The yeast protein Spa2p localizes to growth sites and is important
for polarized morphogenesis during budding, mating, and pseudohyphal
growth. To better understand the role of Spa2p in polarized growth, we
analyzed regions of the protein important for its function and proteins
that interact with Spa2p. Spa2p interacts with Pea2p and Bud6p (Aip3p)
as determined by the two-hybrid system; all of these proteins exhibit
similar localization patterns, and spa2
,
pea2
, and bud6
mutants display similar
phenotypes, suggesting that these three proteins are involved in the
same biological processes. Coimmunoprecipitation experiments
demonstrate that Spa2p and Pea2p are tightly associated with each other
in vivo. Velocity sedimentation experiments suggest that a significant portion of Spa2p, Pea2p, and Bud6p cosediment, raising the possibility that these proteins form a large, 12S multiprotein complex. Bud6p has
been shown previously to interact with actin, suggesting that the 12S
complex functions to regulate the actin cytoskeleton. Deletion analysis
revealed that multiple regions of Spa2p are involved in its
localization to growth sites. One of the regions involved in Spa2p
stability and localization interacts with Pea2p; this region contains a
conserved domain, SHD-II. Although a portion of Spa2p is sufficient for
localization of itself and Pea2p to growth sites, only the full-length
protein is capable of complementing spa2 mutant defects,
suggesting that other regions are required for Spa2p function. By using
the two-hybrid system, Spa2p and Bud6p were also found to interact with
components of two mitogen-activated protein kinase (MAPK) pathways
important for polarized cell growth. Spa2p interacts with Ste11p (MAPK
kinase [MEK] kinase) and Ste7p (MEK) of the mating signaling pathway
as well as with the MEKs Mkk1p and Mkk2p of the Slt2p (Mpk1p) MAPK
pathway; for both Mkk1p and Ste7p, the Spa2p-interacting region was
mapped to the N-terminal putative regulatory domain. Bud6p interacts
with Ste11p. The MEK-interacting region of Spa2p corresponds to the
highly conserved SHD-I domain, which is shown to be important for
mating and MAPK signaling. spa2 mutants exhibit reduced
levels of pheromone signaling and an elevated level of Slt2p kinase
activity. We thus propose that Spa2p, Pea2p, and Bud6p function
together, perhaps as a complex, to promote polarized morphogenesis
through regulation of the actin cytoskeleton and signaling pathways.
INTRODUCTION
In both unicellular and
multicellular organisms, polarized cell growth is crucial for the
formation of precise cell morphologies that allow cells to carry out
their specialized functions (17, 19, 67, 72). For example,
the development of neurites enables nerve cells to carry out sensory
transduction (20), formation of microvilli enables
epithelial cells to absorb nutrients (56), and growth of
pollen tubes in the styles of flowers facilitates plant fertilization
(6). Although the cytological events involved in polarized
cell growth have been well studied, the molecular mechanisms involved
in this process are not well understood.
The budding yeast Saccharomyces cerevisiae undergoes
polarized cell growth in several stages of its life cycle (17, 19, 67, 72). Polarized growth is prominent during budding in
vegetative and pseudohyphal growth and during projection formation in
the mating response. Polarized growth in a vegetative cell begins in
late G1, when a bud emerges from a specific site dictated
by the mating-type locus and the pedigree of the cell (12, 28, 36,
70, 80). Cell growth occurs initially at the tip of the bud
(apical growth) and then continues isotropically as the bud enlarges
(47). Finally, just prior to cytokinesis, new cell wall and
membrane deposition occurs at the mother-bud neck (47). When
limited for nitrogen sources, yeast cells also undergo budding but
adopt an elongated morphology and form chains of connected cells called
pseudohyphae, which allow cells to spread across a surface to gain
access to nutrients (31, 75). During mating, haploid cells
respond to pheromone from cells of the opposite mating type and form
projections toward their mating partners (82); these
projections are important for cell fusion (30, 87).
Polarized cell growth in yeast is a complex process that involves the
polarized organization of the actin cytoskeleton (19), the
coordinated function of many polarity proteins (67, 72), and
the regulation of signal transduction cascades (35, 45). The
actin cytoskeleton appears as distinct structures during polarized cell
growth (1, 41). Cortical actin patches are concentrated at
sites of polarized growth, and actin cables run parallel to the
polarity axis (the mother-bud axis during budding and longitudinal to
the projection during mating). The actin cytoskeleton is thought to
direct secretory vesicles containing growth components (e.g., cell wall
and plasma membrane) to growth sites (4, 58, 60).
Many components that influence cell polarity localize to sites of
polarized growth. The yeast protein Spa2p localizes to growth sites and
is important for polarized morphogenesis (14, 30, 57, 71, 80, 81,
92). Spa2p can be found at the incipient bud sites of unbudded
cells, the bud tips of small budded cells, the necks of cells
undergoing cytokinesis, and the projection tips of mating cells.
spa2
cells are rounder than wild-type cells and are
defective in bud site selection in diploid cells, cytokinesis, projection formation during mating, and pseudohyphal growth. Spa2p is a
large protein of 1,466 amino acids with several domains (30, 71), including a predicted coiled-coil region, a domain with 25 9-amino-acid repeats, and five regions that are conserved with those of
a related yeast protein, Sph1p (3, 71). These five domains
are named SHD-I to -V (SHD stands for Spa2p homology domain). The
N-terminal SHD-I is also present in proteins from a wide variety of
eukaryotes, and approximately half of SHD-II is predicted to be coiled
coil (71). How these domains contribute to the function of
Spa2p is not clear.
Pea2p and Bud6p (Aip3p) exhibit many similarities to Spa2p (2,
86). These proteins localize to growth sites in a fashion similar
to that of Spa2p, and diploid pea2
and bud6
mutants are defective in bud site selection (86, 93). Like
spa2
cells, pea2
mutants are defective in
projection formation and pseudohyphal growth (14, 57);
bud6 mutants form round cells and are defective in
cytokinesis (2, 93). Pea2p and Bud6p are smaller than Spa2p
(420 and 788 amino acids, respectively), and each has a predicted
coiled-coil domain (2, 86). Spa2p fails to localize in
pea2
mutants, and Pea2p is not stably produced in
spa2
mutants, raising the possibility that these proteins
might interact (86). Bud6p interacts with actin
(2), which also participates in diploid bud site selection
and cellular morphogenesis (18, 60, 88, 91). Thus, Spa2p,
Pea2p, and Bud6p represent an important group of proteins that
participate in many common cellular processes; perhaps these proteins
help regulate the actin cytoskeleton during polarized cell growth. The
similar localization patterns of Spa2p, Pea2p, and Bud6p together with
the common phenotypes of cells that lack these proteins suggest that
these components may function very closely, perhaps as a complex, in
the same processes. Direct evidence for such a complex has not been
described.
Like Spa2p, components of several mitogen-activated protein kinase
(MAPK) pathways also participate in the process of polarized cell
growth (35, 46). MAPK pathways are composed of a cascade of
protein kinases which act sequentially to transmit signals upon
perception of external stimuli or internal cues; these signals ultimately result in various cellular responses, such as cell growth or
differentiation (52, 85). The MAPK cascade includes a MAPK;
a MAPK kinase (MEK), which phosphorylates and activates MAPK; and a MEK
kinase (MEKK), which phosphorylates and activates MEK. In the budding
yeast, at least two MAPK pathways participate in cell growth and
differentiation. The mating response requires the Fus3p-Kss1p pathway
(59, 95), which functions downstream of the Ste20p kinase
(43). This pathway is composed of two MAPKs, Fus3p and Kss1p
(21, 22, 29); the MEK Ste7p (25); and the MEKK
Ste11p (83). Ste11p, Ste7p, and Kss1p also function in
pseudohypha formation (48, 51, 69). The other MAPK pathway, the Slt2p (Mpk1p) MAPK pathway, functions downstream of the yeast protein kinase C homolog, Pkc1p, to maintain cellular integrity during
polarized growth (46). Components of this pathway include the MAPK Slt2p (Mpk1p) (53, 84); two homologous and
redundant MEKs, Mkk1p and Mkk2p (37); and the MEKK Bck1p
(Slk1p) (15, 44). Defects in this pathway lead to cell lysis
at elevated temperatures and failure to form proper mating projections
(15, 24, 45). This pathway is also activated in response to
mating pheromone and at the G1-S transition when bud
emergence is initiated, consistent with its role in cell polarity
(24, 94). The concomitant involvement of both Spa2p and
components of these MAPK pathways in mating, pseudohypha formation, and
bud growth raises the possibility that Spa2p and perhaps other polarity
proteins might interact with signaling components. Genetic interactions
between SPA2 and BCK1 (SLK1) and between
SPA2 and SLT2 (MPK1) have been demonstrated (15, 16). Other interactions have not been uncovered.
In this study, we analyzed different SPA2 deletions and
investigated the interactions among a number of the different polarity proteins and signaling components. We provide evidence that Spa2p is a
complex protein with many important domains. Spa2p physically interacts
with Pea2p and Bud6p, and these proteins cosediment at approximate 12S,
suggesting that they form a multiprotein complex. Spa2p interacts with
Pea2p via the conserved SHD-II, which is important for both the
stability and localization of Spa2p and Pea2p. In addition, Spa2p and
Bud6p interact with components of MAPK pathways. The N-terminal
150-amino-acid MEK-interacting region of Spa2p contains a conserved
domain, SHD-I, that is important for mating and other Spa2p functions.
The signaling activities of two MAPK pathways are altered in
spa2 mutants. Taken together, these results suggest that
Spa2p, Pea2p, and Bud6p are part of a large multiprotein complex that
may exert its function through regulation of the actin cytoskeleton;
this complex may link polarity components and signaling pathways during
polarized cell growth.
MATERIALS AND METHODS
Strains, media, and microbiological techniques.
The yeast
strains used in this study are listed in Table
1. Growth media and genetic manipulation
of yeast strains were as described by Sherman et al. (77).
Yeast transformations were performed by the lithium acetate procedure
(38) or the one-step method (13).
Construction of yeast strains.
Y2003
(PEA2::HA) and Y2004
(PEA2::myc) were constructed by epitope
tagging the PEA2 gene in a haploid wild-type yeast strain, Y762, by the strategy described by Schneider et al. (76).
Primers 5'-GAG GCA AAC ACC TCG CTG GCG CTT AAT AGA GAC GAT CCA CCA GAT ATG CTA AGG GAA CAA AAG CTG GAG-3' and 5'-ATT CTT CTT ATT CTA TAT TTA
TAT ATC AAT GTT TTA TAA TAA GAT GTT TAT TCA CTA TAG GGC GAA TTG-3' were
used to amplify a region of pMPY-3XHA (for the hemagglutinin [HA]
tag) or pMPY-3XMYC (for the c-myc tag). The resulting PCR product of
approximately 1.5 kb contains a URA3 gene flanked by direct
repeats encoding three copies of the HA or c-myc epitope
(76). Both ends of the PCR fragment contain about 50 bp of
PEA2 sequences surrounding the site of fragment insertion.
Each DNA fragment was used to transform Y762. Transformants that had
integrated the fragment correctly at the chromosomal locus were
identified by PCR analysis. The integrants were allowed to grow
overnight in YPAD (77) and plated onto 5-fluoro-orotic acid
plates. 5-Fluoro-orotic acid-resistant colonies were checked by PCR for
loss of the URA3 gene through homologous recombination between the two repeated epitope-coding sequences; this leaves a single
in-frame epitope-coding sequence at the site of integration. Proper
formation of the epitope-tagged proteins was further confirmed by
immunoblot analysis. The tagged genes contain approximately 40 additional codons for the epitope plus linker sequences, and the
resulting protein is approximately 5 kDa larger than the wild-type proteins.
The doubly tagged strain Y2009 was constructed by tagging the 3' coding
sequence of BUD6 with HA in strain Y2004 by the procedure described above. The tag is inserted before the last 10 codons of
Bud6p. The primer pair used was 5'-GAA AAT TTT GTG GGT AAT TCA AAC CTG
AAA AAA TCG GGA GGC TTG AAG AAG AGG GAA CAA AAG CTG GAG-3' and 5'-CAA
AAT ATG CTC TCA AAT TTG CTT CAT CCT TCT GCT TTC TTA TCC TTT CTA TCA CTA
TAG GGC GAA TTG-3'. All of the tagged strains used in this study
(Y2003, Y2004, and Y2009) appear normal in growth and form mating
projections similar to those of wild-type cells after pheromone
treatment.
Y1213 was generated by deletion of STE5 from Y864 by a
method previously described (5). Primers 5'-CTA AAA AAG GAA
GAT ACA GGA TAC AGC GGA AAC AAC TTT TAA ATG ATG GAA CTG AGA GTG CAC CAT
AAA-3' and 5'-C GGG ATG CTT TCT TTT TAT TAT TGC ATA AAA TTT AGT GTA TAC
TCT ATA TTG AGC TGA TTT AAC AAA AAT-3' were used to amplify
TRP1 from plasmid pRS314 (78). The resulting PCR
product contained a TRP1 gene flanked by 50 bp of sequences
upstream and downstream of STE5 coding region. This fragment
was transformed into Y864. Correct substitution at the STE5
locus was verified by PCR. The resulting strain (Y1213) does not form
mating projections after
-factor treatment.
Y2002 was constructed by deletion of PEA2 from yeast strain
Y762 as described above with primers 5'-C GAA GTC CTG TGT TCG AGA GGG
AGA ATA ACG GTG GAT ATC ACG TTT CAT AAG CGC GCC TCG TTC AGA ATG-3' and
5'-C TTC TTA TTC TAT ATT TAT ATA TCA ATG TTT TAT AAT AAG ATG TTT ATT
CAC TCT TGG CCT CCT CTA GTA-3' to amplify HIS3 from pRS313
(78). Correct substitution of PEA2 was verified by PCR analysis. This strain (Y2002) forms peanut-shaped mating projections upon exposure to mating pheromone as described for typical
pea2 mutants (86).
The spa2(1-2, 116-1466) alleles in Y2011 and
Y2016 were generated from Y762 and Y2014 (a segregant of 105A-1
[23]), respectively, as described by Schneider et al.
(76). The primers used were 5'-CG AGC CAC CGA AAC AGA ATA
AAC AAA AGA AAA GAA AGA GTA AAC ATG GGT AGG GAA CAA AAG CTG GAG CTC-3'
and 5'-GG GGG CCG TGG AGC ATC CAA ATC CTT GTC GAA CCC TCT TCT CTT GAT
CTC TAG GGC GAA TTG GGT ACC GGG-3'. The resulting spa2
allele has three in-frame copies of HA-coding sequence replacing codons
3 to 115 of SPA2.
HA::SPA2 deletion constructs
and phenotypic assays.
A fragment containing SPA2 with
a NotI site after the first codon (created by K. Madden) was
cloned into pRS315, and a NotI fragment encoding three
copies of an HA epitope was introduced into the NotI site,
creating plasmid pBU4. The ApaI site in pBU4 was destroyed
by filling in with T4 DNA polymerase, and a linker with the
ApaI site and stop codons in all three frames was inserted into the HindIII-XhoI sites of this plasmid,
resulting in pBU19. A series of 3' deletions were made from pBU19 by
using the Double-Stranded Nested Deletion Kit from Pharmacia,
Piscataway, N.J. pHA::spa2(1-481, 1208-1466) was
created by excising the internal SphI fragment of
SPA2. pHA::spa2(1-13, 265-1466) and
pHA::spa2(1-13, 265-552) were created by digesting
pHA::SPA2(1-1466) and
pHA::spa2(1-552), respectively, with
EcoRV and ligating the in-frame sites. The last codon of
each deletion was determined by DNA sequence analysis (W. M. Keck
Foundation Biotechnology Resource Laboratory, Yale University). (The
plasmid constructs generated are listed in Table 3 and diagrammed in
Fig. 3.)
Plasmids were transformed into spa2
strains for
phenotypic analyses. Protein levels in lysates prepared from the
resulting strains were examined by immunoblot analysis with anti-HA
monoclonal antibodies (MAb) (see below). Immunolocalization of the
proteins is described below.
Complementation of the different defects was determined as follows.
Complementation of defects in projection formation was carried out with
yeast strains Y2007 and Y2008. Cells carrying the HA::Spa2p
deletion construct were treated with
-factor mating pheromone (Sigma
Chemical Co., St. Louis, Mo.) for 1.5 h as described previously
(50), fixed, and observed under a light microscope. Complementation of the bud site selection defect was assessed with
strain Y2001. Budding patterns were analyzed by Calcofluor staining
(50). Complementation of the pseudohyphal defect was assayed
in Y1209 (
background) as described by Roemer et al. (71). Complementation of spa2
colethality with
cdc10-10, swi4-100, bem2-101, or
slk1-1 was examined with yeast strains Y831, Y877, Y875, and
Y754, respectively, by using a colony sectoring assay (15).
Two-hybrid interactions.
The plasmids used in the two-hybrid
assay are listed in Table 2. Unless noted
otherwise, the plasmids were constructed by amplifying the
protein-coding sequences by PCR and inserting each fragment into the
BamHI sites of vectors pSH2-1 (27, 32) and pACTII
(22a). Typically the BamHI sites were created on
both ends of the PCR fragment; for fragments containing endogenous BamHI sites, BglII sites were created and used
instead. The orientations of the insert and fusion junctions were
checked by restriction mapping and DNA sequence analysis (W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University).
To test for interactions, plasmid pairs (one LexA fusion and one
activation domain [AD] fusion) were cotransformed into the reporter
yeast strain Y864. Transformants or patches grown from the
transformants were replica plated onto a sterile circle of Whatman
filter paper on fresh SC-His-Leu solid medium to select for the pSH2-1-
and pACTII-derived plasmids. Cells were allowed to grow overnight at
room temperature, and after lysis with chloroform vapor for 15 min, the
filters were transferred onto X-Gal
(5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside) plates
(89). The X-Gal plates with the filters were incubated at
30°C for up to 24 h. Blue color appeared within 2 h for the very strong interactions. The strength of the interactions was determined visually by the intensity of coloration.
For quantitative
-galactosidase (
-Gal) assays most experiments
were performed with strains grown in liquid medium as described by
Erdman et al. (23). However, for some of the two-hybrid
strains, the
-Gal activity was lost after continuous propagation in
liquid medium, presumably because the constructs are deleterious for cell growth. To circumvent this problem, yeast transformants were patched on plates containing selective medium. After incubation at room
temperature for 1 day, cells were scraped from the plate and
transferred to Z buffer for analysis (23).
-Gal activity was normalized to protein concentration in all cases. Three samples were usually analyzed for each interaction.
Antibodies.
MAb against HA (clone 16B12) and c-myc (clone
9E10) were purchased from BAbCO, Richmond, Calif. A MAb against actin
(clone C4) was purchased from ICN Biochemicals, Inc., Aurora, Ohio.
Anti-Swi6p antiserum was obtained from Brenda Andrews' laboratory and
affinity purified in a study by Madden et al. (49).
Affinity-purified anti-Spa2p antiserum was as described by Snyder
(80). Alkaline phosphatase-conjugated goat anti-mouse
immunoglobulin G (IgG) and anti-rabbit IgG and CY3-conjugated goat
antimouse antibodies were purchased from Jackson ImmunoResearch
Laboratory, Inc., West Grove, Pa. Antibodies used in indirect
immunofluorescence were preabsorbed with whole cells and spheroplasts
of untagged wild-type yeast cells to remove nonspecific antibodies
(10).
IP and immunoblot analyses.
Overnight cultures grown in YPAD
or selective media were diluted to an optical density at 600 nm
(OD600) of 0.1 to 0.15 in fresh YPAD and grown to early log
phase (OD600 = 0.3 to 0.5). Cells were then collected,
washed, and lysed with glass beads in lysis buffer as described by
Kamada et al. (40). Lysates were centrifuged for 10 min at
14,000 × g to remove cell debris. The clear lysates
were subjected to immunoprecipitation (IP), or 1/3 volume of 4×
Laemmli sample buffer (73) was added for immunoblot
analysis.
IPs were performed by using the buffer system described by Kamada et
al. (40). For each IP, 100 µg of total yeast protein in
450 µl of IP buffer was incubated with antibodies for 1.5 h at
4°C. For IP with MAb 16B12 or 9E10, a 1:150 dilution of crude ascites
fluid was used. For IP with affinity-purified anti-Spa2p or preimmune
serum, a 1:100 dilution of the affinity-purified antibody was used. To
collect the immunocomplex, 50 µl of a 1:2 IP buffer suspension of
protein A-conjugated Sepharose beads (Pierce, Rockford, Ill.) was
added, and the mixture was incubated for an additional 1.5 h at
4°C with constant mixing. The beads were pelleted and washed four
times with IP wash buffer and then eluted with 50 µl of lysis buffer
plus 25 µl of 4× Laemmli sample buffer.
For immunoblot analysis, cell lysates or immunoprecipitates in Laemmli
sample buffer were fractionated by sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis (SDS-8% PAGE), blotted onto Immobilon (Millipore, Bedford, Mass.), and probed with primary antibody. The reactive bands were visualized with alkaline
phosphatase-conjugated antibodies and CDP-Star (Boehringer Mannheim
Corp., Indianapolis, Ind.) detection reagent or the NBT-BCIP
(nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate) color
detection system as described by Harlow and Lane (34).
In vitro kinase assays.
Slt2p kinase activity was assayed as
described by Kamada et al. (40). Cells containing
pFL44-SLT2HA (94) were grown to early log phase
(OD600 = 0.2 to 0.3), collected, and lysed with glass
beads. For each kinase reaction, 250 µg of total yeast proteins was
preincubated with protein A-conjugated Sepharose beads for 1 h,
and the supernatant was immunoprecipitated with MAb 16B12. IP mixtures
were then incubated with 0.25 mg of myelin basic proteins (MBP) per ml
and 15 µCi of [
-32P]ATP at 30°C for 25 min. The
reaction were stopped by addition of sample buffer. The phosphorylation
of MBP was analyzed by SDS-15% PAGE followed by autoradiography.
Kinase activities were quantified by using Bio-Rad Multi Analyst,
version 1.0.2. As a control, background phosphorylation of MBP was
determined in a parallel assay with strains lacking HA-tagged Slt2p.
Indirect immunofluorescence microscopy.
Indirect
immunofluorescence of yeast cells containing HA::Spa2p
deletions and Pea2p::myc was performed as described
previously (30). Y2007 yeast strains with plasmids were
grown overnight in selective media, diluted to an OD600 of
0.1 in fresh YPAD, and grown to early log phase (OD600 = 0.3).
-Factor was added to a final concentration of 5 µg/ml as
described by Madden and Snyder (50). Cells were fixed in
3.7% formaldehyde for 60 min and spheroplasted. Spheroplasts were
allowed to settle on poly-L-lysine-coated slides and
incubated with preabsorbed primary antibodies (MAb 16B12 for the HA
epitope tag or MAb 9E10 for the c-myc epitope tag) overnight at 4°C.
For detection of primary antibodies, preabsorbed CY3-conjugated goat
anti-mouse IgG was used.
Sucrose gradient velocity sedimentation.
Cell lysates were
prepared from strain Y2009 and subjected to velocity centrifugation.
Cells were grown to early log phase (OD600 = 0.3 to 0.5) in
YPAD and lysed by vortexing in modified phosphate-buffered saline
buffer (0.17 M NaCl, 2.7 mM KCl, 10 mM Na2HPO4,
1.8 mM KH2PO4, pH 7.2) containing glass beads
and protease inhibitors. The crude extract was centrifuged at
16,000 × g at 4°C for 10 min. Prior to loading onto
the gradient, 160 µl of crude extract was centrifuged at 110,000 × g at 4°C for 5 min in an A-100/18 Airfuge rotor
(Beckman, Instruments., Fullerton, Calif.). Molecular size standards
(40 µl of a mixture of thyroglobulin [19.4S], catalase [11.3S],
aldolase [7.4S], and bovine serum albumin [4.4S] at 10 mg/ml in the
same buffer) were mixed with the supernatant and then loaded onto a
5-ml linear sucrose gradient (5 to 20%, wt/vol) in modified
phosphate-buffered saline buffer containing protease inhibitors.
Gradients were centrifuged at 40,000 × g at 4°C for
20 h in an SW55Ti rotor (Beckman). Approximately 175-µl aliquots
were withdrawn from the bottom of each tube by using a peristaltic pump
and analyzed by immunoblot analysis as described above. Proteins on the
immunoblots were quantified by using the National Institutes of Health
Image version 1.59. Molecular size standards were resolved
on 8% polyacrylamide gels stained with Coomassie blue. To test whether
the migration of the 12S complex is affected by detergent, 0.1% Triton
X-100 was added to the protein lysate prior to centrifugation. Analysis
of the 12S complex in pheromone-treated cells was carried out by
incubating yeast cells grown to an OD600 of 0.3 with 5 µg
of mating pheromone per ml for 2 h. Cell lysates were prepared and
analyzed as described above.
RESULTS
Spa2p and Pea2p form a stable complex in vivo.
spa2
and pea2
cells share many phenotypes, and Spa2p and Pea2p
localize to the same regions of the cell (30, 80, 86). To
determine whether Spa2p and Pea2p interact physically, co-IP experiments and two-hybrid analysis were performed. For the co-IP experiments, we tagged the chromosomal PEA2 gene immediately
upstream of the translational stop codon with a DNA fragment encoding
three copies of either an HA epitope or a c-myc epitope, using the
method described by Schneider et al. (76). Yeast cells
expressing the epitope-tagged PEA2 gene
(PEA2::HA or
PEA2::myc) form mating projections that
are indistinguishable from those of isogenic wild-type cells, and the
Pea2p::HA and Pea2p::myc proteins localize to
polarized growth sites in a manner identical to that described for
wild-type Pea2p (86) (see Fig. 4, bottom panel a, which
shows the localization of Pea2p::myc). Thus, the
epitope-tagged proteins are functional. Immunoblot analysis indicates
that the epitope-tagged Pea2 proteins migrate as a doublet at
approximately 60 kDa (see Fig. 1A for Pea2p::HA and Fig. 5
for Pea2p::myc), consistent with the predicted molecular
mass. This protein is not observed in control strains (see Fig. 1A,
lanes 3, 4, and 6).
To test whether Spa2p and Pea2p associate in vivo, protein extracts
prepared from PEA2::HA and
PEA2::myc strains were
immunoprecipitated with either anti-HA MAb, anti-c-myc MAb,
affinity-purified anti-Spa2p antiserum, or preimmune serum. The
resulting immunoprecipitates were then analyzed on immunoblots probed
with anti-HA, anti-c-myc, and anti-Spa2p antibodies. As expected,
Pea2p::HA was immunoprecipitated by an antibody that
recognizes the HA tag but not by an anti-c-myc antibody (Fig.
1A); similarly, Pea2p::myc can
be immunoprecipitated by an anti-c-myc MAb but not an anti-HA MAb (data
not shown). When affinity-purified anti-Spa2p antiserum was used for
immunoprecipitation of the PEA2::HA
lysates, Pea2p::HA was also detected in the precipitate (Fig.
1A, seventh lane from the left). The preimmune serum was unable to
immunoprecipitate Pea2p::HA from the same cell lysate (Fig.
1A, eighth lane from the left). Similarly, affinity-purified anti-Spa2p
antiserum, but not the preimmune serum, can precipitate Pea2p::myc from the PEA2::myc
extract (data not shown; the results were identical to those shown in
Fig. 1A). Anti-Spa2p antiserum failed to precipitate
Pea2p::HA with protein extracts prepared from a
PEA2::HA spa2
strain (Fig. 1A,
rightmost lane). Reciprocally, the anti-HA or anti-c-myc MAb can
specifically precipitate Spa2p from
PEA2::HA and
PEA2::myc lysates, respectively, but
not from extracts of untagged control strains (Fig. 1B). Thus, Spa2p
and Pea2p are tightly associated with each other in a complex in vivo.

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FIG. 1.
Spa2p and Pea2p coimmunoprecipitate. Proteins were
prepared from SPA2 PEA2::HA, SPA2
PEA2::myc, spa2
PEA2::HA, and spa2
PEA2::myc strains (Y2003, Y2004, Y2005, and
Y2006, respectively) and immunoprecipitated with the indicated
antibodies. The total yeast lysates and immunoprecipitates were
analyzed on immunoblots. (A) Immunoblot probed with anti-HA MAb 16B12.
The doublet at approximately 60 kDa corresponds to Pea2p::HA;
it is observed in lysates from the SPA2
PEA2::HA strain but not the SPA2
PEA2::myc strain. The doublet is also
observed in IP with anti-HA antibody or anti-Spa2p antiserum from the
SPA2 PEA2::HA lysate (fifth and seventh
lanes from the left). Anti-Spa2p antiserum failed to precipitate
Pea2p::HA from the spa2
PEA2::HA lysate (last lane). +, presence of
an allele (for PEA2::HA and
PEA2::myc) or wild-type copy of the
gene (for SPA2). (B) Immunoblot probed with
affinity-purified anti-Spa2p antiserum. Spa2p migrates as a 190-kDa
band that is not present in the spa2 lysates. The band
was detected in an IP with anti-HA antibody or anti-Spa2p antiserum
from the SPA2 PEA2::HA lysate or in IP
with anti-c-myc antibody or anti-Spa2p antiserum from the SPA2
PEA2::myc lysate. IgG marks the position of
immunoglobulin heavy chain. Pre-imm, preimmune serum.
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We also tested whether Spa2p and Pea2p interact by two-hybrid analysis.
The coding sequence of SPA2 was fused to the sequence encoding the LexA DNA binding domain (LexA), and the sequence of
PEA2 was fused to the sequence of the Gal4p transcriptional AD. Coexpression of LexA::Spa2p and AD::Pea2p
results in an 18-fold increase in
-Gal activity in a LexA-responsive
lacZ reporter yeast strain (see Table 4). The interactions
between Spa2p and Pea2p fusion proteins are specific, since these
fusions do not activate transcription either on their own, when
coexpressed with other fusions, or in the presence of vectors alone
(Fig. 2A and data not shown). We could
not test whether LexA::Pea2p interacts with
AD::Spa2p because the LexA::Pea2p construct
activates transcription in the absence of AD plasmids. Nevertheless,
the co-IP and two-hybrid results both demonstrate that Spa2p interacts
with Pea2p.

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FIG. 2.
Spa2p interacts with Pea2p through a region containing
SHD-II. (A and B) Filters showing the two-hybrid interactions. The dark
patches indicate protein-protein interactions that result in expression
of -Gal. (A) LexA::Spa2p and AD::Pea2p interact
with each other but not AD::Kar3p or LexA::Cik1p,
two coiled-coil proteins. (B) Examples of interactions between
AD::Pea2p and LexA fusions containing different regions of
Spa2p. (C) Summary of mapping of the Pea2p-interacting region of Spa2p.
Each horizontal bar represent a segment of Spa2p-coding sequence fused
to the LexA plasmid (pSH2-1); the end residues for each Spa2p segment
are labeled. These fusions were tested for interaction with the
AD::PEA2 construct, and the strength of
interaction were assigned as ++++ for the strongest interaction
and for interaction not detectable above background as judged
from replicas of whole transformation plates. The summarized
Pea2p-interacting region is shaded. The structure of Spa2p is presented
above the fusions as described by Roemer et al. (71). CC,
coiled coil A from residue 281 to 428; SHD-II, residue 429 to 535;
a.a., amino acid. The first subregion of SHD-II is predicted to be
coiled coil as well (coiled coil B).
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Spa2p interacts with Pea2p through SHD-II, which contains
coiled-coil sequence.
To delineate the region of Spa2p that
interacts with Pea2p, we constructed LexA fusions containing various
fragments of SPA2 coding sequence and used the two-hybrid
system to test for interaction with AD::Pea2p (Fig. 2B and
C). LexA::Spa2p(1-698), when coexpressed with
AD::Pea2p, resulted in an increase of
-Gal activity,
indicating a Pea2p-interacting region in the first 698 residues of
Spa2p. This interaction was not observed with
LexA::Spa2p(512-1118) or LexA::Spa2p(743-1466).
There is a predicted coiled-coil region in Spa2p from amino acid 281 to
488 (30). Part of this region is unique to Spa2p; however,
residues 429 to 488 also belong to a domain (i.e., SHD-II [residues
429 to 535]) that is shared with a Spa2p-related protein, Sph1p
(71). To find out whether the coiled-coil region of Spa2p is
involved in Pea2p interaction, we further delineated the
Pea2p-interacting region within the first 698 amino acids. Coexpression
of AD::Pea2p with either LexA::Spa2p(1-530) or
LexA::Spa2p(281-530), but not LexA::Spa2p(1-488), resulted in a strong increase in
-Gal (Fig. 2B and C). Thus, the C-terminal boundary for Pea2p
interaction resides between residues 488 and 530. Because
LexA::Spa2p(375-698) interacts with AD::Pea2p, the
N-terminal boundary likely lies after residue 375 (Fig. 2C). The region
from residue 375 to 530 could not be directly tested for interaction in
this assay because a LexA fusion containing only this region activates
transcription in the absence of an AD construct. These results indicate
that the region necessary for Pea2p interaction lies between residues 375 and 530. This region is predicted to have mostly a coiled-coil structure and includes SHD-II.
It is possible that the interaction between Spa2p and Pea2p is through
nonspecific interactions between coiled-coil sequences. This does not
appear be the case, because neither Spa2p nor Pea2p interacts with the
coiled-coil proteins Cik1p and Kar3p in parallel two-hybrid tests
(54, 63) (Fig. 2A and data not shown); furthermore, Spa2p
failed to interact with two other coiled-coil proteins, Nuf1p and NuMA
(42, 55, 81a, 90), and Spa2p failed to interact with itself
in similar assays (data not shown). Thus, Spa2p specifically interacts
with Pea2p through a region predicted to contain coiled-coil sequence.
Deletion analysis reveals multiple regions involved in proper
localization of Spa2p.
Spa2p is a large protein, and
spa2
phenotypes suggest that it is involved in many
aspects of polarized cell growth (30, 80). To determine
which region of Spa2p is important for its localization and whether
there are separable functional domains in the protein, we generated and
analyzed a series of SPA2 deletion mutants. To facilitate
detection of Spa2p, a DNA segment encoding three copies of HA was fused
immediately before the SPA2 coding sequence corresponding to
the N terminus. Full-length HA::Spa2p(1-1466) localizes
in a pattern indistinguishable from that of wild-type Spa2p and is
fully functional because it complements all spa2
defects,
including projection formation, bud site selection, pseudohyphal growth, and spa2
colethality, with cdc10-10,
swi4-100, bem2-101, and slk1-1 (Table
3). A series of deletion constructs were
prepared from the pHA::SPA2(1-1466) construct
(Fig. 3A and Table 3). The levels of
HA::Spa2p produced from these constructs were examined by
immunoblot analysis (Fig. 3B). Constructs lacking either part or all of
the coiled-coil domain and the Pea2p-interacting SHD-II failed to
produce detectable amounts of HA::Spa2p [e.g.,
pHA::spa2(1-430), -(1-193), -(1-99,
537-552), and -(1-99, 537-1466)]. In contrast, pHA::spa2(1-13, 266-552), which contains almost
exclusively the entire coiled-coil domain and SHD-II, produces protein
at a level comparable to that for the full length construct (Fig. 3B).
These observations suggest that an intact coiled-coil domain and SHD-II are important for the stability of Spa2p.

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FIG. 3.
HA::SPA2 deletion
constructs and their relative cellular protein levels. (A) Diagram of
HA::Spa2p deletions. Each horizontal bar represents a segment
of HA::Spa2p. The end residues for each segment are labeled
according to the position in the wild-type protein. A summary of
deletion analysis results is presented to the right of the constructs.
For more detailed phenotypic analysis, see Table 3. The structure of
Spa2p is presented above the deletions as described by Gehrung and
Snyder (30) and Roemer et al. (71). The shaded
regions below SHD-I and SHD-II correspond to the MEK-interacting region
and Pea2p-interacting region, respectively (see also Fig. 2 and 6). (B)
Immunoblot analysis of proteins from HA::Spa2p deletion
strains. Cell lysates were prepared from a spa2 strain
(Y2007) containing the indicated
HA::SPA2 deletion constructs. Equal
amounts of protein were loaded in each well and fractionated by
SDS-8% PAGE. The immunoblot was prepared and probed with anti-HA MAb
16B12. Two separate samples from two different experiments are shown
for the 1-430 and 1-530 constructs. The 110-kDa band is a protein that
cross-reacts with 16B12 ascites; this band is also present in Fig. 1A.
NT, not tested.
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We also determined the regions of Spa2p that are important for its
localization. HA::Spa2p produced from the different
pHA::spa2 deletion constructs was analyzed by
indirect immunofluorescence microscopy in both vegetative and mating
pheromone-treated cells. Cells carrying pHA::spa2
constructs that lacked portions or all of the coiled-coil domain and
SHD-II did not stain above the background level, since these cells
lacked detectable Spa2 protein by immunoblot analysis. As shown in Fig.
4 for pheromone-treated cells,
HA::Spa2p(1-13, 266-552), which contains primarily
the coiled-coil region and the Pea2p-interacting SHD-II, failed to
localize to the projection tips (Fig. 4, upper panel, e), indicating
that these regions are not sufficient for localization to growth sites.
There are two other regions flanking the Spa2p coiled-coil domain that
are also important for localization. All constructs containing residues 1 to 530 localize to projection tips (Fig. 4, upper panels a to c, and
data not shown), although the shortest construct,
HA::Spa2p(1-530), exhibits weaker staining (Fig. 4, upper
panel c). These results indicate that a region from residue 14 to 265, which is N terminal to the coiled-coil domain, contributes to Spa2p
targeting to growth sites. However, this region is not absolutely
required for localization in the presence of another portion of Spa2p;
HA::Spa2p(1-13, 265-1466), which lacks the region from
residue 14 to 265 but has all the sequences C terminal to the
coiled-coil domain, is able to localize to projection tips. Despite
their ability to localize at growth sites, the Spa2p deletion
constructs differ from the full-length protein in that they are more
diffuse along the projection tip region rather than localized as a
tight patch at the very tip (Fig. 4, upper panels, compare panel a to
panels b, c, and f). Identical results for Spa2p localization at the
incipient bud site and bud tip were obtained with vegetative cells
(data not shown). These data indicate that in addition to the
coiled-coil domain and Pea2p-interacting region, at least two other
regions of Spa2p are important and redundant for localizing Spa2p to
polarized growth sites.

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FIG. 4.
Immunolocalization of HA::Spa2p and
Pea2p::myc in strains containing Spa2p deletion constructs.
spa2 PEA2::myc strains containing
the different HA::SPA2 deletion
constructs were treated with -factor and stained with anti-HA
antibodies (top) or anti-c-myc antibodies (bottom). Examples of the
full-length protein (a) and five different constructs, i.e.,
pHA::spa2(1-736) (b),
pHA::spa2(1-530) (c),
pHA::spa2(1-430) (d),
pHA::spa2(1-13, 265-552) (e), and
pHA::spa2(1-13, 265-1466) (f), are shown. Although
each of the different mutants exhibits polarization defects, fields
that contain polarized cells are shown.
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Proper localization of Spa2p is required for the localization of
Pea2p to growth sites.
It was reported previously that in
spa2
cells, Pea2p was not detectable by immunoblot
analysis with anti-Pea2p antiserum (86). Thus, it was not
possible to determine whether Spa2p only affected the stability of
Pea2p or whether Spa2p was directly involved in Pea2p localization.
However, Pea2p::HA and Pea2p::myc are readily
detected by immunoblot analysis when SPA2 is deleted, albeit
at a lower level (Fig. 1A, second lane) (data not shown for
Pea2p::myc). This allows analysis of Pea2p localization in the absence of Spa2p or in the presence of Spa2p deletion constructs.
Pea2p::myc localization in spa2
strains
carrying the pHA::spa2 deletion constructs
described above was analyzed (Fig. 4, bottom panels). All strains that
localize HA::Spa2p properly also localize
Pea2p::myc. For example, a deletion construct with most of
the carboxy terminus of Spa2p removed [Spa2p(1-736)] still localizes
Pea2p::myc to the growth sites (Fig. 4, bottom panel b).
These results indicate that full-length Spa2p is not required for
Pea2p::myc localization. Moreover, constructs that exhibit weak Spa2p localization [e.g., Spa2p(1-530)] at growth sites also exhibit very weak Pea2p::myc staining, suggesting a
correlation between the efficiency of Spa2p localization and that of
Pea2p::myc. Finally, all deletion constructs tested with
either part of the Pea2p-interacting region removed [e.g.,
HA::Spa2p(1-481, 1208-1466)] or the localization of Spa2p
disrupted [e.g., HA::Spa2p(1-13, 265-552)] also fail to
localize Pea2p::myc. These results indicate Pea2p
localization at growth sites is dependent upon both its interaction
with Spa2p and the Spa2p localization sequences.
Full-length Spa2p is required for all aspects of Spa2p
function.
The indirect immunofluorescence analysis showed that
only a portion of Spa2p is required for localization of itself and
Pea2p. We also analyzed the abilities of the truncated Spa2 proteins to
complement various defects of the spa2
mutant. The
results of these studies are summarized in Table 3 and Fig. 3. Only the full-length Spa2 protein is capable of rescuing the different defects
of spa2 mutants, including the bud site selection defect, projection formation defect, pseudohypha formation defect, and colethality with bem2-101, cdc10-10,
slk1-1, and swi4-100. These results suggest that
full-length Spa2p is required for multiple aspects of its biological
functions. Alternatively, because the larger HA::Spa2p
derivatives are present at lower levels (Fig. 3B), the level of Spa2p
may be important for its function.
Spa2p interacts with Bud6p (Aip3p).
We also tested whether
Spa2p interacts with other polarity components by using the two-hybrid
system. LexA::Spa2p(1-1466) was tested for interaction with AD fusions
of Bud6p (Aip3p), Bem1p, Cdc24p, Cdc42p, Chs5p, or Act1p (actin) (see
references 2, 7, 39, 61, 65, 74, and
79 for publications describing these proteins). No
reproducible interaction was observed with Bem1p, Cdc24p, Cdc42p,
Chs5p, or Act1p. However, interaction was observed with the
AD::Bud6p(275-788) and AD::Bud6p(358-768)
constructs. A low level of interaction was observed with a shorter
Bud6p construct, AD::Bud6p(478-788) (Table
4). These results suggest that the region
of Bud6p from residue 275 to 478 contributes to its interaction with
Spa2p. Attempts to coimmunoprecipitate Spa2p and Bud6p epitope tagged
with HA or c-myc at either its N terminus or C terminus were not
successful. However, as demonstrated below, a significant portion of
Bud6p cosediments in a similar-size complex with Spa2p, raising the
possibility that these two proteins associate in vivo.
Sedimentation of Spa2p, Pea2p, and Bud6p.
The interactions
described above suggest that Spa2p, Pea2p, and Bud6p may be part of a
large multiprotein complex. To test this possibility, velocity
sedimentation experiments were performed. For detection of all three
proteins, we constructed an SPA2 BUD6::HA PEA2::myc strain. This strain has a normal
growth rate and forms pointed mating projections similar to those of
wild-type cells after pheromone treatment. Cell lysates were prepared
from this strain, and cell debris was removed. The resulting lysates
containing the soluble material were separated in a 5 to 20% sucrose
gradient. Fractions from the gradient were collected and probed with
anti-Spa2p antiserum, an anti-HA MAb (to detect Bud6p::HA),
an anti-c-myc MAb (to detect Pea2p::myc), and an antiactin
MAb. As shown in Fig. 5, most of the
Spa2p, Pea2p::myc, and Bud6p::HA cosediment and
peak at approximately 12S. A small amount of Pea2p migrates slightly
slower than Spa2p; this is likely due to cosedimentation with Spa2p
degradation products. Most of Bud6p cosediments with Spa2p, although a
small portion sediments at a higher S value (e.g., see fraction 18).
Actin present in these lysates remains at the top of the gradient.
Similar results for Spa2p and HA::Bud6p were observed when
cell lysates from an HA::BUD6 strain
(containing Bud6p tagged at the N terminus) were analyzed. Control
experiments analyzing another complex, the Cik1p-Kar3p microtubule
motor complex (62), have shown that this motor complex
sediments much more slowly than Spa2p, indicating that the 12S complex
is relatively specific for these proteins.

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FIG. 5.
Velocity sedimentation analysis of Spa2p, Pea2p, and
Bud6p. Cell lysates of a PEA2::myc
BUD6::HA strain were prepared in the absence
(A) or presence (B) of detergent and subjected to centrifugation in a 5 to 20% sucrose gradients. Fractions were collected and probed with
anti-Spa2p antibodies, anti-c-myc antibodies (to detect
Pea2p::myc), anti-HA antibodies (to detect
Bud6p::HA), or an anti-actin MAb (C4). The S values of
markers included in the same gradients are indicated at the top; these
are thyroglobulin (19.4S), catalase (11.3S), aldolase (7.4S), and
bovine serum albumin (4.4S). The amount of each immunoreactive protein
was quantified for each fraction and is shown above the immunoblots.
Only the top two-thirds of the gradient is shown, as no immunoreactive
material is detected in the bottom third of the gradient. Note that in
panel A the Pea2p peak contains material with a lower S value than
Spa2p and Bud6p; this is likely to reflect its association with a
degradation product of Spa2p that is not shown.
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The sedimentation of Spa2p, Pea2p, and Bud6p was also analyzed in
protein samples treated with the nonionic detergent Triton X-100. The
sedimentation of Spa2p (Fig. 5B) and Pea2p (not shown) is not affected
by the presence of detergent. However, a portion of Bud6p reproducibly
migrates to low-S fractions (approximately 4.4S) of the gradients,
while the majority cosediments with Spa2p (Fig. 5B) and Pea2p (not
shown). These data suggest that the majority of Spa2p, Bud6p, and Pea2p
cosediment as part of a multiprotein complex that is not associated
with vesicles and that a fraction of Bud6p is associated with vesicles.
The size of the Spa2p-associated complex was similar in cells treated
with mating pheromone. These results are consistent with the hypothesis
that Spa2p, Pea2p, and Bud6p constitute part of a large multiprotein
complex.
Spa2p interacts with components of two MAPK pathways involved in
polarized morphogenesis.
Two MAPK signaling pathways, the Fus3p
Kss1p mating pathway and the Slt2p cell integrity pathway, have been
implicated in yeast polarized cell growth (35, 46). We
tested whether Spa2p interacts with components of these two pathways by
using the two-hybrid system. As shown in Table
5 and Fig.
6A, LexA::Spa2p(1-1466) interacts with AD fusions of Mkk1p and Mkk2p, the MEKs of the Slt2p
pathway, and Ste7p and Ste11p, the respective MEK and MEKK needed for
the mating response and pseudohypha formation. The scaffolding protein
Ste5p (14a, 67a) is not required for the interaction between
Spa2p and Ste11p or Ste7p, since these interactions still occur in a
ste5
strain (Table 5). Spa2p does not interact in this
assay with other signaling components, including Pkc1p, Ste20p, Slk1p,
Slt2p, and Pbs2p (Pbs2p is the MEK of the Hog1p pathway, which mediates
responses to high external osmolarity [8]) (Table 5,
Fig. 6A, and data not shown). Attempts to coimmunoprecipitate Spa2p and
its interacting kinases produced either at wild-type levels (Ste7p,
Mkk1p, and Mkk2p), at high copy (Mkk1p), in either the presence or
absence of mating pheromone, or from a GAL1 promoter (Ste11p) were not successful. This suggests that these interactions may
be either weak or transient in vivo or that they require different physiological conditions.

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FIG. 6.
Spa2p interacts with MEKs through its N-terminal region
containing the conserved domain SHD-I. (A and B) Filters of two-hybrid
assays. (A) Interaction of LexA::Spa2p with AD constructs of
Ste7p, Pbs2p, Mkk1p, and Mkk2p. (B) Different Spa2p fusions were tested
for interaction with each of the AD::MEK constructs in panel
A and with AD vector. (C) Summary of the interaction analysis for the
different fusions. For each construct, the interactions were tested on
colonies of hundreds of transformants and on patches from at least six
transformants. Not shown is the interaction of
AD::Spa2p(1-150) with LexA::Ste7p; these fusions
interact strongly.
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The MEK-interacting domain of Spa2p resides in the conserved
N-terminal region.
To determine the MEK-interacting region of
Spa2p, we tested for interactions between LexA fusions of different
Spa2p domains and the AD fusions of Mkk1p, Mkk2p, and Ste7p. As shown
in Figure 6C, LexA::Spa2p(1-698), which contains the
Pea2p-interacting region, also interacted with all three MEKs. However,
the fusion LexA::Spa2p(1-488), which did not interact with
AD::Pea2p, still interacts with AD fusions of all three MEKs;
another fusion, LexA::Spa2p(112-530), which is positive for
Pea2p interaction, failed to interact with any of these three MEKs. A
shorter fusion, LexA::Spa2p(1-150), containing only the first
150 residues of Spa2p interacts strongly with each of the
AD::MEKs, although this construct activates transcription slightly on its own (Fig. 6B). There was no such increase with LexA::Spa2p(1-150) when the vector control plasmid was tested (Fig. 6B). In addition, AD::Spa2p(1-150), which does not
activate transcription alone, also interacts with
LexA::Ste7p (data not shown). Thus, the MEK-interacting
region of Spa2p lies within the first 150 residues (Fig. 6B). This
region contains a highly conserved sequence (SHD-I) with two repeats
that are shared with Sph1p, a Spa2p homolog, and several unknown
proteins in other organisms (71).
To determine if the MEK-interacting region is important for Spa2p
function, we substituted codons 3 to 115 with a sequence containing
three copies of the HA-coding region by using the PCR method
(76). The resulting strain produces a protein, Spa2p(1-2, 116-1466)::HA, that is expressed at levels indistinguishable
from those produced by wild-type cells (data not shown). In mating assays, strains containing this protein are defective in mating, similar to spa2
cells (Table
6). These cells also display defects in
bud site selection and mating projection formation similar to those of
spa2
cells (data not shown). Thus, the conserved region
of the MEK-interacting domain of Spa2p is important for its function.
The N-terminal nonkinase domains of Ste7p and Mkk1p interact with
Spa2p.
Ste7p and Mkk1p each contain a kinase domain at their C
termini and putative regulatory domain at their N termini (37,
82). To determine the Spa2p-interacting region of each of these
kinases, AD fusions containing N-terminal and C-terminal regions of
both Mkk1p and Ste7p were constructed and tested for interactions with LexA::Spa2p. AD::Ste7p(172-515), which contains the
kinase domain, did not interact with LexA::Spa2p, whereas
AD::Ste7p(1-172) and AD::Ste7p(1-136), which
contain the nonkinase regions of Ste7p, both interacted strongly with
Spa2p (Fig. 7B). Thus, Spa2p interacts with the amino-terminal nonkinase region of Ste7p.

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FIG. 7.
The N-terminal nonkinase domains of MEKs interact with
Spa2p. (A) LexA::Spa2p fusions were tested for interactions
with full-length, N-terminal (N-Term), or C-terminal (C-Term) AD
constructs of Ste7p, Mkk1p, and Mkk2p. (B) Specific constructs from
panel A and all fusions tested. The kinase domains of Ste7p and Mkk1p
are indicated. NT, not tested.
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AD fusions of different Mkk1p regions were also tested for interactions
with LexA-Spa2p. As found for Ste7p, LexA-Spa2p interactions occurred
with fusions containing the nonkinase domain,
AD::Mkk1p(1-214) and AD::Mkk1p(1-179), but not with
AD::Mkk1p(214-508), which contained the kinase domain (Fig.
7B). Therefore, the Spa2p-interacting region of Mkk1p also lies in the
N-terminal noncatalytic region of the kinase.
Effects of Spa2p on the MAPK signaling pathways.
The
interaction of Spa2p with Mkk1p and with Mkk2p raises the possibility
that Spa2p might affect the signaling activity of the Slt2p MAPK. To
test this possibility, we determined the relative Slt2p kinase
activities in wild-type and spa2 mutant strains. An HA
epitope-tagged Slt2p was immunoprecipitated from SPA2,
spa2
, and spa2(1-2, 116-1466) cells
(the latter lack the MEK-interacting domain) and incubated with
[
-32P]ATP and MBP, a MAPK substrate. As shown in Fig.
8, Slt2p proteins immunoprecipitated from
the spa2 mutant strains exhibit a two- to threefold increase
in in vitro phosphorylation of MBP relative to that for wild-type
control strains. These results demonstrate that the activity of the
Slt2p MAPK pathway is elevated in the spa2 mutants.

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FIG. 8.
Slt2p kinase activities in wild-type cells (WT) and
spa2 mutants. (A) In vitro phosphorylation of MBP by Slt2p
immunoprecipitated from spa2 and congenic wild-type
cells. (B) MBP phosphorylation by Slt2p immunoprecipitated from
spa2(1-2, 116-1466) strain (shown as
spa2 SHD-I) and an isogenic wild-type strain.
The relative kinase activities were quantified and are shown below each
autoradiograph.
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To further examine the activity of the Slt2p pathway, the
phosphorylation state of Swi6p, a downstream target of the Slt2p MAPK
(49), was analyzed in wild-type and spa2
cells. As shown in Fig. 9, the
slow-mobility isoform of Swi6p, which is the phosphorylated form of the
protein (49), is more intense in the protein extract prepared from a spa2
strain than in that from wild-type
cells. Quantification indicates that the level of Swi6p
hyperphosphorylation is about 25% in wild-type cells and 50% in
spa2
cells (Fig. 9). The same phenomenon was observed
when the phosphorylation patterns of Swi6p in diploid wild-type and
spa2
/spa2
mutant cells were compared (data not shown).
These results suggest that deletion of SPA2 results in a
higher activity of the Slt2p pathway which causes
hyperphosphorylation of Swi6p. Hyperphosphorylation of Swi6p was
also observed in pea2
cells (Fig. 9). This is
consistent with the observation that Spa2p and Pea2p function as
a complex in the same process.

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FIG. 9.
Levels of hyperphosphorylated Swi6p in wild-type (WT),
spa2 , and pea2 strains. Equal amounts of
proteins from cell lysates of wild-type (Y604 and Y762),
spa2 (Y602), and pea2 (Y2002) strains grown
to early log phase (OD600 = 0.3) were fractionated by
SDS-8% PAGE. The immunoblot was prepared and probed with
affinity-purified anti-Swi6p antiserum. The percentages of
hyperphosphorylated Swi6p are shown below the blots. The relative
amounts of proteins in the upper and lower bands were quantified by
transmittence-reflectance scanning densitometry.
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The interaction of Spa2p with Ste7p and Ste11p also raises the
possibility that Spa2p might affect the Ste11p-Ste7p signaling pathways. Therefore, we analyzed the expression of two mating signaling
reporter constructs, fus2::lacZ and
fig1::lacZ (23), in
spa2
and wild-type strains after treatment with different concentrations of mating pheromone. spa2
fus2::lacZ strains either treated with low
levels of mating pheromone (0.6 nM) or not treated with pheromone
reproducibly exhibit a reduced level of expression (approximately two-
to threefold) relative to that of wild-type cells (Table
7). Treatment of cells with high levels
of mating pheromone or analysis of
fig1::lacZ strains revealed little or no reproducible difference between spa2
and wild-type
cells (Table 7 and data not shown). spa2(1-2,
116-1466) cells, which lack SHD-I, also exhibit reduced
fus2::lacZ expression in the presence of a low concentration of or no mating pheromone, similar to that of a
spa2
mutant (Table 7). These experiments indicate that SHD-I is important for optimal MAPK signaling.
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TABLE 7.
Expression of fus2::lacZ
in wild-type and spa2 cells treated with different
concentrations of mating pheromone
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Bud6p interacts with Ste11p.
We also used the two-hybrid
system to test for interactions between Pea2p, Bud6p, and components of
the signaling pathways described above. AD::Pea2p did not
interact with LexA::Ste11p, LexA::Ste7p, or
LexA::Fus3p (not shown). We could not test for Pea2p
interaction with Mkk1p, since LexA::Mkk1p, like
LexA::Pea2p, activates reporter transcription by itself.
However, AD::Bud6p(358-768) and AD::Bud6p(275-788)
showed strong transcription activation when coexpressed with
LexA::Ste11p (Table 8),
suggesting an interaction between Bud6p and Ste11p. The level of
interaction between AD::Bud6p(478-788) and
LexA::Ste11p was similar to the background level. These
interactions suggest that the region from residue 358 to 478 contributes to the Ste11p interaction. The Bud6p fusions did not
interact, as determined by two-hybrid analysis, with other signaling
components tested, including Ste7p, Fus3p, Mkk1p, Mkk2p, Slt2p, and
Pbs2p. Co-IP between Myc::Bud6p and HA::Ste11p was
not successful, suggesting that the interaction may be transient or
weak in vivo.
DISCUSSION
In this study, we demonstrated that the 1,466-amino-acid protein
Spa2p contains multiple domains that interact with the polarity proteins Pea2p and Bud6p. Together, these polarity proteins may constitute a large multiprotein complex that functions to promote polarized cell growth through regulation of the actin cytoskeleton (Fig. 10). We also showed that
components of this putative multiprotein complex interact with
constituents of two MAPK signaling pathways involved in budding,
mating, and pseudohypha formation (Fig. 10). These different
interactions may provide links by which polarity components and
signaling pathways can coordinate the complex processes of polarized
growth.

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FIG. 10.
Summary of the different interactions between
constituents of the 12S complex and signaling components. Proteins that
contact one another have been shown to be physically associated by
co-IP or sedimentation analysis. Proteins that interact as determined
by the two-hybrid system are indicated by lines with arrowheads at each
end. Components that were analyzed in this study are shaded. For a
further description of the different components, see reference
17.
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Spa2p contains multiple protein-interacting domains that are
important for its function.
Sequence analysis of Spa2p has
revealed a variety of domains within this protein (30, 71).
There are three large regions of over 100 amino acids (SHD-I, -II, and
-V) that are homologous to Sph1p, a related protein in yeast. The
second of these, SHD-II (amino acids 429 to 535), contains a large
putative coiled-coil segment (71). In addition, Spa2p has
two regions that are unique: a coiled-coil domain from residue 281 to
428 as well as a stretch of 9-amino-acid repeats between residues 816 and 1087 (30).
We have found that Spa2p interacts with Pea2p and that the
Pea2p-interacting domain maps to a region between residues 375 and 530. This region is composed mostly of SHD-II, which is primarily coiled
coil, and a small portion of the coiled-coil region that is unique to
Spa2p. Pea2p also possesses a putative coiled-coil domain from residue
236 to 327 (86). The interaction of the Spa2p coiled-coil
segments with Pea2p raises the possibility that the two polarity
proteins might associate with each other via a coiled-coil interaction.
Although much of the Pea2p-interacting region of Spa2p is homologous to
Sph1p, Pea2p and Sph1p do not interact by using the two-hybrid system
(71). Thus, it is likely either that the region outside the
SHD-II region contributes to interaction with Spa2p or that the SHD-II
regions of Spa2p and Sph1p interact with different proteins.
The Pea2p-interacting region of Spa2p is important for stability,
localization, and function of the protein. Deletions that remove most
of this region result in loss of detectable Spa2p (Fig. 3 and Table 3),
and immunolocalization analyses indicate that this region in
conjunction with one of two other regions of Spa2p is required for
localization to growth sites within the cell (Fig. 4). Arkowitz and
Lowe (3) have also mapped a localization domain of Spa2p to
this same region (see below), and Valtz and Herskowitz have found that
loss of Pea2p results in mislocalization of Spa2p (86).
Thus, interaction of this region with Pea2p is critical for Spa2p
localization and function.
We have also found a region within the N-terminal 150 amino acids of
Spa2p that interacts with the yeast MEKs Ste7p, Mkk1p, and Mkk2p (Fig.
6) and an as-yet-unidentified region that interacts with Ste11p. All of
these kinases belong to signaling pathways that have been implicated in
polarized morphogenesis and mating (25, 37, 48, 69, 83, 94).
The MEK-interacting region of Spa2p overlaps with SHD-I, which is
homologous to domains in Sph1p and proteins in other organisms, such as
humans and nematodes (71). In addition, Roemer et al. have
found that Sph1p also interacts with these same MEKs (71).
SHD-I contains two conserved repeated sequences called SDRs (for Spa2
direct repeat). We speculate that the conserved SDRs interact with MEKs
in a wide variety of organisms.
Several other domains exist in the Spa2p protein. SHD-V and at least
two other smaller SHDs are predicted from the Spa2p sequence, in
addition to the Spa2p unique coiled-coil domain segment and 9-amino-acid repeats. We have found that Bud6p and Ste11p interact with
Spa2p as determined by the two-hybrid system, and Roemer et al. found
that Sph1p also interacts with these two proteins (71).
Thus, it is likely that other SHDs (III, IV, and/or V) bind to Bud6p
and Ste11p. Presumably other proteins also bind Spa2p domains. One
additional candidate is Bni1p; bni1
mutants have
phenotypes similar to those of spa2
cells, and Bni1p
localizes to growth sites similarly to Spa2p (26).
Additional localization and functional domains of Spa2p.
In
addition to the Pea2p-interacting domain, our analysis of
HA::Spa2p deletions also revealed two other, less defined
regions that are important for proper Spa2p localization at growth
sites. One contains the sequence located between residues 14 and 264, and the other resides between residues 552 and 1466. Either region, when combined with the Pea2p-interacting SHD-II, is sufficient for
targeting Spa2p to growth sites (Table 3; Fig. 3 and 4). It is likely
that these two regions either contain additional sequences that
facilitate Spa2p targeting or help Spa2p adopt a conformation that is
competent for localization to growth sites. The different sequences
that help localize Spa2p also are critical for localization of Pea2p
(Table 3 and Fig. 4).
As this paper was being prepared, Arkowitz and Lowe reported that a
region of Spa2p from amino acids 397 to 549 is sufficient for targeting
a Spa2-green fluorescent protein (GFP) fusion to growth sites
(3). Although this region overlaps well with the Pea2p-interacting region/localization domain that we identified, they
did not identify the other regions of the proteins that are important
for Spa2p localization. The discrepancy between our results and theirs
may be due to the difference in sensitivity of detection between GFP
and indirect immunofluorescence microscopy or to differences in the
stability of Spa2p-GFP and HA::Spa2p proteins. Regardless,
our data indicate that other regions of Spa2p are important for its
proper localization.
Localization of Spa2p to the growth sites is not sufficient for its
function. Only the full-length HA::Spa2p construct is capable
of rescuing spa2 mutant defects (Table 3 and Fig. 3). Even
the deletions removing fewer than 90 residues at the C terminus [i.e.,
HA::Spa2p(1-~1410) and HA::Spa2p(1-1380)] fail
to complement the spa2 defects. Since this region overlaps
with SHD-V, it is likely that SHD-V interacts with another component
that functions in the same processes as does Spa2p. Alternatively, it
is also possible that SHD-V contributes to the stability of the protein so that proteins lacking this region are present at lower levels [which is the case for HA::Spa2p(1-~1410) and
HA::Spa2p(1-1380)] and are thus unable to provide proper
Spa2p function. It is also important to note that although many Spa2p
deletion constructs are capable of localizing to growth sites, their
localization patterns are more diffuse over a broad region instead of
forming a tight patch at growth sites as does the full-length protein. This is especially evident in pheromone-treated cells, in which the
projection is wider in these mutants (Fig. 4); the wide projection might explain in part the broad distribution of the protein.
Alternatively, as discussed further below, Spa2p is part of a
multiprotein complex that functions early in the process of polarized
cell growth. Thus, multiple regions of Spa2p are essential for the
proper localization and function of Spa2p.
Spa2p interacts with other polarity proteins to form a multiprotein
complex.
The results presented in this study indicate that Spa2p
interacts with at least two other polarity proteins, Pea2p and Bud6p, and that a large portion of these proteins cosediment in sucrose gradients as a 12S complex (Fig. 5), which we call the 12S polarisome. These data are consistent with the similar localization patterns of
these proteins and the similar phenotypes of spa2
,
pea2
, and bud6
cells (2, 14, 30, 81,
86, 93).
A 12S sedimentation coefficient corresponds to a size of approximately
300 kDa, assuming a globular conformation (11). However, if
the complex adopts an asymmetrical conformation, the mass might be
greater. Given the predicted sizes of Spa2p (160 kDa),
Pea2p::myc (53 kDa), and Bud6p::HA (90 kDa), it is
likely that many of the components of the 12S complex are already
accounted for. There may be additi