A positive feedback loop stabilizes the guanine-nucleotide exchange factor Cdc24 at sites of polarization

Abstract

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.

Fig. 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 (Cdc42^C188S 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.

Fig. 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.

Fig. 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).

Fig. 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.

Fig. 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.

Fig. 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).

Fig. 7. bem1-m cells exhibit a bilateral mating defect and a reduced ability to form polarized mating projections. (A) The ability of wild-type (YACB296), bem1Δ (YMP459), bem1-m1 (YACB298) and bem1-m2 (YACB300) cells to mate with each other was examined by patch mating. Note that bem1-m cells exhibit a bilateral mating defect. (B) Cell cycle arrest of wild-type (YACB296), bem1Δ (YMP459), bem1-m1 (YACB298) and bem1-m2 (YACB300) cells in response to pheromones was examined by halo assay. Fifteen micrograms of α-factor were spotted on each filter disc. (C) The ability of orientation-defective far1-c (YACB288), bem1Δ far1-c (YACB36), bem1-m1 far1-c (YACB290) and bem1-m2 far1-c (YACB292) cells to mate with wild-type cells (IH1793) was analyzed by patch mating. (D) The shmoo morphology of wild-type (YMG681), bem1-m1 (YMG682) and bem1Δ (YMP1046) cells (W303 background) was examined by phase-contrast microscopy after addition of 30 nM α-factor for 3 h at 30°C. The actin cytoskeleton was stained with rhodamine–phalloidin and analyzed by fluorescence microscopy. Numbers indicate the percentage of cells with an unpolarized actin cytoskeleton; 300 cells were included in the analysis. (E) The polarized localization of Bem1–GFP and Bem1-m1–GFP was analyzed by GFP fluorescence (left panels) and phase-contrast microscopy (right panels) in the indicated strains (EG123 background) treated with 30 nM α-factor for 3 h at 30°C.

Fig. 8. Recruitment of Bem1 to the incipient bud site requires active Cdc42. (A) cln1, 2, 3Δ pMETCLN2 (YMG258) cells expressing Bem1–GFP from the ADH promoter were arrested in G₁ by depletion of Cln2 in medium containing methionine (–Cln2). Cells were quickly washed and Cln2 was induced in medium lacking methionine but containing the actin polymerization inhibitor Lat-A (+Cln2 + Lat-A) to prevent bud emergence. Localization of Bem1–GFP was determined by fluorescence microscopy. (B) Temperature-sensitive cdc42-27 (left panel) and cdc24-5 (right panel) mutant cells expressing, as indicated, Bem1–GFP, Cdc24–GFP or Gic2–GFP from the ADH promoter were released from their block in G₀ either at 25°C or at the restrictive temperature 37°C. The localization of the proteins was examined by GFP fluorescence after the times indicated and quantified by counting at least 200 cells for each time point. (C) cln1, 2, 3Δ pMETCLN2 (YMG258) cells expressing Bem1–GFP from the ADH promoter were grown at 30°C in 2% raffinose medium and arrested by depletion of Cln2 as described in (A). Expression of Cdc42–G12V from the GAL promoter was induced by addition of 2% galactose for 3 h at 30°C, and the localization of Bem1–GFP was analyzed by fluorescence microscopy.