A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization
2002; Springer Nature; Volume: 21; Issue: 7 Linguagem: Inglês
10.1093/emboj/21.7.1565
ISSN1460-2075
AutoresAnne Christine Butty, Nathalie Perrinjaquet, Audrey Petit, Malika Jaquenoud, Jeffrey E. Segall, Kay Hofmann, Catherine Zwahlen, Matthias Peter,
Tópico(s)Plant Molecular Biology Research
ResumoArticle1 April 2002free access A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization Anne-Christine Butty Anne-Christine Butty Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Nathalie Perrinjaquet Nathalie Perrinjaquet Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Audrey Petit Audrey Petit Institute of Organic Chemistry, University of Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author Malika Jaquenoud Malika Jaquenoud Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Jeffrey E. Segall Jeffrey E. Segall Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Kay Hofmann Kay Hofmann Bioinformatics Group, Memorec Stoffel GmbH, Stöckheimerweg 1, D-50829 Köln, Germany Search for more papers by this author Catherine Zwahlen Catherine Zwahlen Institute of Organic Chemistry, University of Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author Matthias Peter Corresponding Author Matthias Peter Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Anne-Christine Butty Anne-Christine Butty Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Nathalie Perrinjaquet Nathalie Perrinjaquet Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Audrey Petit Audrey Petit Institute of Organic Chemistry, University of Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author Malika Jaquenoud Malika Jaquenoud Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Jeffrey E. Segall Jeffrey E. Segall Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA Search for more papers by this author Kay Hofmann Kay Hofmann Bioinformatics Group, Memorec Stoffel GmbH, Stöckheimerweg 1, D-50829 Köln, Germany Search for more papers by this author Catherine Zwahlen Catherine Zwahlen Institute of Organic Chemistry, University of Lausanne, CH-1015 Lausanne, Switzerland Search for more papers by this author Matthias Peter Corresponding Author Matthias Peter Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland Search for more papers by this author Author Information Anne-Christine Butty1, Nathalie Perrinjaquet1, Audrey Petit2, Malika Jaquenoud1, Jeffrey E. Segall3, Kay Hofmann4, Catherine Zwahlen2 and Matthias Peter 1 1Swiss Institute for Experimental Cancer Research, Chemin des Boveresses 155, CH-1066 Epalinges/VD, Switzerland 2Institute of Organic Chemistry, University of Lausanne, CH-1015 Lausanne, Switzerland 3Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461 USA 4Bioinformatics Group, Memorec Stoffel GmbH, Stöckheimerweg 1, D-50829 Köln, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1565-1576https://doi.org/10.1093/emboj/21.7.1565 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In Saccharomyces cerevisiae, activation of Cdc42 by its guanine-nucleotide exchange factor Cdc24 triggers polarization of the actin cytoskeleton at bud emergence and in response to mating pheromones. The adaptor protein Bem1 localizes to sites of polarized growth where it interacts with Cdc42, Cdc24 and the PAK-like kinase Cla4. We have isolated Bem1 mutants (Bem1-m), which are specifically defective for binding to Cdc24. The mutations map within the conserved PB1 domain, which is necessary and sufficient to interact with the octicos peptide repeat (OPR) motif of Cdc24. Although Bem1-m mutant proteins localize normally, bem1-m cells are unable to maintain Cdc24 at sites of polarized growth. As a consequence, they are defective for apical bud growth and the formation of mating projections. Localization of Bem1 to the incipient bud site requires activated Cdc42, and conversely, expression of Cdc42–GTP is sufficient to accumulate Bem1 at the plasma membrane. Thus, our results suggest that Bem1 functions in a positive feedback loop: local activation of Cdc24 produces Cdc42–GTP, which recruits Bem1. In turn, Bem1 stabilizes Cdc24 at the site of polarization, leading to apical growth. Introduction The establishment of cell polarity and polarized growth is crucial for the development and functioning of both uni- and multi-cellular organisms (Drubin, 2000). On a cellular level, polarity results in the generation and maintenance of shape, directional movement, phagocytosis, adhesion and motility. All these changes require dynamic assembly and rearrangements of the actin cytoskeleton, which are orchestrated by local activation and inactivation of Rho-type GTPases (Hall, 1998). However, the mechanisms underlying their spatial and temporal regulation remain poorly understood. The yeast Saccharomyces cerevisiae exhibits polarized growth at several stages of its life cycle (Pruyne and Bretscher, 2000). During the cell cycle, activation of the Cdc28-ln kinase triggers polarization of the actin cytoskeleton towards the incipient bud site in the late G1 phase, while during mating, cells polarize towards their mating partner along a pheromone gradient (Gulli and Peter, 2001). Upon nitrogen starvation, cells elongate from one pole, forming chains of linked cells that spread across the substratum (Kron and Gow, 1995). Central to the initiation of actin polarization is the local activation of the small GTPase Cdc42. Like all members of the Ras superfamily, Cdc42 cycles between an inactive GDP-bound and an active GTP-bound state. GDP/GTP cycling is regulated by the guanine-nucleotide exchange factor (GEF) Cdc24, which promotes GDP dissociation and facilitates GTP binding, whereas the GTPase-activating proteins Rga1 and Bem3 have the opposite effect and stimulate the intrinsic GTPase activity of Cdc42. In the active GTP-bound state, Cdc42 interacts with its downstream effectors, which in turn control the assembly of actin filaments and their organization into complex structures (Johnson, 1999). Cortical actin patches congregate at the site of polarization and actin cables become orientated towards that site, resulting in polarized secretion of vesicles and hence polarized surface growth. We are interested in the spatial and temporal regulation of Cdc42 during polarized growth. Previous studies suggested that the small GTPase Rsr1/Bud1 targets Cdc24 to the incipient bud site (Bender and Pringle, 1989; Park et al., 1997), while, in a pheromone gradient, Far1 delivers Cdc24 to the site of receptor activation marked by Gβγ (Butty et al., 1998; Nern and Arkowitz, 1999). Cdc24 is stabilized at the site of polarization, presumably by binding to the adaptor protein Bem1 (Gulli et al., 2000). Cells deleted for BEM1 are viable, but exhibit severe defects in actin organization and polarized growth (Bender and Pringle, 1991; Chenevert et al., 1992). After bud emergence, Cdc24 is hyperphosphorylated by Cla4 (Bose et al., 2001), and this phosphorylation is thought to trigger its release from Bem1 at the polarization site and thus limit polarized growth (Gulli et al., 2000). In addition to Cdc24 (Peterson et al., 1994), Bem1 interacts with numerous other proteins including Cdc42 (Butty et al., 1998; Bose et al., 2001), the PAK-like kinases Ste20 (Leeuw et al., 1995) and Cla4 (Gulli et al., 2000; Bose et al., 2001), Rsr1/Bud1 (Park et al., 1997), Boi1 and Boi2 (Bender et al., 1996), Far1 (Butty et al., 1998) and Ste5 (Lyons et al., 1996), and has thus been implicated in many cellular pathways such as signal transduction and morphogenesis. Boi1 and Boi2 interact with the C-terminal SH3 domain of Bem1 (Bender et al., 1996), while Cdc24 binds to its C-terminus (Peterson et al., 1994). However, it is not known whether all binding partners directly associate with Bem1, or whether they bind in a mutually exclusive manner. To investigate the role of Bem1 during polarized growth, we characterized the molecular interaction between Bem1 and Cdc24 in vitro and in vivo. We demonstrate that Bem1 and Cdc24 interact directly through two conserved motifs, while the interaction between Far1 and Bem1 is bridged by Cdc24. Importantly, our results suggest that the critical role of Bem1 in vivo is to stabilize Cdc24 at sites of polarized growth. Results Isolation of BEM1 alleles unable to interact with Far1 and Cdc24 To isolate Bem1 mutants defective for binding to Far1, we designed a two-hybrid screen (see Materials and methods) in which Bem1 was fused to the DNA-binding domain and tagged at is C-terminus with green fluorescent protein (GFP). The GFP fusion did not affect the in vivo function of Bem1, and in the two-hybrid assay, Bem1–GFP interacted with Far1 and Cdc42 to the same extent as the non-tagged control (data not shown). A library of randomly mutagenized two-hybrid plasmids was transformed into EGY48 and screened for loss of interaction with Far1. Promising candidates were re-screened for GFP fluorescence to eliminate mutants that failed to express Bem1 or harbored truncated forms of Bem1. Two Bem1 mutants (referred to as Bem1-m1 and Bem1-m2) were defective for binding to Far1 but interacted efficiently with Cdc42–GTP and thus were used for further analysis. The interaction between Far1 and Bem1 requires Cdc24 We compared the ability of wild-type Bem1, Bem1-m1 and Bem1-m2 to interact with Far1, Cdc24, Cdc42, Boi1 and Cla4 by two-hybrid analysis (Figure 1; data not shown). In addition, we included the C-terminally truncated Bem1-s1 and Bem1-s2 proteins (Chenevert et al., 1992), as these mutant proteins have been shown previously to be defective for their interaction with Ste20 (Leeuw et al., 1995). All Bem1 mutant proteins interacted efficiently with Cdc42–GTP (Figure 1), confirming that it binds to the N-terminal domain of Bem1 (Bose et al., 2001). Bem1-s1 and Bem1-s2 were both defective for their interaction with Far1, Cdc24 and Cla4, indicating that all these proteins interact with the C-terminal domain of Bem1. As expected, Bem1-m1 was unable to bind to Far1, whereas the interaction of Bem1-m2 with Far1 was strongly reduced (Figure 1). Surprisingly, however, the interaction between Bem1-m1 and Cdc24 was also abolished, and Bem1-m2 binding to Cdc24 was strongly decreased. Co-immunoprecipitation experiments confirmed that Bem1-m1 and Bem1-m2 were defective for binding to Cdc24 (Figure 2A). In contrast to wild-type Bem1 (lane 4), only small amounts of Bem1-m1–GFP (lane 6) or Bem1-m2–GFP (lane 8) co-immunoprecipitated with Cdc24-myc. Finally, the interaction between Bem1 and Cdc24 was also observed in far1Δ strains (Figure 1), demonstrating that Far1 is not required for their binding. Figure 1.Two-hybrid analysis of wild-type and mutant Bem1. (A) Wild-type and various mutants of Bem1 were tested by two-hybrid analysis in EGY48 cells for their ability to interact with Far1, Cdc24, Cla4 and Cdc42–G12V/C188S (Cdc42C188S GTP-bound). Expression of the β-gal reporter was quantified and is shown as Miller units ± SD. (B) Schematic summary of the interactions between Bem1 and Cdc42, Cdc24 and Far1. Cdc42–GTP binds to the N-terminal part of Bem1 and requires an intact second SH3 domain (Bose et al., 2001). The C-terminal PB1 domain interacts with Cdc24. The interaction between Far1 and Bem1 is likely to be bridged by Cdc24. Download figure Download PowerPoint Figure 2.Interactions between wild-type and mutant Bem1 with Cdc24 and Cla4. (A) Cdc24-myc was immunoprecipitated with 9E10 antibodies from cells harboring, as indicated, an empty control vector (lanes 1 and 2) or plasmids allowing expression of wild-type Bem1–GFP (lanes 3 and 4), Bem1-m1–GFP (lanes 5 and 6) or Bem1-m2–GFP (lanes 7 and 8) from the GAL promoter. The immunoprecipitates (labeled IP) and cleared cell lysates before immunoprecipitation (labeled SN) were analyzed for the presence of Bem1–GFP (upper panel) and Cdc24-myc (lower panel) by immunoblotting with GFP or 9E10 antibodies, respectively. The asterisk marks an unspecific protein recognized by the GFP antibodies. (B) Cla4-myc (lanes 1–8) or extracts from untagged controls (lanes 9–12) were immunoprecipitated with 9E10 antibodies from wild-type cells harboring, as indicated, either an empty control vector (lanes 1 and 2) or a plasmid allowing expression of wild-type Bem1–GFP (lanes 3 and 4), Bem1-m1–GFP (lanes 5 and 6) or Bem1-m2–GFP (lanes 7 and 8) from the GAL promoter. The immunoprecipitates and cleared cell lysates were analyzed for the presence of Bem1–GFP (upper panel) and Cla4 (lower panel) by immunoblotting with GFP or Cla4 antibodies, respectively. (C) Hyperphosphorylation of Cdc24 was examined in the indicated strains by immunoblotting. The arrowhead points to the position of unphosphorylated Cdc24; the bracket marks the position of hyperphosphorylated Cdc24. (D) Wild-type (lane 1) or bem1Δ cells (lanes 2–5) were transformed with a plasmid expressing Cdc42–G12V and either an empty control vector (lane 2) or plasmids expressing, as indicated, wild-type or Bem1-m mutant proteins fused to GFP. Hyperphosphorylation of Cdc24 was analyzed by immunoblotting. For control, the extracts were also examined for the presence of Bem1–GFP with GFP antibodies (middle panel) and actin (lower panel). The asterisk marks an unknown protein recognized by the GFP antibody. (E) The levels of Cdc24 were compared by immunoblotting of extracts prepared from far1Δ (lane 1) and cdc24-5 far1Δ cells (lane 2) shifted to 37°C for 3 h. Immunoblotting with antibodies against actin confirmed equal loading (lower panel). Note that cdc24-5 cells express very low levels of Cdc24, and were thus used as a host for the two-hybrid experiments described in Table I. Download figure Download PowerPoint Because both Far1 and Cdc24 were unable to interact with Bem1-m1 and Bem1-m2, they may share the same binding site, or Cdc24 may bridge binding of Far1 to Bem1. To distinguish between these two possibilities, we tested the interaction of Far1 and Bem1 in cdc24-5 cells, which failed to express detectable amounts of Cdc24 after shifting them to a restrictive temperature for 3 h (Figure 2E). Strikingly, the interaction between Far1 and Bem1 was strongly reduced in cdc24-5 cells (Table I), suggesting that the interaction between Far1 and Bem1 requires Cdc24 (Figure 1B). Table 1. Two-hybrid analysis Activation domain fusion DNA-binding domain fusion Miller units ± SD cdc24-5 far1Δ Far1 Bem1 64 ± 16 Far1 Cdc24 2085 ± 542 Far1 vector 3 ± 1 vector Bem1 1 vector Cdc24 6 ± 1 far1Δ Far1 Bem1 902 ± 136 Far1 Cdc24 2473 ± 321 Far1 vector 6 ± 2 vector Bem1 2 ± 2 vector Cdc24 10 Two-hybrid analysis of wild-type Far1 fused to an activation domain and Bem1 and Cdc24 fused to the LexA-DNA-binding domain performed at 37°C in either cdc24-5 far1Δ (YNP39) or far1Δ cells (YNP35). Note that cdc24-5 cells express very low levels of Cdc24, as shown in Figure 2E. Expression of the β-gal reporter was quantified and is shown as Miller units ± SD. bem1-m cells are unable to phosphorylate Cdc24, although Bem1-m1 and Bem1-m2 interact with Cla4 Phosphorylation of Cdc24 in vivo is mediated by Cla4, and requires activated Cdc42 and Bem1 (Gulli et al., 2000; Bose et al., 2001). Unlike wild-type cells, both bem1-m1 and bem1-m2 cells failed to phosphorylate Cdc24 (Figure 2C), even after overexpression of Cdc42–GTP (Figure 2D). Because Cla4 was able to co-immunoprecipitate efficiently with both the wild type and the Bem1-m mutants (Figure 2B), and also interacts with Bem1-m1 by two-hybrid analysis (Figure 1), these results demonstrate that binding of Cdc24 to Bem1 is required for its phosphorylation by Cla4 in vivo. Two conserved protein–protein interaction motifs mediate binding of Bem1 to Cdc24 Sequencing of the bem1-m1 and bem1-m2 alleles revealed single non-conservative amino acid substitutions located within the C-terminal domain of Bem1 (Figure 3A), consistent with the previous finding that Cdc24 interacts with the C-terminal domain of Bem1 (Figure 1; Peterson et al., 1994; Ito et al., 2001). bem1-m1 harbors an A to C conversion, changing lysine 480 to glutamic acid, while bem1-m2 contains a C to G mutation that changes serine 547 to a proline residue. Sequence alignment of the C-terminal region of Bem1 from S.cerevisiae, its Schizosaccharomyces pombe homolog Scd2 (Chang et al., 1994) and Bem1 from Ashbya gossipii (Figure 3A) revealed a conserved motif, which was recently termed PB1 for phox and Bem1 (Ito et al., 2001). The K480E mutation of Bem1-m1 is part of the conserved PB1 core, while the S547P mutation of Bem1-m2 lies further downstream, suggesting that the flanking regions may contribute to efficient binding to Cdc24. Besides fungal Bem1 proteins, the PB1 motif is found in the N-terminal region of several mammalian signaling proteins including human MEK3, isoforms of protein kinase C and several hypothetical proteins of Arabidopsis thaliana (Ito et al., 2001). Figure 3.Identification of a conserved motif in Bem1 sufficient to directly bind to Cdc24. (A) Schematic representation of Bem1 with its different domains. PX, PX domain of Bem1; SH3, Src homology domain. The amino acid sequence of a C-terminal motif of Bem1 (termed PB1) from S.cerevisiae (Bem1) was aligned with homologs from A.gossipii (AgBem1) and S.pombe (SCD2). The numbers indicate the amino acids in Bem1 starting from the N-terminal methionine. Identical amino acids are shown with black boxes; similar amino acids are shaded. The mutations found in bem1-m1 (K480E) and bem1-m2 (S547P) are indicated by the arrowheads. The mutation determined by Ito et al. (2001) is marked by an arrow. (B) The PB1 and OPR motifs of Bem1 (amino acids 463–551) and Cdc24 (amino acids 781–854) were expressed in E.coli and purified to homogeneity (input). The purified domains were mixed in a 1:1 ratio, separated on a superose 12 gel filtration column, and the fractions examined by spectrometry (right panel). The fractions marked by the arrows were analyzed by SDS–PAGE followed by silver staining (output). Download figure Download PowerPoint Two-hybrid assays demonstrated that a small fragment (amino acids 463–551) containing the PB1 domain of Bem1 was sufficient to interact with Cdc24 and Far1 (Table II; data not shown). In contrast, this fragment was unable to interact with Cla4 (data not shown), indicating that this interaction requires determinants outside the PB1 domain. Interestingly, the C-terminus of Cdc24 contains an evolutionarily conserved motif termed octicos peptide repeat (OPR; Ponting, 1996), which is also present in the C-terminus of Scd1, the Cdc24 homolog of S.pombe. Constructs encompassing the OPR domain of Cdc24 readily interacted with the PB1 domain of Bem1 by two-hybrid assay (Table II; Ito et al., 2001), suggesting that the OPR and PB1 domains may mediate specific binding. Indeed, purified OPR and PB1 domains quantitatively formed heterodimers in vitro, as assayed by gel filtration on a superose 12 column (Figure 3B). Thus, the OPR domain of Cdc24 directly interacts with the PB1 domain of Bem1, suggesting that the two motifs comprise novel protein–protein interaction domains. Table 2. Two-hybrid analysis in EGY48 cells Activation domain fusion DNA-binding domain fusion Miller units ± SD Cdc24 Bem1 888 ± 29 Cdc24 Bem1 (463–551) 938 ± 132 Cdc24 (781–854) Bem1 1220 ± 111 Cdc24 (781–854) Bem1 (463–551) 1729 ± 85 Cdc24 vector 2 ± 1 Cdc24 (781–854) vector 2 Vector Bem1 2 Vector Bem1 (463–551) 51 ± 13 Two-hybrid analysis of full-length and the indicated fragments of Bem1 and Cdc24. The interaction was quantified as described in the footnote to Table I. Note that the PB1 domain of Bem1 is sufficient to interact with the OPR domain of Cdc24. Cdc24–GFP is recruited to the incipient bud site, but is not maintained at bud tips in bem1-m cells To determine whether binding of Cdc24 to Bem1 affects the subcellular localization of Cdc24, wild-type and bem1-m cells expressing Cdc24–GFP were released from a nutritional block in early G1, and the localization of Cdc24–GFP was examined by GFP microscopy. In both strains, ∼35% of Cdc24–GFP initially localized to the incipient bud site (Figure 4A). However, as in bem1Δ cells (Gulli et al., 2000), Cdc24 was not detectable at tips of small buds in bem1-m cells (arrow), while it was maintained at sites of polarized growth in wild-type cells. The localization of Cla4–GFP was not affected in bem1-m cells (data not shown), supporting the observation that the Bem1-m mutant proteins efficiently bind Cla4. Based on the localization and degradation of the Cdc42 effector Gic2 (Figure 4B), bem1Δ cells fail to keep activated Cdc42 at the incipient bud site (Figure 4B; Jaquenoud et al., 1998). We conclude from these results that binding of Cdc24 to Bem1 is not required for initial membrane recruitment but to maintain active Cdc24 at the site of polarized bud growth. Figure 4.Binding of Cdc24 to Bem1 is required to maintain Cdc24 at bud tips. (A) The localization of Cdc24–GFP expressed from the ADH promoter was examined in either wild-type (YNP63; left panel) or bem1-m1 mutant cells (YNP64; right panel) after synchronization by nutritional starvation. The experiment was quantified (graphs) by counting at least 200 cells every 30 min by fluorescence and phase-contrast microscopy. Note that Cdc24–GFP is recruited to the incipient bud site in bem1-m1 cells, but it is not found at tips of small buds (arrow). (B) The localization of the Cdc42 effector Gic2 (left panel) was examined by GFP microscopy after G0 release at 37°C in the presence of Lat-A in wild-type (YMP288) or bem1Δ (YMP1046) cells. Gic2 and Cdc24 were also analyzed by immunoblotting (right panel). Note that Bem1 is required to concentrate activated Cdc42 at the site of polarization. (C) The subcellular localization of Bem1–GFP (YACB302; upper row) and Bem1-m1–GFP (YACB303; lower row) was analyzed by GFP microscopy. Download figure Download PowerPoint To examine the subcellular localization of Bem1 wild type and Bem1-m, we expressed functional GFP fusions from the endogenous BEM1 promoter in bem1Δ cells. Like wild-type Bem1–GFP (Ayscough et al., 1997; Gulli et al., 2000), Bem1-m1–GFP was distributed throughout the cytoplasm in early G1 cells and localized to the incipient bud site later in G1 (Figure 4C). Bem1-m1–GFP was found as a crescent at the tip of small and medium budded cells, and remained all over the cortex in large budded cells, although the staining became progressively weaker. Finally, Bem1–GFP and Bem1-m1–GFP localized to the mother bud neck during mitosis. Therefore, the ability of Bem1 to interact with Cdc24 is not required for its subcellular localization, and the failure of Cdc24–GFP to localize to bud tips in bem1-m cells is not caused by a localization defect of Bem1-m1. bem1-m cells have a growth defect but exhibit a correct haploid budding pattern To examine the physiological consequences of cells harboring a Bem1 mutant protein unable to interact with Cdc24, we constructed bem1Δ cells expressing wild-type Bem1, Bem1-m1 or Bem1-m2 from the endogenous promoter. Immunoblotting confirmed that all Bem1 proteins were expressed at similar levels (data not shown). We first determined whether bem1-m cells exhibit a bud site selection defect when grown at 25°C. As shown in Figure 5A, staining of bud scars by calcofluor white revealed that wild-type as well as bem1-m mutant cells in the EG123 background position their buds in the characteristic axial pattern (Chant, 1999), demonstrating that bem1-m cells are not defective for bud site selection. Figure 5.Budding pattern and growth properties of bem1-m mutant cells. (A) The budding pattern was quantified in wild-type (YACB302), bem1-m1 (YACB303) and bem1-m2 (YACB304) cells in the EG123 background by staining the bud scars with calcofluor white. (B) Growth of wild-type, bem1Δ and bem1-m mutant cells in either the EG123 (upper plates) or W303 (lower plates) background was compared after 2 days on rich medium (YPD) at 25°C (left plates) or 37°C (right plates). (C) Wild-type (YMG681), bem1Δ (YMP1046) and bem1-m1 (YMG682) mutant cells (W303 background) were shifted to 37°C for 3 h, and the actin cytoskeleton was examined by fluorescence microscopy after staining with rhodamine–phalloidin. Numbers indicate the percentage (%) of cells with an unpolarized actin cytoskeleton; 300 cells were included in the analysis. Download figure Download PowerPoint Like bem1Δ cells (Chenevert et al., 1992), the growth of bem1-m1 cells was impaired at 37°C, and many cells accumulated with an unpolarized actin cytoskeleton (Figure 5B and C). However, we observed a striking difference of this growth defect depending on the genetic background. For example, while bem1-m1 in the W303 background was barely viable at 37°C (lower plates), the same mutation in the EG123 background only slightly impaired colony formation (upper plates). The explanation for this difference is not clear at present. We conclude that binding of Cdc24 to Bem1 is not required to interpret the correct positional information during budding, but may be necessary to establish or maintain actin polarization at elevated temperatures. bem1-m cells are defective for apical bud growth To examine whether bem1-m cells are defective for polarized growth, we first compared the morphology of wild-type and bem1-m cells overexpressing the G1 cyclin Cln2, which is known to trigger an extended phase of apical growth (Lew and Reed, 1993). Strikingly, overexpression of Cln2 in bem1-m cells did not cause elongated buds (Figure 6A), although the level of Cln2 was comparable with wild-type strains (right panel). Likewise, cells defective for the E2 ubiquitin-conjugating enzyme Cdc34 exhibited hyperpolarized bud growth, and Cdc24–GFP, Bem1–GFP and Cla4–GFP remain at their tips (Figure 6B; Gulli et al., 2000). This phenotype was not reversed by deletion of SWE1, suggesting that it is not caused by activation of the morphogenesis checkpoint (Lew and Reed, 1995). Importantly, cdc34-2 bem1-m double-mutant cells displayed normal bud morphology (Figure 6B), and Cdc24–GFP was distributed through out the cytoplasm. In contrast, both Bem1–GFP and Bem1-m1–GFP as well as Cla4–GFP remained at bud tips in the majority of cdc34-2 and cdc34-2 bem1-m mutant cells (Figure 6B), demonstrating that binding of Cdc24 to Bem1 is required to keep Cdc24 at bud tips. Expression of wild-type Bem1 in cdc34-2 bem1-m1 mutant cells restored polarized growth (data not shown), demonstrating that the Bem1-m1 mutant protein is recessive. These data strongly argue that bem1-m cells are defective for apical bud growth because they are unable to maintain Cdc24 at the site of polarization. Figure 6.bem1-m cells are defective for apical bud growth. (A) Hyperpolarized growth was induced in wild-type (YACB341) or bem1-m1 (YACB342) cells by overexpression of Cln2 from the GAL promoter for 3 h. The localization of Bem1–GFP or Bem1-m1–GFP was visualized by GFP fluorescence (right images). The numbers represent the percentage (%) of hyperpolarized cells (phase) or Bem1–GFP localized at bud tips (GFP). Expression of Cln2 (arrow) was controlled by immunoblotting with Cln2 antibodies (right panel). (B) The localization of Bem1–GFP, Bem1-m1–GFP, Cdc24–GFP or Cla4–GFP was analyzed by GFP microscopy (upper rows) in cdc34-2 or cdc34-2 bem1-m1 cells after 3 h at 37°C. The phase-contrast images are shown below to visualize the bud morphology. The experiment was quantified as described in (A). Download figure Download PowerPoint bem1-m cells exhibit a bilateral mating defect, but are able to orientate correctly in a morphogenetic gradient Because Bem1-m failed to interact with Far1, we tested whether bem1-m cells are defective for orientating cell growth towards their mating partner. Indeed, bem1-m cells exhibited a bilateral mating defect (Figure 7A), and both bem1-m1 and bem1-m2 cells showed a reduced ability to mate with orientation-defective far1-c cells (data not shown). bem1-m cells were able to efficiently arrest the cell cycle in response to pheromones (Figure 7B), implying that the signal transduction and cell cycle arrest machinery are intact. To directly examine polarization in a morphogenetic gradient, we compared the behavior of wild-type and bem1-m1 cells in a pheromone gradient using time-lapse microscopy (Segall, 1993). Clearly, >60% of the bem1-m1 cells were able to polarize towards the position of the highest α-factor concentration compared with 75% of wild-type controls (data not shown), demonstrating that binding of Bem1 to Far1 is not essential for orientating cell polarity in a mo
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