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First published online 16 June 2005
doi: 10.1242/jcs.02418

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Formin-induced actin cables are required for polarized recruitment of the Ste5 scaffold and high level activation of MAPK Fus3

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, MA 02115, USA

View larger version (43K): [in a new window]   Fig. 1. bni1 mutant cells are defective in mating pathway responses. (A) Schematic diagram of yeast mating MAPK pathway. Binding of mating pheromone ( factor) to its seven-transmembrane receptor (Ste2) induces dissociation of a heterotrimeric G protein, creating free Gß (Ste4/Ste18) dimer. Gß associates with Ste5 scaffold allowing activation of associated Ste11 (MAPKKK) by Ste20 (PAK), which is anchored at the plasma membrane through Cdc42 GTPase. Ste20 may also associate with and be stimulated by Gß. Ste20 phosphorylates Ste11, allowing sequential phosphorylation of Ste7 (MAPKK) and Fus3/Kss1 (MAPK). The MAPKs (mainly Fus3) activate Ste12 (transcription factor) and Far1 (cyclin-dependent kinase inhibitor) to trigger growth arrest and shmoo formation. (B) Morphology of bni1 cells before and after factor treatment. WT (EY699) and bni1 (EYL427) cells were grown to A600 0.6 at 30°C then treated with 5 µM factor for 2 hours. The cells were fixed with 4% formaldehyde, briefly sonicated and then observed under the microscope. (C) bni1 cells do not undergo G1 arrest when exposed to factor. Shown here is a halo assay of WT and bni1 SST1 strains. Photo was taken after 36 hours. (D) Quantification of bni1 cell defects in factor-induced G1 arrest, shmoo formation and mating. For G1 arrest and shmoo formation, cells were treated with factor as in B. Over 300 cells were counted. For quantitative mating, WT and bni1 cells were mated to a MAT strain for 6 hours on YPD plates. Diploid formation was quantitated as the percentage of prototrophs formed between equal number of MATa and MAT cells. (E) factor-activated transcription activation is suppressed in bni1 cells. EY699 and EYL427 strains harboring FUS1-lacZ (EBL95) were treated with factor for 1.5 hours. ß-Galactosidase assays were performed as described. Values (Miller units of ß-galactosidase activity) are means ± s.d. of triplicate transformants. Error bars are too small to be seen for the bni1 values.

View larger version (68K): [in a new window]   Fig. 2. bni1 cells are defective in factor-induced Fus3 activation. (A) Dose dependence. WT (EY957) and bni1 (EYL917) cells harboring FUS3-HA (PYEE1102) were treated with different concentrations of factor for 15 minutes and frozen at –80°C. (B) Time course. Cells were treated with 25 nM factor for different lengths of time as indicated. Fus3 activation was monitored by in vitro kinase assay. Casein was used as a substrate. Upper bands are endogenous substrates that co-precipitate with Fus3-HA. (C) Effect of bni1 deletion on Fus3 and Kss1 activation. bni1 strain (EYL917) was transformed with CEN plasmids harboring WT BNI1 or bni1 domain deletion mutants. The deletions are the RBD (Rho), both FH1 and FH2 domains (FH), the FH1 domain (FH1) and the FH2 domain (FH2). Empty vector was also transformed as control. Cells were stimulated with 25 nM factor for 15 minutes.

View larger version (55K): [in a new window]   Fig. 3. bni1 functions at or upstream of Ste5 in Fus3 activation. (A) Ste11-4-induced Fus3 activation of WT and bni1 cells. STE11-4 was expressed in a CEN plasmid (EBL284) and empty vector was used as control. (B) Ste5-CTM-induced Fus3 activation. Ste5-CTM (EBL204) was expressed from the GAL1 promoter. After shifting from raffinose to galactose medium, cells were collected at 0, 1, 2, 3 and 5 hours.

View larger version (48K): [in a new window]   Fig. 4. bni1 is required for Ste5 recruitment to the cell cortex. (A) Time course of Ste5-GFP localization in WT and bni1 cells. Cells (EY699 and EYL427) expressing Ste5-GFP (EBL367) from the CUP1 promoter were induced with 500 µM CuSO4 for 2 hours, then treated with 50 nM factor. An aliquot of cells was fixed at 15, 45 and 120 minutes in 4% formaldehyde. Cells were briefly sonicated and then observed under the microscope. Nomarski images (Nom) show cell morphology. (B) Ste5-Myc9 localization in WT (filled circles) and bni1 (open circles) cells. EY957 and EYL917 expressing Ste5-Myc9 (EBL453) were treated with 50 nM factor and fixed in 4% formaldehyde and indirect immunofluorescence was conducted to detect Myc. (C) Quantitation of Ste5-GFP localization in WT and bni1 cells. Cells were treated as in A. Values represent averages of two independent experiments. Up to 200 cells were counted for each condition. (D) Quantitation of Ste5-Myc9 localization in WT and bni1 cells. Values represent averages of two independent experiments. Up to 300 cells were counted for each condition. (E) Ste5-CTM cannot induce Ste5-Myc9 translocation in bni1 cells. WT (EY957) and bni1 (EYL917) cells were transformed with Ste5-CTM (EBL204) and Ste5-Myc9 (EBL365). Cells were pregrown in 2% raffinose overnight, shifted to 2% galactose to induce Ste5-CTM expression. Aliquots of cells were fixed at 2, 3 and 6 hours and Myc was detected by indirect immunofluorescence. (F) Quantitation of Ste5-Myc9 cortical localization in Ste5-CTM-induced cells. Ste5-Myc9 rim staining in WT (filled circles) and bni1 (open triangles) were counted in over 300 cells. (G) Quantitation of Ste5-Myc9 nuclear localization in Ste5-CTM-induced cells. Ste5-Myc9 nuclear accumulation in WT (filled circles) and bni1 (filled triangles) were counted in over 300 cells.

View larger version (56K): [in a new window]   Fig. 5. bni1 is required for Fus3, but not Ste20 recruitment. (A) Recruitment of Fus3 and Ste20 in bni1 strain. Upper panels: actin staining. WT (EY957) and bni1 (EYL917) cells were treated with 50 nM factor for 2 hours, fixed, and then stained with Rhodamine-phalloidin. Middle panels: Fus3-GFP. Lower panels: Ste20-GFP. Strains expressing genomic Fus3-GFP (EYL1096 and QMY27) or strains (EY957 and EYL917) transformed with GFP-Ste20 (EBL511) were treated with 50 nM factor for 2 hours, fixed with 4% formaldehyde, sonicated and then mounted for microscopy. (B) Quantitation of Fus3-GFP and Ste20-GFP localization in WT and bni1 cells. Values represent mean ± s.d. of two transformants. (C,D) Bni1 is recruited for Cdc24 recruitment during mating. WT (EY957) and bni1 (EYL917) strains were transformed with CDC24-GFP (EBL664). Cells were pregrown overnight in medium containing 0.19 mM methionine, then treated with 50 nM factor for 2 hours. Note that Cdc24 localizes at bud tips (arrowheads) and shmoo tips (arrows). Over 200 cells were counted. Values in D represent mean ± s.d. of two transformants.

View larger version (51K): [in a new window]   Fig. 6. Rho1 is involved in Bni1-mediated Ste5 recruitment. (A) Schematic structure of Bni1. RBD, Rho-binding domain; SBD, Spa2-binding domain; BBD, Bud6-binding domain. (B) Rho-binding domain is required for Bni1 polarization. WT cells were transformed with bni1RBD-GFP (QMB79) or BNI1-HA (EBL332); bni1 cells were transformed with BNI1-GFP (EBL334) or bni1RBD-GFP (QMB79). Cells were treated with 50 nM factor for 2 hours, then direct fluorescence of GFP was observed. Nomarski images (Nom) show cell morphology. (C) Bni1 polarization is defective in rho1-TS mutants. WT (QMY550), rho1-2 (QMY553) and rho1-104 (QMY551) cells expressing Bni1-GFP were grown at room temperature. Cells were shifted to 37°C for 1 hour, then induced with 5 µM factor at 37°C or 2 hours. (D) Ste5 recruitment is defective in rho1-TS mutants. WT, rho1-2 and rho1-104 cells expressing TAgNLSK128TSte5-Myc9 (EBL444) were treated as in C, except the factor stimulation time was 30 minutes. Cells were fixed and Myc was detected by indirect immunofluorescence microscopy. Values represent mean ± s.d. of two transformants.

View larger version (44K): [in a new window]   Fig. 7. bni1 regulates Ste5 relocalization by controlling actin cable formation. (A) The bni1TS mutant cannot recruit Ste5. WT (EYL1765) and bni1TS bnr1 (EYL1748) cells expressing TAgNLSK128TSte5-Myc9 were grown at room temperature. Cells were centrifuged, resuspended in pre-warmed medium (34°C) containing 5 µM factor, and then incubated at 34°C for 10 minutes. (B) The tpm1TS mutant cannot recruit Ste5. TAgNLSK128TSte5-Myc9 was expressed in tmp2 (EYL1736) and tpm1TS/tmp2 (EYL1735) mutant cells. Cells were then treated as in A, except at a nonpermissive temperature of 34.5°C. Values in A and B represent mean of two independent experiments. (C) Ste5 recruitment is inhibited in myo2-66 cells at nonpermissive temperature. MYO2 (QMY458) or myo2-66 (QMY459) cells expressing TAgNLSK128TSte5-Myc9 (EBL444) were grown at room temperature, then incubated at 37°C for 2 hours, treated with 5 µM factor for 30 minutes, then fixed for indirect immunofluorescence. (D) Overexpression of Myo2 tail domain suppresses Ste5 recruitment. Cells harboring MYO2DN (QMB75) and TAgNLSK128TSte5-Myc9 (EBL444) were pre-grown in medium containing 2% raffinose overnight. The cells were shifted to medium containing 2% galactose for 1 hour, treated with 5 µM factor for 15 minutes, then fixed and Myc was detected by indirect immunofluorescence microscopy. (E) Ste5 co-immunoprecipitates with Myo2 in factor-treated cells. EY1775 (ste5) expressing Ste5-Myc9 (from EBL358) and/or Myo2-HA (from QMB80), with or without control vectors, were grown to early logarithmic phase. Where indicated (+), cells were treated with 50 nM factor for 45 minutes prior to collecting cells for extract preparation. 12CA5 and 9E10 monoclonal antibodies were used to detect Myo2-HA and Ste5-Myc9, respectively. Note the nonspecific binding of Ste5 to Sepharose beads.

View larger version (36K): [in a new window]   Fig. 8. Actin is not required to keep Ste5 at the plasma membrane. (A) Dynamics of Ste5 localization in bni1TS mutants. WT (EYL1765) and bni1TSbnr1 cells (EYL1748) expressing TAgNLSK128TSte5-Myc9 were treated with 5 µM factor at room temperature for 30 minutes, and then shifted to 34°C for 10 minutes and 20 minutes. Values are means of two independent transformants. Two separate experiments gave comparable results. (B) Actin filaments are required to recruit Ste5. WT (EY957) cells harboring TAgNLSK128TSte5-Myc9 were treated with 100 µM latrunculin A (Lat A) for 15 minutes, then treated with 50 nM factor for 15 minutes. (C) Ste5 dynamics in Lat A-treated cell. Cells as used in B were treated with 50 nM factor for 30 minutes, then Lat A was added to the medium at a final concentration of 100 µM. The cells were further incubated for 15 minutes before fixed for immunofluorescence. Values are mean ± s.d. from three independent experiments.

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