INTRODUCTION

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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

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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.

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.)

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).

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.

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.

RESULTS

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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).

View larger version (36K): [in this window] [in a new window]   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.

View larger version (33K): [in this window] [in a new window]   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).

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).

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.

View larger version (31K): [in this window] [in a new window]   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.

View larger version (51K): [in this window] [in a new window]   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.

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.

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.

View larger version (23K): [in this window] [in a new window]   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.

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.

View larger version (34K): [in this window] [in a new window]   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.

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).

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.

View larger version (22K): [in this window] [in a new window]   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.

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.

View larger version (29K): [in this window] [in a new window]   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 spa2SHD-I) and an isogenic wild-type strain. The relative kinase activities were quantified and are shown below each autoradiograph.

spa2/spa2

View larger version (27K): [in this window] [in a new window]   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.

spa2 fus2

View this table: [in this window] [in a new window]   TABLE 7.   Expression of fus2::lacZ in wild-type and spa2 cells treated with different concentrations of mating pheromone

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.

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.

View larger version (32K): [in this window] [in a new window]   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.

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).

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).

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).