Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, CB2 2QH, United Kingdom
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Abstract |
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Oriented cell growth requires the specification of a site for polarized growth and subsequent orientation of the cytoskeleton towards this site. During
mating, haploid Saccharomyces cerevisiae cells orient
their growth in response to a pheromone gradient overriding an internal landmark for polarized growth, the
bud site. This response requires Cdc24p, Far1p, and a
heterotrimeric G-protein. Here we show that a two-
hybrid interaction between Cdc24p and G requires
Far1p but not pheromone-dependent MAP-kinase signaling, indicating Far1p has a role in regulating the association of Cdc24p and G
. Binding experiments demonstrate that Cdc24p, Far1p, and G
form a complex in
which pairwise interactions can occur in the absence of
the third protein. Cdc24p localizes to sites of polarized
growth suggesting that this complex is localized. In the
absence of CDC24-FAR1-mediated chemotropism, a
bud site selection protein, Bud1p/Rsr1p, is essential for
morphological changes in response to pheromone.
These results suggest that formation of a Cdc24p-Far1p-G
complex functions as a landmark for orientation of the cytoskeleton during growth towards an external signal.
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Introduction |
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EUKARYOTIC cells are able to polarize their growth in
response to both external and internal signals. Polarization to external signals plays a crucial role in
development and tissue formation. During yeast mating,
cells of opposite mating type secrete peptide pheromones
and respond to pheromone from their mating partner (for
review see Sprague and Thorner, 1992; Chenevert, 1994
;
Leberer et al., 1997a
). Mating pheromone binds to specific
G-protein-coupled receptors on cells of opposite mating
type (Bender and Sprague, 1989
; Blumer et al., 1988
). Receptor activation results in cell cycle arrest, transcriptional
activation, morphological changes, and polarized growth
towards a partner cell (Sprague and Thorner, 1992
; Chenevert, 1994
; Leberer et al., 1997a
).
Cells respond to a gradient of mating pheromone by
oriented growth along this gradient (Segall, 1993). Such
chemotropic growth is essential for efficient mating (Dorer et al., 1995
; Valtz et al., 1995
; Nern and Arkowitz,
1998
). During oriented growth, the actin cytoskeleton and
secretory apparatus polarize towards the tip of the mating
projection (Baba et al., 1989
; Read et al., 1992
). As a result
cell wall and plasma membrane material is deposited at
the tip of this pear-shaped cell known as a shmoo (Lipke
et al., 1976
; Tkacz and MacKay, 1979
). Pheromone receptors and the heterotrimeric G-protein composed of G
(GPA1), G
(STE4), and G
(STE18) are required for
chemotropic growth (Jackson et al., 1991
; Schrick et al.,
1997
; Xu and Kurjan, 1997
). Certain alleles of the cyclin-dependent kinase inhibitor FAR1, such as far1-H7 (Valtz
et al., 1995
), and of the GDP-GTP exchange factor for the small GTPase Cdc42p CDC24, such as cdc24-m1 (Nern
and Arkowitz, 1998
), are specifically defective in chemotropic growth. These mutants are unable to orient in a
pheromone gradient and select a site for mating projection
growth adjacent to their previous bud site. Similarly, in the
presence of saturating uniform concentrations of mating
pheromone, shmoo formation occurs next to the previous bud site (Madden and Snyder, 1992
; Dorer et al., 1995
).
The latter process has been referred to as default mating
(Dorer et al., 1995
).
During vegetative growth, haploid cells bud at a specific
site next to their previous bud site, resulting in a characteristic axial budding pattern (Chant and Pringle, 1995). The
BUD genes are required for this budding pattern (Chant
and Herskowitz, 1991
; Drubin and Nelson, 1996
). During
budding, cells polarize their actin cytoskeleton (Adams
and Pringle, 1984
; Kilmartin and Adams, 1984
) and secretory apparatus towards the bud site (Tkacz and Lampen,
1972
; Field and Schekman, 1980
). However, this internal signal generated during budding is overridden upon exposure to a mating pheromone gradient, allowing cells to orient growth towards their mating partner (Madden and
Snyder, 1992
). How cells switch from an internally programmed polarized growth process to a process dictated
by an external cue is unknown.
The pheromone receptors and the heterotrimeric G-protein are also required for cell cycle arrest, mitogen-activated
protein (MAP)1-kinase-mediated gene induction, and cell
morphological changes during mating (Sprague and Thorner, 1992; Chenevert, 1994
; Leberer et al., 1997a
). Genetic
studies indicate that G
activates all these processes
(Whiteway et al., 1990
) with G
having a negative regulatory role (Dietzel and Kurjan, 1987
; Miyajima et al., 1987
). By analogy to other G-protein coupled receptors, receptor
activation results in dissociation of G
from G
. G
is
found as a complex at the plasma membrane (Hirschman
et al., 1997
).
Previously we have shown that an association between
G and Cdc24p is involved in oriented growth during
mating (Nern and Arkowitz, 1998
). We now show this
Cdc24p-G
complex also contains Far1p. Genetic studies are consistent with the involvement of Far1p in this
complex. Cdc24p localizes to sites of polarized growth in
shmooing cells, suggesting that the complex is localized. In
the absence of growth orientation mediated by this complex, cells form a mating projection adjacent to the bud
site in a manner that is dependent on BUD1, suggesting
Bud1p can regulate Cdc24p when chemotropic signaling is
blocked. Together our results suggest that Cdc24p-Far1p-G
acts as a landmark for cytoskeleton orientation in response to a pheromone gradient.
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Materials and Methods |
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General Techniques
Standard techniques and media were used for growth and genetic manipulation of yeast (Rose et al., 1991). Unless otherwise indicated, yeast cells
were grown at 30°C.
Strains and Plasmids
The yeast strains used in this study are described in Table I. In general, deletion mutants were constructed by PCR-based gene disruption as described (Arkowitz and Lowe, 1997; Nern and Arkowitz, 1998
).
Far1
strains were constructed either by PCR-based gene disruption (
-1) or
with a knockout cassette (
-2). This cassette contained the FAR1 ORF
followed by 100 bp 3' sequence with URA3Kl replacing all but the first
109 codons. Far1-H7, a far1 allele with a truncated COOH terminus
(Valtz et al., 1995
), was constructed by replacing codons 757-830 of FAR1
with a stop codon followed either by HIS5Sp or URA3Kl. Gene disruptions were confirmed by PCR and phenotype including mating defects
with wild-type and enfeebled testers, mating pheromone growth arrest,
and budding pattern. The
bud1 cdc24-m1 double mutant was crossed
with appropriate wild-type haploid and random spore analyses demonstrated that the inability to shmoo in the presence of pheromone specifically segregated with
bud1 cdc24-m1 and this mating defect was observed in both mating types.
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A single HA epitope (amino acids YPYDVPDYA) was added to the
NH2 terminus of STE4 by PCR. HASTE4 (including 394 bp 5' upstream of the ATG) was cloned into pRS406 and two-step gene replacement (Scherer and Davis, 1979) of STE4 was used to construct RAY910. Protein A-tagged Far1p (RAY1258 and RAY1336) and Ste4p (RAY1276) strains were constructed by PCR-based gene replacement using pZZ-His5 (Rayner and Munro, 1998
) as template and oligonucleotides with 60 nucleotides 5' and 3' of the termination codon. Myc epitope-tagged Cdc24p
and cdc24-m1p strains were constructed by PCR-mediated gene replacement as described (Nern and Arkowitz, 1998
) except the sequence encoding a triple myc tag (MEQKLISEEDL MEQKLISEEDL MEQKLISEEDL) was directly fused to the Cdc24p NH2 terminus. Gene replacements were confirmed by PCR and expression of epitope-tagged proteins of the correct size by immunoblotting using either 12CA5 (anti-HA) mAB tissue culture supernatant at 1:40 dilution, anti-protein A mAb
(Sigma) at 1:2,000 dilution, or anti-myc polyclonal serum (Santa Cruz) at
1:500 dilution followed by ECL (Amersham). Strains with tagged proteins
mated with wild-type mating efficiencies and arrested growth normally in
response to
-factor.
Cdc24HAGFP was constructed by fusing an HA epitope followed by
PacI, SphI, NotI, and SacII restriction sites to the COOH terminus of
Cdc24p using PCR and p414Cdc24 (Nern and Arkowitz, 1998) as a template. This resulted in p414Cdc24HA which had the amino acids YPYDVPDYAGLIKHARPPPRG fused to the COOH terminus. Yeast enhanced green fluorescent protein (GFP; Cormack et al., 1997
) followed by
the ADH terminator was PCR amplified from pMK199 (a gift from E. Schiebel) with an oligonucleotide that added a PacI site at the 5' end and a
NotI site at the 3' end. This PCR product was cloned into p414Cdc24HA
using PacI and NotI sites resulting in Cdc24p followed by YPYDVPDYAGLIKGSGAGAGAGAGA fused to GFP followed by the ADH terminator (p414Cdc24HAGFP). p416GalHASte4 was constructed by cloning
HASTE4 into pRS416 containing the Gal1/10 promoter.
The ADE2 gene from pSP73Ade2 (cloned by PCR from genomic DNA with oligonucleotides that added an EcoRI site at the 3' end and a XhoI site at the 5' end) was released by digestion with EcoRI and BsrGI followed by blunting. This fragment was cloned into pRS425 in which the LEU2 gene had been removed by digestion with Tth111I and NaeI followed by blunting resulting in p2µA. TPI-STE18 (triose phosphate isomerase promoter) from p416TSte18 (pRS416 with TPI cloned into the SacII EagI sites and STE18 cloned into BamHI EcoRI sites) was cloned into the SacI EcoRI sites of p2µA resulting in p2µATSte18. An oligonucleotide encoding the GAL4 nuclear localization signal (NLS) MDKAELIPEPPKKKRKVEL followed by a NcoI restriction site was cloned into EagI BamHI sites of p2µATSte18 yielding p2µATNLSSte18. Subsequently, an oligonucleotide encoding an HA epitope tag was cloned into the NcoI BamHI sites resulting in the following NLS-HA sequence,
MDKAELIPEPPKKKRKVELPWMYPYDVPDYA fused to the NH2 terminus of Ste18p yielding p2µATNLSHASte18. An EcoRI SacI fragment of p2µATNLSHASte18 containing TPI-NLSHA-STE18 was then cloned into pRS413 resulting in p413TNLSHASte18. STE18 was removed from this vector by digestion with BamHI and EcoRI and replaced with the coding sequence of FAR1 from pGAD424Far1 (see below) yielding p413TNLSHAFar1.
The coding sequences of the entire FAR1 ORF and far1-H7 were amplified by PCR from genomic DNA and cloned into pGAD424. SpeI PstI
fragments of FAR1 and far1-H7 from pGAD424 plasmids were cloned
into pMal-c2 (New England Biolabs) resulting in pMFar1 (amino acid residues 133-831) and pMFar1H7 (amino acid residues 133-756). pMFar1N
(amino acid residues 638-831) and pMFar1
C (amino acid residues 133-
297) were derived from pMFar1 by removal of a BamHI or HindIII fragment, respectively. pMFar1H7
N (amino acid residues 638-756) was derived from pMFar1H7 by removal of a BamHI fragment. GSTCdc24 is
comprised of the NH2-terminal 472 amino acids of Cdc24p fused to GST
as described (Nern and Arkowitz, 1998
).
Two-Hybrid
Two-hybrid interactions were tested by growth on SC-leu-trp-his as described (Nern and Arkowitz, 1998). Identical results were obtained with at
least three transformants.
Expression of a LacZ reporter from Y187 derived two-hybrid strains
was quantified by -galactosidase assays (Miller, 1972
). An EcoRI site
was inserted by oligonucleotide-directed mutagenesis after amino acid 153 of Spa2p (Arkowitz and Lowe, 1997
). This 153-amino acid Spa2p fragment was then cloned into pGAD424. PJ69-4A cdc24-m1 (RAY1449) was
constructed by PCR-mediated gene replacement as described (Nern and
Arkowitz, 1998
) and confirmed by PCR and mating defect phenotype.
Three independent PJ69-4A cdc24-m1 strains were used for two-hybrid
analyses. Because TRP1 is used to replace CDC24 with cdc24-m1, STE4
cloned into the 2µ URA3 GAL4 DBD vector pGBDU-C1 (James et
al., 1996
) was used in this strain. Diploid two-hybrid strains were constructed by transformation of either DBD fusions or AD fusions along
with p2µAT and p413T plasmids into SFY526 or Y187 and crossing these
strains. After two-hybrid assays, phenotypes (diploid state and sterility) of
diploid and haploid deletion two-hybrid strains were confirmed. Expression of NLSHAFar1p and NLSHASte18p in two-hybrid strains were confirmed by analysis of yeast extracts using SDS-PAGE, immunoblotting, probing with 12CA5 mAb, and ECL visualization.
Immunoprecipitation
RAY1254, RAY1258, RAY1260, and RAY1336 cells carrying p416GalHASte4 were grown to an OD600 of 0.5 in SC-ura with 2% (wt/vol) raffinose, galactose was added to a final concentration of 2% (wt/vol) and the
cultures grown for 4 h. All subsequent steps were carried out at 4°C. Cells
were harvested by centrifugation, and lysed by agitation with glass beads
in buffer A (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 40 µg/ml
each of leupeptin, chymostatin, pepstatin A, aprotinin, and antipain) containing 0.1% Triton X-100. Before use IgG-Sepharose was cross-linked
with dimethylpimelimidate (Sigma; Harlow and Lane, 1988). Cell extracts
were clarified by two centrifugations (10,000 g for 10 min). Supernatants,
which contained the majority of the tagged proteins, were incubated with
20 µl of IgG-Sepharose (Pharmacia) equilibrated in buffer A containing
0.1% Triton X-100 for 1 h. Resin was then washed four times with buffer
A containing 0.1% Triton X-100 and Far1-protein A fusions were specifically eluted by incubation with 20 U of TEV-protease (Boehringer Mannheim) for 4 h at 16°C in the same buffer. Eluates were analyzed by SDS-PAGE and immunoblotting using polyclonal sera against myc and Far1p
(a gift from M. Peter) at 1:1,000 dilution followed visualization with ECL.
Protein Purification
All purification steps were carried out at 4°C. MBP and GST fusion proteins were expressed in E. coli with MBPFar1 and MBPFar1-H7 bacteria
grown at 30°C. Cells were resuspended in buffer B (PBS, 1 mM DTT,
0.1% Triton X-100), frozen in liquid N2 and stored at 70°C. Cells were
lysed by sonication in buffer B with 1 mM PMSF. Extracts were clarified
by centrifugation (10,000 g for 10 min) and fusion proteins were isolated
using glutathione-agarose (Sigma) or amylose resin (New England
Biolabs). MBP fusion proteins were eluted with 10 mM maltose in buffer
B and dialyzed against buffer C (50 mM Tris-HCl pH 7.4, 10 mM
MgCl2, 1 mM DTT, 10% [vol/vol] glycerol). Protein concentrations were
determined by the Bradford method or by comparing intensities of bands
on Coomassie stained SDS-PAGE gels with BSA (Sigma) as a standard.
For both MBPFar1 and MBPFar1-H7, concentrations used refer to the
full-length protein and not proteolytic breakdown products.
HASte4-(TEV)-protein A was purified from RAY1276 cells using IgG-Sepharose under conditions similar to those described in (Song et al.,
1996). Cells were grown in YEPD to an OD600 of ~3, harvested by centrifugation, resuspended in 20 mM Tris-HCl pH 7.4 with 50 mM NaCl at
~300 OD600/ml, snap frozen in liquid N2, and stored at
70°C. Typically
2,500 OD600 of cells were broken in buffer D (buffer A containing 2 mM
EDTA and 3 mM MgCl2) by agitation with glass beads. Triton X-100 was
added to cell extracts at a final concentration of 1%. After 1 h incubation
the extract was centrifuged at 10,000 g for 20 min. The supernatant was incubated overnight with 250 µl IgG-Sepharose equilibrated in buffer D
containing 1% Triton X-100. The resin was collected by centrifugation,
washed once with buffer D containing 1% Triton X-100 and twice with
buffer D containing 0.1% Triton X-100. HASte4p was specifically eluted by incubation with 20 U of TEV-protease in 400 µl buffer D containing 0.1% Triton X-100 for 5 h. Comparison of the amounts of total protein
and HASte4p in yeast extracts (treated with TEV-protease) and eluted
HASte4p preparations indicated that HASte4p was enriched over 1,000-fold in comparison to cell extracts. By immunoblotting both 3xmycCdc24p
and Far1p were undetectable in HASte4p preparations (<0.01% of the
starting level).
Binding Studies
Binding experiments were all carried out at 4°C. For binding of GSTCdc24 and MBP fusion proteins ~10 µg of GSTCdc24 bound to glutathione-agarose was incubated with respective MBP fusion proteins in 100 µl of buffer C overnight. Glutathione-agarose samples were washed twice with 1 ml of buffer C and once with 1 ml of buffer B. Proteins were eluted with SDS-PAGE sample buffer and analyzed by SDS-PAGE followed by Coomassie blue staining or transfer to nitrocellulose, probing with anti-MBP mAb (Sigma) at 1:4,000 dilution and visualized by ECL. For binding experiments with yeast HASte4p, the HASte4p preparation was diluted 10-fold into buffer C and 100 µl was incubated with either resin bound GST or MBP fusions. MBP fusions were bound to amylose resin by incubation of ~5 µg of each protein with 20 µl of amylose resin for 1 h. GSTCdc24-MBPFar1 was prepared by passing a bacterial extract (from 100 ml of cells) containing GSTCdc24 over a column with ~500 µg of MBPFar1 bound to amylose resin. The column was washed with buffer B and then GSTCdc24-MBPFar1 was eluted with buffer B containing 10 mM maltose. The eluate was incubated with glutathione-agarose for 30 min which was then washed three times with buffer B. Proteins bound to the resin were analyzed by SDS-PAGE and Coomassie blue staining or used for HASte4p binding.
Mating and Pheromone Response Assays
Quantitative matings were carried out as described in (Arkowitz and
Lowe, 1997; Nern and Arkowitz, 1998
) with Mata cells as indicated and
Mat
RAY1135 cells. Pheromone induced cell cycle arrest and induction
of a Fus1LacZ reporter were assayed as described (Nern and Arkowitz,
1998
). For pheromone treatment ~0.2 OD600 of log-phase cells were collected by centrifugation, resuspended in 2 ml YEPD containing 12 µM
-factor (synthesized by David Owen, MRC LMB) and incubated for 3 h.
Cells were fixed with formaldehyde and actin was visualized as described
(Nern and Arkowitz, 1998
) using rhodamine phalloidin (Molecular
Probes). To examine cell morphologies in mating mixtures, Mata cells were stained with 10 µg/ml Calcofluor white (Pringle, 1991
) (Sigma) in
YEPD for 5 min at rt and subsequently washed extensively with YEPD.
Approximately 5 × 106 stained cells were then mixed with unstained Mata
(RAY1135) cells and incubated on filters. After 2 h cells were washed
from the filters, briefly sonicated, resuspended in PBS and fixed with formaldehyde. Images of cells were taken using a Zeiss Axioskop microscope with either a NA 1.4 ×63 or NA 1.3 ×100 objective and recorded
with a Princeton Instrument Micromax CCD camera. Fluorescence and
differential interference-contrast (DIC) images were merged to permit
identification of Mata cells.
Localization of Cdc24p
Cdc24HAGFP (p414Cdc24HAGFP) was transformed into RAY931 which
is deleted for CDC24 and kept alive by the rescuing plasmid pEG(KT)Cdc24 (Nern and Arkowitz, 1998). This strain was able to lose the rescuing plasmid (both by extensive growth in SC-trp media or counter-selection on 5-FOA) as determined by markers and PCR, resulting in
RAY1360, indicating that Cdc24HAGFP was functional. RAY1360 grew
normally at 22°C, 30°C, and 37°C on YEPD plates. Budding patterns were
determined as described (Arkowitz and Lowe, 1997
) and mating efficiency was determined as described above. Cdc24HAGFP expression and
size was verified by SDS-PAGE, immunoblotting, probing with 12CA5
mAb and ECL visualization. Confocal microscopy was carried out as described (Arkowitz and Lowe, 1997
) except cells were grown in SC supplemented with 55 µg/ml adenine to reduce fluorescence due to ade2. Pheromone treatment was with 140 µM
-factor. Cells were imaged after 1 h in
order to observe early localization. For latrunculin A treatment of budding cells 2 µl of either 10 mM latrunculin A (Molecular Probes) in
DMSO or DMSO was added to 200 µl of log-phase cells (final concentration latrunculin A 0.1 mM) and cells were incubated for 3 h (Ayscough et
al., 1997
). For latrunculin A treatment of shmoos, cells were incubated
with 140 µM
-factor for 1 h and then 0.1 mM latrunculin A or DMSO
was added to cells which were incubated for 2 h. After observation by confocal microscopy, actin depolymerization was confirmed by staining fixed
cells with rhodamine phalloidin as described above.
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Results |
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The Cdc24p-G Interaction Requires FAR1 but Not
Pheromone-dependent Signaling
Cdc24-m alleles are defective in growth orientation along
a pheromone gradient, yet do not affect pheromone-dependent MAP-kinase pathway signaling (Nern and Arkowitz,
1998). These mutants are also unable to interact with G
(Ste4p) in two-hybrid assays. In Cdc24p-G
two-hybrid
assays G
was overexpressed, which has been shown to activate the MAP-kinase pathway (Whiteway et al., 1990
).
Hence it was possible that MAP-kinase signaling is required for this interaction. Two-hybrid experiments revealed that while Cdc24p and G
interact in a haploid
strain, no detectable interaction was observed in a diploid
(compare Fig. 1, A and B), in which several mating specific proteins, including G
(Ste18p), are not expressed
(Sprague and Thorner, 1992
). G
is required for this interaction in a haploid (Nern and Arkowitz, 1998
). Surprisingly, overexpression of G
in a diploid did not restore
the Cdc24p-G
interaction, whereas overexpression of
G
in a
ste18 haploid restored this interaction (Table II).
This result is consistent with the notion that either a haploid specific component and/or pheromone-dependent signaling is required for the Cdc24p-G
interaction.
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To examine the role of the pheromone-dependent MAP-
kinase pathway in the Cdc24p-G interaction, two-hybrid
strains were constructed in which each component of this
pathway was deleted. The MAP-kinase scaffolding protein Ste5p, the PAK kinase Ste20p which phosphorylates
Ste11p, the MAPKKK Ste11p, the MAPKK Ste7p, the
MAPK Fus3p or Kss1p, and the transcription factor
Ste12p were each individually disrupted in a two-hybrid
strain. In addition, the Ste20p homologue Skm1p, the
cyclin-dependent kinase inhibitor required for
-factor
cell cycle arrest Far1p, the polarity establishment protein
Bem1p, the G
effector Akr1p, and the bipolar bud site selection protein Bud6p were deleted from this strain. Several of these proteins including Ste5p, Fus3p, and Far1p
are only expressed in haploids (Sprague and Thorner,
1992
) and thus are candidates for haploid specific components required for the Cdc24p-G
interaction. Deletion
of SKM1, BEM1, AKR1, or BUD6 had no effect on the
Cdc24p-G
interaction. In contrast, removal of any component of the pheromone-dependent MAP-kinase pathway (with the exception of Fus3p and Kss1p which are
functionally redundant for mating) resulted in the loss of the Cdc24p-G
two-hybrid interaction. Because G
appeared to be required for the Cdc24p-G
interaction (Nern
and Arkowitz, 1998
), we examined whether overexpression of G
was able to restore the Cdc24p-G
interaction
in these strains. Table II shows that overexpression of G
partially restored the Cdc24p-G
interaction in
ste5,
ste11, and
ste7 strains and to a lesser extent in
ste20
and
ste12 strains. These results indicate that signaling
through the pheromone-dependent MAP-kinase cascade
per se is not required for the Cdc24p-G
interaction. However, deletion of FAR1 resulted in a loss of the
Cdc24p-G
interaction which was not restored upon overexpression of G
(Table II and Fig. 1 C), suggesting that
Far1p may be essential for this interaction.
The requirement for G and Far1p in the Cdc24p-G
two-hybrid interaction suggested an explanation for the
low level of LacZ reporter activity observed in the haploid
Y187 two-hybrid strain and the absence of an interaction
in the diploid two-hybrid strain, namely that these two
proteins were limiting in haploids and absent in diploids.
To test this possibility, we overexpressed G
and Far1p individually and together in the Y187 haploid two-hybrid strain. Fig. 1 A shows that overexpression of G
in the
presence of pAS1Cdc24 and pGAD424Ste4 resulted in an
approximately twofold increase in LacZ activity, whereas
the additional overexpression of Far1p resulted in a further increase in LacZ activity by ~3.5-fold. In diploids,
overexpression of G
did not result in a Cdc24p-G
interaction. However overexpression of Far1p resulted in LacZ reporter activity (Fig. 1 B) and this was further increased
by additional overexpression of G
, suggesting that in the
absence of pheromone-dependent signaling Far1p is sufficient for restoring the Cdc24p-G
interaction.
FAR1 is necessary for both pheromone-dependent growth
arrest and oriented growth towards a pheromone gradient
(Chang and Herskowitz, 1990; Valtz et al., 1995
). These
two functions of FAR1 can be separated, with the Far1p
NH2 terminus necessary for cell cycle arrest and the COOH
terminus necessary for growth orientation. Our two-hybrid
results indicate FAR1 is necessary for the Cdc24p-G
interaction, yet it is unclear which function of FAR1 this corresponds to. Because cdc24-m and far1-s appear phenotypically identical and both exhibit orientation defects
(Valtz et al., 1995
; Nern and Arkowitz, 1998
), we examined the effect of the far1-s allele far1-H7 on this interaction. This far1 mutation results in a COOH-terminal 75-
amino acid deletion and despite its orientation defect is normal for cell cycle arrest. Fig. 1 C shows that a far1-H7
mutation prevents the Cdc24p-G
interaction. These results suggest that the FAR1 orientation function is required for the Cdc24p-G
association, consistent with the
role of this interaction in growth orientation.
We next investigated whether Far1p interacted with
Cdc24p and G. Fig. 1 and Table III show that in two-hybrid assays Far1p can interact with both Cdc24p and
G
. The Cdc24p-Far1p interaction was observed in strains
deleted for STE4, STE18, FUS3, or STE12, indicating that
it does not require G
nor pheromone-dependent MAP-kinase signaling (including Fus3p-dependent phosphorylation of Far1p). Similarly, the Far1p-G
interaction did not
require STE18, FUS3, or STE12. The Far1p-G
interaction also did not require the CDC24 orientation function
as we observed this interaction in a cdc24-m1 two-hybrid
strain. In addition we examined Cdc24-m1p, which we had
previously shown does not interact with G
, and found that Cdc24-m1p also did not interact with Far1p (data not
shown). Together these results suggest that Far1p and
Cdc24p can associate and this association is independent
of pheromone signaling.
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Far1p Binds Cdc24p
To further investigate these interactions epitope-tagged
versions of Far1p and Cdc24p were constructed. Myc and
protein A domains were fused to Cdc24p and Far1p, respectively and these fusions were used to replace wild-type
genes. Far1-protein A fusions had a tobacco etch virus
(TEV) protease cleavage site between Far1p and protein
A to allow specific elution. These strains grew normally
and exhibited normal vegetative morphology. Furthermore, both fusions mated with similar efficiencies as a
wild-type strain when crossed to a wild-type tester or to
an enfeebled tester. Far1-protein A fusions promoted normal cell cycle arrest and cells carrying this fusion formed
shmoos that appeared normal upon exposure to mating
pheromone. Together these results indicated that the fusion proteins were functional. Fig. 2 shows that when Far1-protein A was isolated with IgG-Sepharose, myc-tagged
Cdc24p was bound (compare lanes 1 and 2 with 3 and 4).
When Cdc24p or Far1p orientation mutants were used, a
substantial decrease in the amount of Cdc24p bound to
Far1p was observed in both cases (lanes 5-8). These results reveal the molecular basis for the similar phenotypes
of cdc24-m1 and far1-H7 mutants. Although two-hybrid
results indicated a Far1p-G association, this was apparently not stable enough to observe by immunoprecipitation.
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To address whether these protein interactions were direct, binding experiments were carried out using purified
proteins. Far1p and the NH2-terminal half of Cdc24p
(amino acids 1-472; Nern and Arkowitz, 1998) were purified from bacteria as fusions to maltose binding protein
(MBP) and glutathione-S-transferase (GST), respectively. Fig. 3 A shows that MBPFar1 bound GSTCdc24 but not
GST alone. MBPFar1-H7 (Fig. 4 A) does not significantly
bind GSTCdc24p, consistent with immunoprecipitation results above (Fig. 2). In these binding experiments an excess of GSTCdc24 is used and an increase in MBPFar1 binding occurs as its concentration in the binding reaction
is increased (Fig. 3 B). These results demonstrate that
Far1p can bind Cdc24p directly in the absence of other
proteins.
|
|
These binding studies demonstrated that the COOH
terminus of Far1p is necessary for GSTCdc24 binding,
hence we examined if this region was also sufficient for
binding. Fig. 4 B shows that a 200-amino acid Far1p
COOH-terminal fragment (lane 10) is not sufficient for
GSTCdc24 binding and furthermore an NH2-terminal Far1p fragment did not bind GSTCdc24 (lane 9). MBPFar1-H7 and MBPFar1C, which do not bind GSTCdc24
are unlikely to be grossly misfolded as they retain the ability to bind G
(see below). These results indicate that although the COOH terminus of Far1p is necessary for
binding Cdc24p it is not sufficient. An immunoblot of the MBPFar1 bound to GSTCdc24 (Fig. 4 B, lane 7) revealed
that proteolytic fragments of Far1 with as little as 25 kD of
the NH2-terminus (approximately residues 133-350) are
coprecipitated with MBPFar1 and GSTCdc24. This region
of Far1p, which includes a Lim domain (Sanchezgarcia
and Rabbitts, 1994
), does not bind Cdc24p directly (Fig. 4
B, lane 9), suggesting that this region may mediate Far1p multimerization.
Far1p Binds G
Hemagglutinin (HA)-tagged G (Ste4p) was purified from
yeast in order to examine its binding to MBPFar1 (Fig. 4
C). For this purpose a strain in which the wild-type copy of
STE4 was replaced with HASte4-(TEV)-protein A was
used. This fusion was functional for mating and cell cycle
arrest. The Ste4p fusion was isolated with IgG-Sepharose
and eluted by specific cleavage between Ste4p and the
protein A domains using TEV protease, yielding HASte4p which was over 1,000-fold enriched compared with cell extracts. This HASte4p preparation had undetectable levels
of Far1p and Cdc24p (<0.01% of the starting level, data
not shown). Aliquots of HASte4p were incubated with
various MBPFar1 fragments immobilized on amylose resin
and bound HASte4p was analyzed by SDS-PAGE and immunoblotting. Fig. 4 C shows that HASte4p bound equally
well to MBPFar1 and MBPFar1-H7 (lanes 2 and 3),
whereas both NH2- and COOH-terminal Far1p fragments
bound substantially less HASte4p (lanes 4-6). Of these
smaller Far1p fragments, only Far1
C (amino acid residues 133-297) bound substantial amounts of HASte4p,
suggesting that this region which includes the Lim domain
is involved in G
binding. These experiments show that
G
can bind Far1p in the absence of Cdc24p and suggest
that perhaps G
and Cdc24p bind to different regions of Far1p.
A Cdc24p-Far1p-G Complex
Since both Cdc24p and Far1p bind G, we addressed
whether the addition of MBPFar1 to GSTCdc24 bound
to glutathione-agarose could compete for HASte4p (G
)
binding. Fig. 5 A shows that addition of MBPFar1 did not
prevent HASte4p binding to GSTCdc24 (compare lanes 1 and 2 with 3 and 4), but rather increased binding by about twofold. Coomassie blue staining of glutathione-agarose
eluates revealed that MBPFar1 bound GSTCdc24. These
results indicate that Far1p binding to Cdc24p does not displace G
, and are consistent with the formation of a complex of all three proteins.
|
To directly test whether a complex of all three proteins
could form we determined whether a stoichiometric complex of Cdc24p-Far1p could bind G (Ste4p). GSTCdc24-
MBPFar1 was isolated by sequential purification using
amylose and glutathione resin. Fig. 5 B shows that
GSTCdc24-MBPFar1 contained roughly equal amounts
of these two fusion proteins. Purified HASte4p was then
incubated either with this complex or GSTCdc24 alone.
Densitometric quantification showed that twofold more
HASte4p bound to Cdc24p-Far1p (Fig. 5 B compare lanes
1 and 2) than to a similar amount of Cdc24p alone, demonstrating that trimeric Cdc24p-Far1p-G
can form. This increase in G
binding does not appear to be cooperative
and is more likely to be the sum of contributions from
Cdc24p and Far1p. Because both Far1p and Cdc24p can
individually bind each other or G
it is likely that in a trimeric complex each protein contacts the other two proteins. These binding studies together with the two-hybrid results suggest that Cdc24p-Far1p-G
is necessary for mating projection orientation.
Cdc24p and Far1p Function in the Same Shmoo Orientation Process
To examine if CDC24 and FAR1 function in the same process we compared the mating efficiencies of both single
and double far1 and cdc24-m1 mutants. Fig. 6 shows that
the presence of a cdc24-m1 mutation in a
far1 background did not result in a further decrease in mating efficiency, suggesting that FAR1 and CDC24 function in the
same orientation process. The mating defect of the double mutant is closer to that of the
far1 mutant that in addition to a chemotropism defect does not arrest growth in
response to mating pheromone. If cdc24-m1 affected chemotropism similarly to
far1, then a double mutant with
spa2, a gene required for the default mating pathway
(Dorer et al., 1995
), should have a mating defect greater
than the product of the individual mating defects, a phenomenon known as synthetic sterility (Dorer et al., 1995
).
Fig. 6 shows that
spa2 cdc24-m1 mutants exhibited synthetic sterility.
|
We also examined genetic interactions between cdc24-
m1 and bem1 or
ste20, two genes involved in polarized
growth and mating. Bem1p is associated with the cytoskeleton (Leeuw et al., 1995
), binds Cdc24p (Peterson et al.,
1994
; Zheng et al., 1995
), and Far1p (Lyons et al., 1996
).
Bem1 mutants are unable to form shmoos and instead
form round cells in the presence of mating pheromone (Chenevert et al., 1992
).
Bem1 cdc24-m1 cells showed
similar temperature sensitive growth and morphological
defects (large round cells) as
bem1 cells, providing further evidence that cdc24-m1 has no effect on vegetative
growth. Even in cells lacking BEM1 which cannot form
shmoos, cdc24-m1 resulted in a substantial decrease in
mating efficiency (Fig. 6), i.e., synthetic sterility. Because deletion of the PAK kinase STE20 in our strain background did not result in complete sterility, we were able to
examine the mating defect of
ste20 cells in the presence
and absence cdc24-m1. In the absence of STE20, cdc24-m1
resulted in a further decrease in mating efficiency. Together these results suggest that in
bem1 and
ste20 mutants, which are unable to form shmoos, polarization may
still be necessary for mating perhaps for the localization of
proteins necessary for cell fusion. Furthermore, because
BEM1 and STE20 are not required for default mating
(Dorer et al., 1997
), such synthetic mating defects with
cdc24-m1 are consistent with a genetic linkage between
shmoo formation and orientation.
Cdc24p Localization
If Cdc24p transmits signals from bud site selection proteins or G, it might be localized to regions of polarized
growth. We therefore examined the localization of a
Cdc24p green fluorescent protein (GFP) fusion. Cdc24HAGFP expressed from its own promoter on a CEN plasmid complemented
cdc24 as determined by growth at different temperatures, budding patterns, and mating efficiencies (data not shown). Fig. 7 A shows the localization
of Cdc24HAGFP in living cells at different stages in the
cell cycle. In unbudded cells Cdc24p localized as a tight
patch at the membrane, and in cells with small buds at the
growing end. In larger buds, this localization became more
spread out. Finally, during cytokinesis Cdc24p generally
localized to the mother-bud neck. Curiously, a preliminary
report showed that an overexpressed GSTCdc24 fusion protein had a circumcellular distribution in budding cells
(Pringle et al., 1995
). Cdc24HAGFP also localized to sites
of polarized growth after
-factor treatment. Fig. 7 B
shows different shmoos in which Cdc24HAGFP is observed as a patch at the tip of the mating projection.
Cdc24HAGFP was localized similarly in mating mixtures (data not shown). Furthermore, as the sole copy of Cdc24p
in a
cdc24 strain, Cdc24-m1HAGFP also localized to
sites of polarized growth in budding and mating cells (data
not shown), indicating that this mutant is not defective in
its localization to sites of polarized growth. These data
demonstrate that Cdc24p localizes to sites of polarized
growth.
|
The early localization of Cdc24p in the cell cycle and its
localization to the shmoo tip are consistent with its function in polarity establishment. The localization of Cdc24p
is similar to that of its substrate Cdc42p (Ziman et al.,
1993). To determine whether the actin cytoskeleton was
necessary for polarized Cdc24p localization, budding and
shmooing cells were treated with the actin depolymerizing
drug latrunculin A (Ayscough et al., 1997
). Fig. 8 A shows
that even in the absence of actin polymerization, Cdc24p is
localized to sites of polarized growth in budding cells. In
contrast, latrunculin A treatment of shmoos resulted in a
substantial decrease in Cdc24p localization (Fig. 8 B).
Upon latrunculin A treatment the number of cells with
Cdc24p localized to the shmoo tip decreased by fivefold (n = 100) and in cells that exhibited localized Cdc24p, there appeared to be a decrease in the amount of Cdc24HAGFP
localized to the shmoo tip and an increase in fluorescence throughout the cell. These results are consistent with the
effects of latrunculin A on Cdc42p localization in budding
and shmooing cells (Ayscough et al., 1997
; Ayscough and
Drubin, 1998
).
|
In the Absence of CDC24- or FAR1-mediated Chemotropism the Bud Site Selection Machinery Is Essential for Shmoo Formation
During mating, a pheromone gradient serves as the external cue for growth orientation. This external signal allows
haploid cells to orient growth in a pheromone gradient
emanating from any direction (Madden and Snyder, 1992),
whereas the site for bud formation in haploids is fixed adjacent to the previous bud site (Chant and Pringle, 1995
).
The selection of a site for the mating projection must override the fixed location of the bud. If Cdc24p acts as a
switch between internal signals during budding and external signals during mating (Nern and Arkowitz, 1998
), we
would predict that bud site selection proteins become important for cell mating when the capacity for shmoo orientation is lost in mutants such as cdc24-m1.
The ras related small G-protein Bud1p/Rsr1p is essential for bud site selection, yet is not required for chemotropic or default mating in saturating pheromone (Roemer
et al., 1996; Dorer et al., 1997
). However Bud1p can directly associate with Cdc24p (Zheng et al., 1995
; Park et al.,
1997
) and this association is likely to be functionally important (Bender and Pringle, 1989
; Michelitch and Chant,
1996
). We therefore examined the phenotype of
bud1 cdc24-m1 double mutants to determine if the loss of
CDC24-mediated chemotropism caused a BUD1-dependent mating defect. Both
bud1 and
bud1 cdc24-m1 cells
grew normally, were not temperature sensitive for growth,
and had the expected random budding pattern (data not
shown). Strikingly, the
bud1 cdc24-m1 double mutant
showed a stronger mating defect (an eightfold further decrease in mating efficiency) than cdc24-m1 alone (Fig. 9
A). In contrast,
bud1 alone had no effect on mating efficiency in agreement with previous studies (Chant and
Herskowitz, 1991
; Dorer et al., 1997
). Microscopic observation of
bud1 cdc24-m1 double mutants treated with a
high concentration of mating pheromone (Fig. 9 B) or exposed to pheromone gradients in mating mixtures (Fig. 9
C) revealed that these cells were defective in shmoo formation. Instead of forming typical pear-shaped shmoos
most cells were enlarged and round. On closer inspection a
small protrusion was occasionally observed on these cells.
Furthermore, the actin cytoskeleton in the double mutants was depolarized, with actin cortical patches and cables disorganized (Fig. 9 D). In contrast, both
bud1 and cdc24-m1 single mutants formed shmoos. Otherwise
bud1
cdc24-m1 double mutants responded normally to pheromone by undergoing cell cycle arrest and pheromone-dependent gene induction (data not shown). These results
suggest that in the absence of chemotropism, BUD1 and
perhaps the bud site selection machinery becomes essential for shmoo formation. Surprisingly, in saturating uniform concentrations of mating pheromone
bud1 does not
result in a mating defect (Dorer et al., 1997
), raising the
possibility that this novel role of BUD1 is revealed specifically when signaling from G
to Cdc24p is blocked.
|
Our results indicate that Far1p is required for signaling
from G to Cdc24p. If the shmoo formation defect of
bud1 cdc24-m1 cells is due to a defect in this signaling, a
bud1
far1 double mutant should show an analogous decrease in mating efficiency.
Bud1
far1 cells had a stronger mating defect (an eightfold decrease in mating efficiency) than
far1 cells. As a control the effect of
bud1
was examined in a
sst2 strain.
Sst2 cells are supersensitive to mating pheromone as SST2 negatively regulates
the heterotrimeric G-protein (Dohlman et al., 1996
).
Therefore
sst2 cells mate as though they are saturated
with mating pheromone, mating by the default pathway
(Dorer et al., 1997
).
Bud1
sst2 cells had a similar mating
defect as
sst2 alone, indicating that the absence of
chemotropic mating by itself is not sufficient to reveal
BUD1 function in mating. These synthetic mating defects
of
bud1 with cdc24-m1 or far1 show that Bud1p, which
normally functions in bud site selection, can play a role in
shmoo formation, presumably by regulating Cdc24p.
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Discussion |
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During mating yeast cells grow in a polarized fashion towards their mating partner (Segall, 1993; Dorer et al.,
1995
; Valtz et al., 1995
; Nern and Arkowitz, 1998
). Yeast
cells are able to sense pheromone gradients and orient
their actin cytoskeleton and secretion towards such a gradient. Here we show that a complex comprised of Cdc24p,
Far1p, and G
can form and is likely to be required for
orientation towards a mating partner. The formation of
this complex does not directly require signaling via the
pheromone-dependent MAP-kinase pathway. Analyses of
mating defects of double mutants indicate that FAR1 and
CDC24 both function in the same cell orientation process.
Cdc24p localizes to sites of polarized growth suggesting that Cdc24p-Far1p-G
is localized. Cdc24p localization
does not depend on the actin cytoskeleton during budding
but does depend on the actin cytoskeleton during shmooing. In the absence of signaling from G
to Cdc24p, the
bud site selection protein Bud1p is required for shmoo formation, demonstrating a molecular link between growth site selection in mating and budding. Together these results suggest that binding of G
to Far1p and Cdc24p creates an internal landmark for growth towards an external signal.
A Complex Comprised of Cdc24p, Far1p, and G
Links External Signals to Cytoskeleton Orientation
Detection of a pheromone gradient and orientation of
growth in such a gradient is a process central to yeast mating and is analogous to Dictyostelium chemotaxis and
nerve cell chemotropism (Arkowitz, 1999). Alleles of both
far1 (Valtz et al., 1995
) and cdc24 (Nern and Arkowitz,
1998
) are specifically defective in orientation towards a
pheromone gradient. Cells mutant for the
-factor pheromone receptor (Ste2p) or the heterotrimeric G-protein, discriminate poorly between pheromone signaling and nonsignaling mating partners suggesting that these components are also required for chemotropism (Jackson et al.,
1991
; Schrick et al., 1997
). Cdc24-m mutants are unable to
interact with the G
subunit of the heterotrimeric G-protein (Nern and Arkowitz, 1998
). These results led to a
model in which G
locally activates or recruits Cdc24p,
which could then activate Cdc42p and other downstream targets required for cytoskeleton orientation. We conclude
from two-hybrid, binding, and genetic data that Far1p is
involved in signaling from G
to Cdc24p by forming a
complex with these proteins.
Our two-hybrid results suggest that the Far1p-G interaction does not require the CDC24 orientation function,
yet Far1p is essential for the Cdc24p-G
interaction. In
contrast, in vitro binding experiments show that Cdc24p
is able to bind to G
purified from bacteria (Nern and
Arkowitz, 1998
) and yeast in the absence of Far1p. We attribute this difference between two-hybrid and in vitro
binding results to the different methods used. For example, in the two-hybrid experiments interactions occur in
the nucleus and on the other hand the in vitro binding
studies are carried out with high concentrations of purified
proteins. We suggest that although Far1p is important for
the Cdc24p-G
interaction it is not absolutely essential,
whereas Cdc24p is not necessary for the Far1p-G
interaction. We have demonstrated that a triple complex comprised of Cdc24p-Far1p-G
can form using purified proteins and believe that in vivo this complex links receptor activation to cytoskeleton organization (Fig. 10). These results show at a molecular level the role of Far1p in growth
orientation. Consistent with the specific phenotype of far1
and cdc24 orientation alleles, we find that pheromone-dependent MAP-kinase cascade signaling is not necessary
for the association of this complex. This result indicates
that the MAP-kinase cascade is not directly required for
chemotropic growth, in agreement with recent mating
partner discrimination studies (Schrick et al., 1997
). Furthermore, the formation of this complex does not require
FUS3, which normally phosphorylates Far1p in a pheromone-dependent fashion (Chang and Herskowitz, 1992
;
Elion et al., 1993
). This phosphorylation of Far1p is necessary for cell cycle arrest, indicating that the cell cycle arrest
function of FAR1 is not required for interactions between
Cdc24p, Far1p, and G
.
|
How is the formation of this protein complex regulated
by pheromone activation of the receptor? Pheromone
binding to the receptor is believed to trigger dissociation
of G from G
. Recent studies suggest G
binds the
pheromone receptor (Kallal and Kurjan, 1997
; Medici et al.,
1997
) and that G
must be membrane associated in order
to function (Pryciak and Huntress, 1998
). GFP fused to G
is localized preferentially to the plasma membrane of
the mating projection after pheromone treatment (Nern
and Arkowitz, unpublished observation). Upon pheromone stimulation, Far1p levels increase (Chang and Herskowitz, 1992
; Valtz et al., 1995
) resulting in increased levels of Cdc24p-Far1p (Nern and Arkowitz, unpublished observation). As Far1p localizes to the nucleus during vegetative growth (Henchoz et al., 1997
), it would appear
likely that Far1p must exit the nucleus in order to carry
out its mating orientation function. We envision that released G
recruits Cdc24p-Far1p to the vicinity of activated receptors and Cdc24p-Far1p-G
ultimately directs
the cytoskeleton towards this internal landmark. Such a
mechanism provides a means of translating local activation of pheromone receptors to cytoskeletal orientation.
We would predict that Far1p, like Cdc24p, localizes to the
tip of the mating projection in pheromone treated cells.
While this work was being reviewed a paper examining
the role of Far1p in polarized growth during mating was
published (Butty et al., 1998). In general, our results agree
with the findings of this work. The authors postulate that
Far1p functions as an adaptor or linker between G
and
polarity establishment proteins including Cdc24p. Our in
vitro binding results indicate that even in the absence of
Far1p, G
can still bind Cdc24p, suggesting that perhaps
Far1p is not simply a physical adaptor but may have more
complex functions. Overexpressed GFPFar1 was shown to
relocalize from the nucleus to the cytoplasm upon treatment with a saturating uniform concentration of pheromone for two hours. In these conditions GFPFar1 does not
appear to accumulate at shmoo tips. It will be interesting
to determine whether wild-type levels of Far1p localizes similarly in cells exposed to a pheromone gradient for various times.
How are these protein interactions involved in transmitting spatial information? Previous studies have indicated
that in a pheromone gradient, shmoo orientation improves
as a function of time (Segall, 1993). This appears to be due
to reorientation of the shmoo tip as it grows (Segall, 1993
;
Nern and Arkowitz, unpublished observation), indicating
that shmoo orientation is a continuous process unlike bud
site selection. Perhaps Cdc24p-Far1p-G
dissociates reasonably fast such that this complex is continually dissociating and forming. Such a dynamic process would provide a
means for continuous reorientation during mating and
could play a central role in translating initial small differences in receptor occupancy into oriented growth.
Another difference between bud and shmoo formation
is that in budding the polarity establishment proteins
Cdc42p and Bem1p localize independent of the actin cytoskeleton (Ayscough et al., 1997) whereas in the latter
process the actin cytoskeleton is necessary for the efficient
localization of these proteins (Ayscough and Drubin,
1998
). During shmoo formation the actin cytoskeleton requirement for localization of Cdc24p and these other polarity establishment proteins appears to be similar. Why is
the actin requirement for localization of this group of proteins different in budding and shmooing cells? Perhaps the
continuous nature of the shmooing process compared with
the committed directional growth required for budding
underlies this different dependence on the actin cytoskeleton. It will be important to examine the role of the actin
cytoskeleton in cells responding to a pheromone gradient.
Coordination of Different Pheromone Responses
Pheromone stimulation results in gene induction, cell cycle
arrest, and morphological changes (Sprague and Thorner,
1992; Chenevert, 1994
; Leberer et al., 1997a
). The timing
and coordination of these different responses is important
for efficient mating. Our genetic studies are consistent
with Cdc24p and Far1p being part of the same protein
complex functioning in growth orientation and we examined two additional genes that might have a role in coordinating various pheromone responses.
The PAK kinase Ste20p is important for MAP-kinase
signaling during mating. It interacts with Bem1p, Ste4p,
Ste5p, and Cdc42p (Leeuw et al., 1995; Zhao et al., 1995
;
Peter et al., 1996
; Leberer et al., 1997b
; Leeuw et al., 1998
).
Recent mating partner discrimination studies (Schrick et al.,
1997
) suggest that STE20 may not be required for chemotropism. Our two-hybrid results suggest that STE20 has some effect on the Cdc24p-G
interaction, however cdc24-
m1 results in a further mating defect in
ste20 cells. While
Ste20p binds G
, it is unclear how this association relates
to the Far1p, Cdc24p, G
interaction. Further studies will
be necessary to elucidate the roles of STE20 in various aspects of mating.
Bem1p is required for polarized growth both during
mating and budding (Bender and Pringle, 1991; Chenevert
et al., 1992
).
Bem1 cells are defective in shmoo formation, mating pheromone-dependent cell cycle arrest and
efficient signaling via the MAP-kinase cascade (Chenevert
et al., 1992
; Lyons et al., 1996
). At the molecular level
Bem1p interacts with many components required for polarized growth such as the G-protein Bud1p (Zheng et al.,
1995
; Park et al., 1997
), Cdc24p (Peterson et al., 1994
;
Zheng et al., 1995
), Far1p (Lyons et al., 1996
), actin
(Leeuw et al., 1995
), Ste5p (Leeuw et al., 1995
; Lyons et al.,
1996
), and Ste20p (Leeuw et al., 1995
). Although Bem1p
binds both Cdc24p and Far1p, Bem1p is not required for
the formation of Cdc24p-Far1p-G
. Results from
bem1
cdc24-m1 mutants suggest that even for cells unable to
form shmoos, polarization is important. Perhaps this is because the molecules necessary for cell fusion must be correctly localized. What is the molecular function of Bem1p
in mating? We favor the idea that Bem1p acts as a scaffolding component linking pheromone-dependent MAP-kinase signaling, shmoo formation, and shmoo orientation.
Cdc24p as a Switch between Growth Site Selection in Mating and Budding
An attractive model (Fig. 10) is that Cdc24p acts as a selector switch that responds to input signals from bud site selection (Sloat et al., 1981) and mating projection orientation (Nern and Arkowitz, 1998
). We envision that the
localization and activation of Cdc24p is essential for its
function in both bud site selection and mating projection
orientation. During budding it is likely that local activation
of the G-protein Bud1p marks the site for bud formation
(Chant et al., 1991
; Michelitch and Chant, 1996
). The GTP
bound form of Bud1p binds Cdc24p (Zheng et al., 1995
; Park et al., 1997
) and this interaction may be required for
Cdc24p localization to the bud site. Interactions of Cdc24p
with the bud site selection machinery dictate the site of
mating projection growth in the absence of local activation
of Cdc24p by G
, such as in the case of far1 (Valtz et al.,
1995
) or cdc24 (Nern and Arkowitz, 1998
) mutations or in
the presence of saturating mating pheromone (Madden
and Snyder, 1992
; Dorer et al., 1995
), wherein the mating
projection forms adjacent to the previous bud. We show Bud1p becomes essential for shmoo formation specifically
in the absence of signaling from G
to Cdc24p. This demonstrates that the bud site selection machinery can function in shmoo formation. It is surprising that under these
conditions, BUD1 functions in shmoo formation, while
during budding it appears only to function in bud site selection and not bud formation. Interestingly, a specific role
for BUD2 in bud formation has been observed in triple mutant combinations with
cln1 and
cln2 (Benton et al.,
1993
; Cvrckova and Nasmyth, 1993
). A possible explanation for these different functions of BUD1 is that mating
projection orientation is a continuous process, in contrast
to bud site selection in which once a site for growth is chosen, subsequent directed growth is fixed to this site and
there may no longer be a requirement for BUD genes.
We attribute the role of BUD1 in shmoo formation to a
synthetic effect with cdc24/far1 suggesting this function of
BUD1 is normally redundant yet revealed in the absence
of G-mediated chemotropism. Recently it has been
proposed that BUD1 is involved in cell fusion (Elia and
Marsh, 1998
), yet the effects of
bud1 we observe in
cdc24-m1 mutants, i.e., the inability to form a shmoo, are
unlikely to be a result of its role in fusion as we observe
this morphological defect in response to mating pheromone without a mating partner. Furthermore, in contrast
to the results of Elia and Marsh (1998)
but in agreement
with previous studies (Dorer et al., 1997
),
bud1 does not
result in a mating defect in our strain background. In addition,
bud1 does not affect mating in the presence of saturating uniform mating pheromone concentration (Dorer
et al., 1997
). Therefore while both mating in the presence of saturating pheromone or mating in a cdc24 or far1 mutant block chemotropic growth, at the molecular level
these two situations are not equivalent and this difference
is consistent with the suggestion that Cdc24p must be localized or locally activated to function properly (Fig. 10).
We imagine that during mating in saturating uniform
pheromone concentrations, the Cdc24p-Far1p-G
linkage is intact, but the external spatial signal is absent. In
contrast, in a cdc24-m1 or
far1 mutant while the external signal is present, signaling from G
to Cdc24p is prevented. Furthermore, the early localization of Cdc24p during shmoo and bud formation supports the proposed role
of Cdc24p in linking a spatial landmark to polarity establishment.
A simple mechanism for growth site selection during
mating and budding is that a threshold level of locally activated Cdc24p is necessary to catalyze the GDP-GTP exchange of Cdc42p. This activation of Cdc24p is presumably
generated in part by Bud1p during budding and switched
to the region of the cell adjacent to the pheromone source
by released G during mating. In such a mechanism, it
would not be necessary to inhibit or erase the incipient
bud site during mating as previously suggested (Dorer et al.,
1995
). It is, however, possible that the binding of Cdc24p to Far1p results not only in an increased level of interaction with G
but also a decrease in the amount of Cdc24p
at the bud site, perhaps by decreasing its affinity for Bud1-GTP. We favor the notion of a balance between Cdc24p
activation at the new bud site and at the region of the
plasma membrane adjacent to pheromone source. We propose that Far1p serves to bias this equilibrium, i.e., shift
the balance, towards the site for shmoo formation.
Cells from a variety of organisms undergo polarized
growth in response to external signals. For example, in C.
elegans embryonic development it is the sperm entry site
that determines antero-posterior axis (Goldstein et al.,
1993). In Dictyostelium, cell aggregation occurs via cAMP-mediated chemotaxis (Parent and Devreotes, 1996
) and
local activation of G-protein signaling events occurs in the absence of cell movement (Parent et al., 1998
). Chemotaxis is necessary for cell migration responses for example
of lymphocytes (Arkowitz, 1999
). Chemotropism is also
essential for axonal guidance and neuronal growth cone
remodeling and extension (Tessier-Lavigne and Goodman, 1996
). Such processes are crucial for tissue and organ
development. Many of these chemotactic and chemotropic
processes appear similar to chemotropism during yeast
mating, in that they depend on chemoattractant gradients
that are recognized and transmitted by a molecular machinery including G-protein coupled receptors, rho-family
GTPases, and their exchange factors. Chemotropic growth in yeast is therefore a suitable model for understanding
the molecular basis of many different chemotropic and
chemotactic processes.
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Footnotes |
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Address correspondence to R.A. Arkowitz, Division of Cell Biology, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom. Tel.: (44) 1223 402229. Fax: (44) 1223 412142. E-mail: ra2{at}mrc-lmb.cam.ac.uk
Received for publication 13 November 1998 and in revised form 21 January 1999.
We thank S. Munro, M. Peter, and E. Schiebel for plasmids and antibodies. We are grateful to M. Bretscher, A. Gonzalez-Reyes, S. Munro, and H. Pelham for their critical reading of the manuscript.
This work was supported by the Medical Research Council. A. Nern was supported by a Marie Curie fellowship of the European Commission.
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Note Added in Proof: |
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We have now demonstrated that in the absence of CDC24-mediated chemotropism, in addition to Bud1p, other components of the general bud site selection machinery are important for shmoo formation (Nern, A., and R.A. Arkowitz, manuscript submitted for publication).
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Abbreviations used in this paper |
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GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; MAP, mitogen-activated protein; MBP, maltose binding protein; TPI, triose phosphate isomerase; TEV, tobacco etch virus.
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