orientation.
Fig. 2. ste2-T326 cells lose orientation in a pheromone gradient at
increased frequency. Data from Table IIIA were examined to determine the percentage of wild-type and ste2-T326 cells that fail to
maintain orientation in a pheromone gradient. The percentage of cells
that lose orientation (more than 10° away from the pheromone source)
as a function of pheromone concentration tested is shown. Wild-type
cells lose orientation at a low frequency and this frequency increases
as the pheromone concentration is increased; ste2-T326 cells exhibit a
rate of orientation loss almost threefold higher than wild type.
258
Vallier et al.
TABLE IV. ste2-337A,345Stop Mutants Are Defective in the Reorientation Assay*
␣-factor
(nM)
Ability to reorient in an ␣-factor gradient
ste2-345Stop
24
70
ste2-337A,345Stop
Cosine at 0 h
Cosine at 4 h
Time to
initiate
projection
nd
⫺0.19 ⫾ 0.09
nd
0.47 ⫾ 0.08
nd
198 ⫾ 13
N
Cosine at 0 h
—
46
⫺0.13 ⫾ 0.09
⫺0.18 ⫾ 0.12
Cosine at 4 h
Time to
initiate
projection
N
⫺0.02 ⫾ 0.13
0.14 ⫾ 0.13
278 ⫾ 11
237 ⫾ 17
31
29
* Cells whose projections at 0 h initiate at a point on the cell surface greater than 45° away from the pheromone source were analyzed for the
ability to reorient their projections toward the pheromone source. Data are the time of projection initiation at 0 hs and the average cosine of the
angle formed between the tip of the pipet to the tip of the mating projection and the line formed by the actual direction of projection growth
(average ⫾ standard error of the mean). ste2-345Stop, RH1860; ste2-337A,345Stop, RH1906 (see Table I for complete genotype); time is in
minutes; nd, not determined; N, number of cells analyzed in reorientation assay.
Endocytosis of the Receptor Is Needed for
Reorientation
The carboxy-terminal tail of the oligomerized Ste2p
receptor is important for endocytosis of the pheromoneactivated receptor [Jenness and Spatrick, 1986; Overton and
Blumer, 2000; Yesilaltay and Jenness, 2000]. A domain
beginning at residue 331 (SINNDAKSS) within the tail that
is necessary for Ste2p endocytosis has been identified previously [Rohrer et al., 1993]. Because the ste2-T326 mutation truncates the receptor protein prior to this domain, it is
possible that endocytosis of the activated receptor contributes to the ability to track the gradient. Two strains were
tested: one carrying a mutation in STE2 that truncates the
gene product at residue 345 (ste2-345Stop), and the other
carrying the same truncation at residue 345 with a second
mutation of Lysine to Alanine at residue 337 within the
endocytosis domain (ste2-337A,345Stop) [Rohrer et al.,
1993]. The mutant carrying only the ste2-345Stop allele
displays proper endocytosis of the receptor while the
ste2–337A,345Stop mutant abolishes endocytosis [Rohrer et
al., 1993].
We examined these two mutants in the SGA and
the reorientation assay for their ability to orient in a
pheromone gradient. In the reorientation assay, the
ste2-345Stop mutant was able to improve its orientation
in the gradient whereas the ste2–337A,345Stop mutant
failed to improve projection orientation (Table IV). We
note that under SGA conditions, both ste2-345Stop and
ste2-337A,345Stop mutants were unable to maintain projection orientation (data not shown). We also note that
cells carrying the wild-type STE2 allele at the same
ectopic site do not show improved orientation in the SGA
4 h after projection initiation (data not shown).
The reorientation data suggest that endocytosis is
an important mechanism for the reorientation of mating
projections and that an intact SINNDAKKS domain in
the Ste2p carboxyl terminus is necessary. Although in the
reorientation assay ste2-337A,345Stop mutants display a
more severe reorientation defect than ste2-T326 mutants,
ste2-337A,345Stop mutants do not completely mimic the
spectrum of phenotypes observed in ste2-T326 mutants:
the timely initiation of projection formation is unimpaired and the morphology of the projection appears wild
type. Thus, endocytosis is likely to be an important but
not exclusive mechanism in the orientation or reorientation of mating projections but is not involved in projection initiation or morphology.
Mutants Lacking the Entire Carboxy-Terminal Tail
of STE2p Are Unable to Efficiently Form
Projections in an ␣-Factor Gradient
Because the Ste2-T326p mutant receptor retains 30
amino acids at the carboxyl terminus that are still present
in the cytoplasm, we determined whether the residual
orientation is due to the presence of these residues. A
receptor mutant truncated at residue 296 (ste2-⌬296) that
removes the entire cytoplasmic tail of STE2 [Blumer et
al., 1988] was constructed and integrated at the ura3-52
locus. In parallel, we integrated the wild-type STE2 gene
with its full-length tail at the same locus in the identical
background (see Materials and Methods). We examined
the ability of these mutants to orient in the spatial gradient assay. Cells carrying the ste2-⌬296 mutation have
been reported to be 100-fold hypersensitive to pheromone [Blumer et al., 1988] and both wild-type and mutant constructs are in a bar1 background, which increases
sensitivity to pheromone [Segall, 1993]. Therefore, we
lowered the concentration of pheromone to compensate
for this increased sensitivity. Surprisingly, we found that
although ste2-⌬296 cells arrested budding, they failed to
initiate projections and grew isotropically in diameter,
thereby forming larger round cells (data not shown).
We then raised the pheromone concentration to see
if these cells could form projections with higher amounts
Ste2p C-Terminus Role in Mating Polarity
259
Fig. 3. ste2⌬296 mutants rarely initiate projection formation in a
pheromone gradient but form projections in an isotropic pheromone
assay. A: STE2 (left) and ste2⌬296 mutant (right) strains were photographed after 20 or 15 h, respectively, in a pheromone gradient at
25–27°C. Pipet containing ␣-factor is indicated by black arrowhead.
Pheromone concentrations in the pipet are 7 nM for the STE2 strain
and 5 nM for the ste2⌬296 strain. White arrows mark the direction of
growth. Scale bar ⫽ 10 ␮. B: STE2 and ste2⌬296 strains were
photographed after treatment with isotropically added ␣-factor pheromone (5 mg/ml; see Materials and Methods). Scale bar ⫽ 10 ␮.
STE2, Y1200; ste2–⌬296, Y971.
of pheromone. We found that as the pheromone concentration increased, ste2-⌬296 mutants displayed an increased, although still extremely diminished, ability to
form shmoos (Fig. 3A). Lack of projection initiation in
these cells was not due to insufficient pheromone concentration because the cells still underwent G1 arrest. We
quantified the ability to form projections at varying concentrations of pheromone in Table VA. Wild-type cells
formed projections efficiently at the expected pheromone
concentration. We found that ste2-⌬296 cells were unable to form projections efficiently over a broad range of
pheromone concentrations in the gradient (Table VA). At
the highest concentration tested, less than half of the
cells responded; at concentrations closer to ideal for
ste2-⌬296 arrest (0.2– 0.75 nM), projection initiation
drops to less than 10%. In contrast, almost all wild-type
cells showed projection initiation. Therefore, the 30 residues between amino acid 296 and 326 in Ste2p seem to
be important for projection initiation but not for cell
cycle arrest.
Because neither the Ste2-T326p nor the Ste2⌬296p
mutant receptors are internalized, we determined whether
those few projections formed by the ste2-⌬296 cells had
a similar or more severe orientation defect than that
260
Vallier et al.
TABLE V. ste2⌬296 Cells Are Able to Make Projections Only Rarely and Are Unable to Reorient Them in a Pheromone Gradient*
A. Projection formation
Number of shmoos/ total number of cells scored
␣-factor (nM)
0.2
0.5
0.75
1.0
5–10
WT
ste2-⌬296 (%)
nd
nd
nd
nd
106/109 (97%)
4/55 (7)
6/48 (13)
2/25 (8)
10/41 (24)
45/113 (40)
B. Reorientation assay
Ability to reorient in an ␣-factor gradient
WT
ste2-⌬296
␣-factor
(nM)
Cosine at 0 h
Cosine at 4 h
Time to
initiate
projection
0.2–1
5–10
nd
0.17 ⫾ 0.07
nd
0.51 ⫾ 0.07
nd
347 ⫾ 22
N
Cosine at 0 h
—
43
⫺0.37 ⫾ 0.15
⫺0.42 ⫾ 0.10
Cosine at 4 h
Time to
initiate
projection
N
⫺0.29 ⫾ 0.17
0.08 ⫾ 0.14
906 ⫾ 84
666 ⫾ 78
14
21
* A: The cells able to initiate projection formation were counted at each pheromone concentration and were expressed as a fraction of the total
number of cells retaining the plasmid during the experiment. Percentage is in parentheses. B: Ability of cells to reorient in a pheromone gradient.
Data are the average cosine of all cells that formed and maintained projections for 4 or more hours and the average time to initiate projection
formation (average ⫾ the standard error of the mean). Cells that initiated a projection in A but did not maintain it for at least 4 h due to the
termination of the experiment or loss of plasmid construct were not analyzed in B. ste2-⌬296, Y971; STE2, Y1200 (see Table I for complete
genotypes); nd, not determined; N, number of cells analyzed in the reorientation assay.
observed in ste2-T326 cells. We found that those
ste2–⌬296 cells able to form a projection initiated them
more slowly than ste2-T326 cells and over twice as slow
as the STE2 control cells (Table VB). For ste2-⌬296
mutants, the average time of projection initiation was 761
min (ranging from 666 to 1,230 min; n ⫽ 35) whereas for
ste2-T326 mutants, the average time of onset was 425
min (ranging from 370 to 473 min; n ⫽ 215) as seen in
Tables IIIB and VB. In comparison, wild-type cells in
this strain background initiated projection orientation
with an average time of 347 min (n ⫽ 43) (Table VB).
Wild-type cells arrested growth and then produced mating projections in 88% of the cells; 12% of the cells
budded with an average doubling time of 3.5 h (and
presumably lost the construct in the non-selective medium). In contrast, 23% of the ste2-⌬296 cells arrested
growth and formed mating projections (ranging from 6 to
39% depending on concentration) (see Table VA); 55%
of the cells budded (ranging from 39 to 71%) with an
average budding time of 9.3 h (ranging from 5.3 to
12.5 h) (data not shown); the remainder (22%) arrested
and swelled but neither budded or formed projections.
However, the budding by the ste2-⌬296 mutants in a
pheromone gradient does not seem likely to be due to
loss of the ste2-⌬296 construct because ste2-⌬296 cells
tend to bud at a slower rate than that observed for cells
without the construct, which are unable to respond to a
pheromone gradient. We note that ste2-⌬296 cells are
unimpaired for vegetative growth. Therefore, at this time
it is unclear what role ste2-⌬296 may have in promoting
this slow budding. Additionally, ste2-⌬296 cells are defective in the reorientation assay (average cosine after 4 h
is 0.08 ⫾ 0.14 vs. ⫺0.42 ⫾ 0.10 at onset of projection
initiation); in contrast, STE2 cells exhibit marked improvement in the reorientation assay (average cosine
after 4 h is 0.51 ⫾ 0.07 vs. 0.17 ⫾ 0.07 at onset of
projection of initiation) (Table VB). Thus, mating projection initiation and reorientation in an ␣-factor gradient
is partially dependent on residues 296 to 326 in Ste2p.
ste2-⌬296 Mutants Form Projections in an
Isotropic ␣-Factor Assay
In a standard shmoo assay, MATa cells are treated
with a vast excess of ␣-factor pheromone (5 ␮g/ml) for
a short period of time (2 h). Log-phase wild-type and
ste2-⌬296 cells were treated identically with ␣-factor for
2 h and their response was photographed. Both wild-type
and ste2-⌬296 cells were able to form normal projections
in isotropic pheromone at a high concentration (Fig. 3B).
The ability of the ste2-⌬296 mutant to form normal
projections indicates first that the mutant is capable of
forming mating projections and, second, that the mating
response pathway is intact.
DISCUSSION
In this study, we have examined the function of
carboxy-terminus of the Ste2p ␣-factor receptor in mat-
Ste2p C-Terminus Role in Mating Polarity
ing polarized cell growth using the spatial gradient assay,
an assay that approximates physiological conditions for
mating projection formation in MATa yeast cells [Segall,
1993]. We presented evidence indicating that the formation of mating projections is a complex, separable process involving the selection of the site of projection
formation, and the formation and orientation of the mating projection toward the pheromone of the mating partner. By analyzing mutations within the cytoplasmic carboxy-terminal tail of the Ste2p ␣-factor receptor, we
have shown definitively that the cytoplasmic tail is necessary for both the initiation and the maintenance of
orientation of the mating projection toward a mating
partner. We further present data that endocytosis of the
activated receptor contributes to reorientation of the projection toward a pheromone source.
Budding Polarity Components Are Not Required
for Mating Polarity
We have shown that components required for polarized cell growth during vegetative growth are not
necessary for polarized cell growth during the mating
response when the Ste2p receptor is intact. Mutants lacking RSR1 (BUD1) appear to orient projections as well as
wild type. Thus, a subset of proteins is specifically dedicated to vegetative polarity function. This is not surprising because of the different nature of the signals: vegetatively growing cells use an intrinsic, internal signal to
mark the site of the next budding event while cells
responding to a mating partner need to utilize a more
adaptable way to organize growth components to form a
projection toward the projection of the partner [for reviews see Madden and Snyder, 1998; Roemer et al.,
1996]. However, the process may be more complex because Nern and Arkowitz [1999] showed that in the
absence of FAR1-CDC24-mediated morphological
changes, RSR1/BUD1 was essential for pheromone-induced polarized cell growth.
Mutants lacking Spa2p, which is required for polarized growth in budding diploid cells, exhibit a minor
but consistent reduction in the ability to orient projections. One possibility is that the Spa2p homolog, Sph1p,
has a redundant function with Spa2p. [Arkowitz and
Lowe, 1997; Roemer et al., 1998]. In the spatial gradient
assay, mutants deleted for SPH1 do not show any reduction in the ability to orient mating projections and the
double mutant, spa2⌬ sph1⌬, does not further enhance
the weak spa2 defect. Thus, SPA2 and SPH1 are not
required for projection orientation but may assist in the
process presumably by concentrating growth at the tip
[Sheu et al., 2000].
261
ste2-T326 Mutants Show Defects in Mating
Orientation
Previous data had shown that the shmoo morphology was blunted in ste2-T326 mutants in a shmoo assay
but its role in projection orientation had not been assessed [Konopka et al., 1988]. We have shown that this
mutant is defective for several aspects of mating projection initiation and orientation. Mutants carrying the
ste2-T326 allele require a longer time to initiate projection formation; the projections have a larger volume and
often turn away from the pheromone gradient. These
results are in agreement with a previous analysis of Ste2p
in which ste2-T326 cells exposed isotropically to pheromone for 6 h exhibit abnormal shmoo morphology
[Konopka et al., 1988]. However, our analysis reveals
new functional defects in these mutants. Analysis of
ste2-T326 mutant cells in the spatial gradient assay using
increasing concentrations of ␣-factor pheromone revealed that regardless of concentration, the initial direction of projection formation is equivalent to that in wildtype cells. However, cells carrying these mutant
receptors tend to lose orientation in a pheromone gradient, such that some cells are oriented toward the pheromone gradient then turn away from the gradient.
Wild-type cells rarely lose orientation at a normal concentration of pheromone but as the concentration of
␣-factor increases, loss of orientation increases, consistent with loss of wild-type mating efficiency in saturating
concentrations of ␣-factor [Dorer et al., 1995]. In contrast, almost half of the ste2-T326 mutant cells lose
orientation at all pheromone concentrations. The hypersensitivity to pheromone exhibited in cells carrying the
ste2-T326 allele is unlikely to be the reason for the loss
of orientation because when the concentration of pheromone is lowered to compensate for the hypersensitivity,
the defect remains: mutant cells continue to lose orientation at a similar frequency; at the same concentration of
pheromone, wild-type cells do not arrest growth. Thus,
this work demonstrates for the first time that the cytoplasmic carboxy-terminal tail of the Ste2p receptor appears to be required for efficient initiation as well as for
the orientation of mating projections in yeast.
The Role of Endocytosis
The carboxyl terminus of Ste2p is predicted to
contain 135 amino acids located intracellularly [Burkholder and Hartwell, 1985; Cartwright and Tipper, 1991].
The Ste2p tail has many potential phosphorylation sites
and an endocytosis motif, which contribute to the internalization of the receptor [Chen and Konopka, 1996;
Hicke et al., 1998; Rohrer et al., 1993]. The endocytosis
motif, 331 SINNDAKSS 339, is required for both constitutive and ligand-mediated internalization of the pher-
262
Vallier et al.
omone receptor, which is signaled by the ubiquitination
of the lysine at residue 337 [Hicke and Riezman 1996].
The ste2-T326 mutation truncates the last 105 amino
acids of the receptor, which removes the endocytosis
motif; 30 amino acids still remain in the cytoplasm
[Konopka et al., 1988]. To test whether endocytosis of
the receptor plays a role in projection orientation, we
examined two alleles of the STE2. Cells carrying the
ste2-345Stop allele have normal internalization of the
receptor while cells carrying the additional alteration of
the lysine to alanine at residue 337 in the endocytosis
motif (ste2-337A,345Stop) lack internalization of the receptor [Rohrer et al., 1993]. When the cells were subjected to the reorientation assay, we found that the
ste2-337A,345Stop mutants were more defective in reorienting than the ste2-345Stop cells (Table IV). Thus,
endocytosis of the activated receptor contributes to reorientation of the mating projection.
In contrast to ste2-T326 cells, the time of projection
initiation of the ste2-337A,345Stop cells was similar to
that seen for the ste2-345Stop cells as well as for cells
carrying STE2 at its endogenous locus. This suggests that
the cause for the delayed onset of projection initiation in
ste2-T326 cells may be the loss of the amino acids
between 326 and 345. We note that in the spatial gradient
assay, both the ste2-345Stop and the ste2-337A,345Stop
strains showed a defect in orientation.
Tailless Receptor Mutants Are Defective in
Projection Formation
Both ste2-⌬296 and ste2-T326 mutants are defective in the endocytosis of activated pheromone receptors
[Blumer et al., 1988; Konopka and Jenness, 1991; Rohrer
et al., 1993]. However, the phenotype of ste2-⌬296 is
much more severe than ste2-T326: when the residues
after amino acid 296 are removed, cells can no longer
efficiently establish projection formation and those rare
projections that do occur are misshapen and are not
directed toward the pheromone source. These results
are consistent with the drop in mating efficiency in
ste2-⌬296 mutants compared to ste2-T326 mutants
[Konopka et al., 1988; Reneke et al., 1988]. Therefore,
other mechanisms besides endocytosis must contribute to
projection formation and orientation.
The distal half of the carboxy-terminus of the pheromone receptor has been shown to form preactivation
complexes with its G-proteins [Docil et al., 2000]. Also,
Cdc24p, Far1p, and Ste4p (G-beta) form a complex as
gauged by the two-hybrid assay and localization of
Cdc24p to the site of receptor activation occurs in the
absence of the other factors in the complex [Nern and
Arkowitz, 1999; Toenjes et al., 1999]. Furthermore, shuttling of the Cdc24p-Far1p complex by Msn1p from the
nucleus to the site of polarized cell growth occurs in
response to mating pheromones [Blondel et al., 1999;
Butty et al., 1998; Shimada et al., 2000]. These varied
mechanisms could rely on the Ste2p carboxy-terminus to
localize the growth components to the site of activated
pheromone receptors and together provide a timely and
efficient response to mating pheromone.
In a pheromone gradient, most ste2-⌬296 mutants
arrest growth or bud at an extremely slow rate and
continue to swell. The slow budding may reflect the
weakening or loss of a budding checkpoint that is maintained during mating through a component that is associated directly or indirectly with the Ste2p cytoplasmic
tail. Alternatively, it is possible that this is a secondary
effect due to the increased cell volume diluting the concentration of a critical repressor of cell division. Finally,
the slow budding kinetics of ste2⌬296 cells might reflect
a differential progression to a signaling threshold in
which adaptation mechanisms to the pheromone signal
are activated.
Surprisingly, we found that ste2-⌬296 cells are able
to form a seemingly normal mating projection in an
isotropic shmoo assay at high concentrations of ␣-factor.
This is probably due to the scavenging of bud site selection machinery already in place for the next cell division
that is seen for wild-type cells [Madden and Snyder,
1992]. Madden and Snyder [1992] previously demonstrated that in an isotropic shmoo assay at high concentrations of ␣-factor, cells use the previous site of cytokinesis to form the projection.
The defect observed in ste2-⌬296 cells in the spatial gradient assay might be due to the inability to select
a site of projection formation (shmoo site selection) after
the initial loss or masking of the bud site tags, which
occurs when the cell is exposed to lower physiological
concentrations of pheromone [Roemer et al., 1996]. This
would explain why cells exposed to a pulse of high
pheromone concentration can form a mating projection
while those exposed to a physiological mating pheromone gradient can not. We favor this possibility. We
note that the defect seen in the spatial gradient assay does
not seem to be a secondary consequence of the low
concentrations of pheromone used to compensate for the
hypersensitivity of this mutant because in these assays
ste2-⌬296 cells arrest comparably to wild-type cells, thus
indicating that the G-protein coupled MAP kinase cascade is functional in these mutants. However, it does not
rule out that these cells may arrest normally but require
a higher concentration of pheromone to form a projection. Another possibility is that upon severely truncating
the receptor, its conformation might be altered such that
proteins normally associating with other parts of the
receptor that are required for mating polarized cell
growth now can no longer associate with it. This does not
Fig. 4. Model. A: Endocytosis contributes to projection orientation.
Cartoons represent how endocytosis of activated receptors might target
the actin cytoskeleton to the site of highest density. MATa cells express
the Ste2p receptor (Y) uniformly over the cell surface; the cartoon
draws them only on the mating partner side for simplicity. When
␣-factor pheromone binds to the Ste2p receptor and the activated
receptor is endocytosed, the actin cytoskeleton (thin lines) is directed
to this region, presumably that of the highest receptor activation. In
conjunction with the recycling of unoccupied receptors through constitutive endocytosis, this would serve as a positive feedback loop to
concentrate newly synthesized Ste2p to the site receiving the maximal
signal from the mating partner. Cells then would continually improve
the direction of growth toward the pheromone source. B: Schematic
cartoon of the regions of the Ste2p receptor tail and summary of
experimental findings.
264
Vallier et al.
appear to be likely because of normal projection formation in the isotropic pheromone assay.
We note that the misorientation and loss of orientation that we have observed in the receptor mutants do
not appear to be due to protein expression or instability.
Cells carrying either ste2-T326 or ste2-⌬296 were examined for protein expression in the original studies and
found to be indistinguishable from wild-type strains
[Konopka et al., 1988; Reneke et al., 1988]; GFP- or
HA-tagged versions of ste2-T326p were found to be
comparable to tagged Ste2p levels by Western blot analysis [Yesilaltay and Jenness, 2000]. Strains carrying
ste2-345Stop or ste2-337A,345Stop mutations were also
tested for protein stability in the original endocytosis
study and found to be similar [Rohrer et al., 1993]. Thus,
protein expression and protein folding do not appear to
be grossly aberrant in the ste2 C-terminal truncation
mutants.
Model
Based on our observations, an improved model for
how cells track pheromone gradients can now be proposed. First, we suggest that the receptor tail is required
for projection formation and selecting the site of projection formation in pheromone gradients. Cells that lack
the entire tail (ste2-⌬296 cells) generally do not form
mating projections and those that do form them do so at
a random location. Since ste2-⌬296 cells treated with
isotropic levels of pheromone form mating projections, it
is likely that they use bud site tags that are already in
place at the previous site of cytokinesis [Madden and
Snyder, 1992]. Docil et al. [2000] demonstrated that the
distal half of the Ste2p carboxy-terminal tail is required
for the formation of preactivation complexes with their
G-proteins. Our data indicate that a region in the proximal half of the carboxy-terminal domain between amino
acids 296 and 326 is required for projection formation
and initial orientation because cells that contain these
thirty residues form projections that are often oriented
properly toward a pheromone source (Fig. 4B). Perhaps
this region interacts with Cdc42p, Cdc24p, or one of the
polarity establishment proteins to help direct polarized
cell growth toward this site; such mutants have a polarized growth defect similar to the ste2-⌬296 cells [Chenevert et al., 1994].
Second, we propose that endocytosis of the receptor is required for tracking pheromone gradients. Cells
mutant for the endocytosis signal lose orientation and do
not track gradients as well as wild-type cells. We presume that endocytosis is useful in several respects. As the
receptor binds pheromone, intracellular signaling presumably helps to preferentially direct the actin cytoskeleton and polarized secretion toward the region of the cell
receiving maximal receptor activation. By constantly
turning over the receptor and directing new receptors
toward the stimulated sites, a positive feedback loop is
established in which increasing number of receptors are
directed toward the region of the cell receiving the maximal signal. This will lead to a constant improvement of
growth toward the pheromone source (Fig. 4A). Cells
that cannot endocytose the receptor will not be able to
reinforce polarized secretion and growth toward the maximally stimulated region and thus will not track pheromone gradients efficiently. In addition, in the absence of
endocytosis, in principle, all of the cell surface receptors
over the entire surface of the cell will ultimately bind
ligand and potentially direct signaling in many locations.
Conclusions
In summary, these results directly demonstrate that
the cytoplasmic tail of the Ste2p receptor is critical for
mating projection site selection, projection formation,
and orientation in a pheromone gradient and these processes have been localized to regions within the carboxyterminus of the receptor. It will be interesting to further
define the domains and the mechanisms responsible for
each of the processes. Given the high degree of conservation of signaling mechanisms between yeast and other
eucaryotes, we expect that similar mechanisms are likely
to exist in other organisms.
ACKNOWLEDGMENTS
We thank Michael Cammer at the Albert Einstein
Imaging Center for invaluable assistance; James
Konopka, Howard Riezmann, Ken Blumer, and C. Stefan
for strains; and Jeremy Thorner for plasmids. This work
was supported in part by an Individual National Research
Service Award (GM15937-01) to L.G.V., a National
Science Foundation grant to J.E.S. (MCB 9304992), and
a National Institute of Health grant to M.S. (GM36494).
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