SteC inhibits signaling through the yeast pheromone response pathway. (A) Left, scheme illustrating pheromone stimulation of the mating pathway that results in Fus3 and Kss1 MAPK phosphorylation and Ste12-mediated FUS1 transcriptional activation. Right, wild-type YPH499 cells bearing pEG(KG) or pEG(KG)-SteC plasmids, expressing GST or GST-SteC respectively, were transformed with the plasmid pRS423-FUS1-lacZ, containing the pheromone-inducible FUS1 promoter and lacZ reporter gene. Cells were grown to mid–log phase in raffinose-based medium, with galactose added for a final 2% for 4 h, and then treated with the indicated concentration of α-factor for additional 90 min. β-Galactosidase activity was expressed as arbitrary fluorescence units. Data shown are the average of three independent experiments performed in triplicate. Error bars indicate standard deviations (SD). (B) Left, scheme illustrating how MATα itc1Δ yeast cells produce a-factor, leading to autocrine stimulation of the mating pathway. Middle, Western blotting analysis of cell extracts from the Y04500 MATα itc1Δ mutant strain transformed with the empty vector pEG-(KG) or pEG-(KG)-SteC, expressing GST or GST-SteC, respectively. Cells were grown as in A. Protein extracts were prepared, and the levels of phospho-Fus3 or phospho-Kss1, GST or GST fusions, and actin as loading control were detected by immunoblot analysis with anti–phospho-p44/42, anti-GST, and anti-actin antibodies, respectively. Right, DIC photographs showing the cell morphology of representative Y04500 MATα itc1Δ cells as in the middle. Bars, 5 μm. (C) Left, a schematic representation of the expressed fragments of SteC. The SteC region containing protein kinase subdomains is shown in gray, and the region showing similarity to RGS domains is indicated as white boxes. Right, percentage of cells displaying aberrant morphology from cultures of Y04500 MATα itc1Δ yeast cells expressing the indicated GST-tagged SteC fragments from pEG-(KG)–derived plasmids. Cells were observed 6 h after induction in galactose. Data are means of three experiments, and at least 100 cells were counted in each experiment. Error bars correspond to the SD. (D) Western blotting analysis of cell extracts from similar transformants as in C. Cells were cultured in absence (–) or presence (+) of 3 μM of α-factor, and protein extracts were prepared and analyzed as in B. Fus3 and Kss1 were detected with anti-Fus3 and anti-Kss1 antibodies, respectively. Reproducible results were obtained in different experiments, and selected images correspond to representative blots.

SteC binds Gpa1 but acts in the mating pathway independently of both the G_α subunit and the MAPK module. (A) Left, main components that operate in the yeast mating pathway. Right, immunoblot analysis of the copurification of Gpa1-6Myc with the indicated GST-tagged SteC fragments. Transformants of the YPH499 (GPA1-6Myc) strain with the plasmids pEG(KG) (GST), pEG-(KG)-SteC (SteC), pEG-(KG)-SteC^1-229 (1–229), pEG-(KG)-SteC^1-156 (1–156), or pEG-(KG)-SteC^157-457 (157–457) were grown to mid–log phase in raffinose-based selective medium and then galactose was added for a final 2% for an additional 6 h. Cell extracts (input) and GST complexes obtained by purification with glutathione-Sepharose (pull down) were analyzed by immunoblotting with anti-Myc and anti-GST antibodies. (B) Transcription activity in response to distinct α-factor concentrations was measured as indicated in Figure 1A in strains YPH499 (WT) and YDM400 (sst2Δ) bearing the plasmid pRS423-FUS1-lacZ and transformed with pEG(KG) or pEG(KG)-SteC plasmids. Data shown are the average of three independent experiments performed in triplicate. Error bars indicate standard deviations. (C) Western blotting analysis of cell extracts from BY4741 (WT) and BYgpa1 (gpa1Δ) strains transformed with the empty vector pEG-(KG) or pEG-(KG)-SteC expressing GST (–) or GST-SteC (SteC), respectively. Cells were cultured and cell extracts analyzed as in Figure 1B. (D) Western blotting analysis of cell extracts from YPH499 (WT) cells expressing GST (–) or GST-SteC (SteC) from pEG-(KG)–derived plasmids and transformed with empty vector pEG(KG)H (–) or plasmids pRS315-GAL-STE4, pEG(KG)H-cdc42^G12V, and pRS425-ste11-4 in order to overproduce Ste4 or the corresponding hyperactive versions of Cdc42 and Ste11-4. In the case of cells bearing pEG(KG)-ste20ΔN, which overproduce the corresponding hyperactive version of Ste20, SteC was expressed from plasmid YCpLG-SteC. Cells were cultured and extracts were analyzed as in Figure 1B. (E) Western blotting analysis of cells extracts from YPH499 (WT) cells expressing GST (–) or GST-SteC (SteC) from pEG-(KG)–derived plasmids and transformed with empty vector pRS316GAL or plasmid pGFP-G-STE5, pG-STE5-CTM, pT-G-STE11-Cpr, or pCUGF-STE5Q59L. Cells were cultured and extracts were processed and analyzed as in Figure 1B.

SteC inhibits signaling through the Cdc42-dependent branch of the HOG pathway. (A) Scheme illustrating the main components that operate in the HOG pathway, including the Sln1-mediated (1) and Sho1-mediated (2) branches of the pathway and the cross-talk that takes place in the absence of Hog1 (3), leading to the spurious phosphorylation of mating MAPKs after high salt stress. (B) Western blotting analysis of cell extracts from the Y02724 (hog1Δ) strain bearing the empty vector pEG-(KG) or pEG-(KG)-SteC, expressing GST or GST-SteC, respectively. Cells were grown to mid–log phase in raffinose-based selective medium, galactose was added for a final 2% for additional 6 h, and then cells were treated with 0.7 M NaCl for the indicated number of minutes. Protein extracts were prepared and analyzed as in Figure 1B. (C) Western blotting analysis of cell extracts from the TM141 (WT) and the TM257 (ssk2Δ ssk22Δ) strains bearing the empty vector pEG-(KG) or pEG-(KG)-SteC. Cells were cultured and extracts were processed and analyzed as in Figure 1B. The level of phospho-Hog1 was detected with anti–phospho-p38 antibodies. Reproducible results were obtained in different experiments, and selected images correspond to representative blots.

(A) Growth on SD (glucose) and SG (galactose) plates of serial dilutions of wild-type YPH499 yeast cells harboring the following plasmids, as indicated: YCpLG (–), YCpLG-SteC (SteC), pEG(KG) (GST), pEG(KG)-SteC (GST-SteC), pYES2 (–), pYES2-Cdc42-L (Cdc42-L), BG1805-Cdc42 (ScCdc42), and pYES2-HsCdc42 (HsCdc42), and therefore expressing under the control of the GAL1 promoter the corresponding proteins on galactose-based medium. (B) DIC and fluorescence microscopy photographs showing the cell morphology (Nomarski), SteC-GFP localization, and internal membrane localization (FM4-64) of YPH499 strain harboring the plasmid pGFP-CFUS-SteC expressing SteC-GFP. Cells were grown to mid–log phase in complete SD medium, washed, and then transferred to SD medium lacking methionine for 6 h and then treated with the vacuolar–membrane staining FM464. Bars, 5 μm. (C) Top, fluorescence microscopy photographs showing the in vivo localization of SteC^1-229 and SteC^1-157. YPH499 cells transformed with either pGFP-CFUS-SteC^1-229 or pGFP-CFUS-SteC^1-157 were grown to mid–log phase in complete SD medium and transferred to a SD medium lacking methionine for 6 h. Bottom, fluorescence microscopy photographs showing the in vivo localization of GFP-Cdc42 in cells expressing GST or GST-SteC, as indicated. YPH499 cells bearing pRS415-GFPCDC42 and pEG-KG or pEG-KG-SteC were grown to mid–log phase in raffinose-based selective medium lacking methionine, and then galactose was added for a final 2% for additional 6 h. Bars, 5 μm.

SteC interacts with the Cdc42 GEF Cdc24. (A) Copurification assays of HA-HsCdc42 with GST (–), GST-SteC (SteC), or GST-SopB^R468A. Transformants of the YPH499 strain with plasmid pYES3HA-Cdc42 and pEG(KG) (–), pEG(KG)-SteC (SteC), or pEG(KG)-SopB^R468A (SigD^R468A) were grown to mid–log phase in raffinose-based selective medium, and then galactose was added for a final 2% for an additional 6 h. Cell extracts (input) and GST complexes obtained by precipitation with glutathione-Sepharose (pull down) were analyzed by immunoblotting with anti-HA and anti-GST antibodies as indicated. (B) Copurification assays of Cdc24-TAP with, as indicated, either yeast- or E. coli–produced GST (–), GST-SteC (SteC), or GST-SteC^1-229 (SteC^1-229). For yeast expression, transformants of the YPH499 strain with plasmid BG1805-Cdc24 and pEG(KG) (–), pEG(KG)-SteC (SteC), or pEG(KG)- SteC^1-229 (SteC^1-229) were grown to mid–log phase in raffinose-based selective medium, and then galactose was added for a final 2% for an additional 6 h. Cell extracts (input) and GST complexes obtained by precipitation with glutathione-Sepharose (pull down) were analyzed by immunoblotting with anti-TAP and anti-GST antibodies as indicated. For in vitro copurification assays of recombinant SteC, E. coli extracts containing GST, GST-SteC, or GST-SteC^1-229 were incubated with yeast extracts containing Cdc24-TAP and glutathione-Sepharose to pull down GST complexes. Immunodetection was performed using anti-TAP (top) and anti-GST (bottom) antibodies. Reproducible results were obtained in different experiments, and selected images correspond to representative blots.

SteC binds S. pombe Scd1 and human Vav1 GEFs. (A) Copurification assays of HA-Scd1 with recombinant GST (–) or GST-SteC (SteC). E. coli extracts containing GST or GST-SteC were incubated with S. pombe extracts containing HA-Scd1 and glutathione-Sepharose to pull down GST complexes. Immunodetection was performed using anti-HA (top) and anti-GST (bottom) antibodies. (B) Copurification assays of Myc-Cdc24 and Myc-tagged Cdc24^287-473 (Cdc24(DH)) with GST (–) or GST-SteC (SteC). Transformants of the YPH499 strain with plasmid p414-3xmycCdc24Fl or p414-3xmycDH-3xmyc and pEG(KG) (–) or pEG-(KG)-SteC (SteC) were grown to mid–log phase in raffinose-based selective medium, and then galactose was added for a final 2% for an additional 6 h. Cell extracts (input) and GST complexes obtained by precipitation with glutathione-Sepharose (pull down) were analyzed by immunoblotting with anti-Myc and anti-GST antibodies as indicated. (C) Copurification assays of the DH domain of human Vav1 with recombinant GST (–), GST-SteC (SteC), GST-SteC^1-229 (SteC^1-229), or GST-Cdc42. E. coli extracts containing GST or GST-fused proteins were incubated with E. coli extracts containing His-tagged Vav1(DH) and glutathione-Sepharose to pull down GST complexes. Immunodetection was performed using anti-His (top) and anti-GST (bottom) antibodies. Reproducible results were obtained in different experiments, and selected images correspond to representative blots. (D) Purified SteC does not modulate the Vav1-catalyzed exchange of guanine nucleotides bound to Rac1. Top, purified Rac1 (400nM) was incubated with Bodipy FL GDP (80 nM) before addition (arrow) of Vav1 preincubated with indicated forms of SteC. Final concentrations of Vav1 and SteC were 5 and 50 nM, respectively. Bottom, purified Rac1 (400 nM) preloaded with Bodipy FL GDP was incubated with excess GDP (20 μM) before addition (arrow) of Vav1 preincubated with indicated forms of SteC. Final concentrations of Vav1 and SteC were 10 and 100 nM, respectively.

SteC modifies Cdc24 localization and does not affect Cdc24 phosphorylation and oligomerization. (A) Western blotting analysis of cell extracts from YPH499 cells harboring plasmids p414-3xmycCdc24Fl and pEG(KG) (–) or pEG(KG)-cla4ΔN (Cla4ΔN) and cotransformed with pEG(KG)H (–) or pEG(KG)H-SteC (SteC). Cells were grown to mid–log phase in raffinose-based selective medium, and then galactose was added for a final 2% for an additional 6 h. The 3xmycCdc24 was detected with 9E10 antibodies, and anti-G6PDH was used to detect G6PDH as a loading control. (B) Fluorescence microscopy photographs showing in vivo localization of Cdc24 and colocalization with 4′,6-diamidino-2-phenylindole staining. YKT725 cells expressing GFP-tagged Cdc24 and transformed with pEG(KG) (GST), pEG(KG)-SteC (GST-SteC), or pEG(KG)- SteC^1-229 (GST-SteC^1-229) were cultured as in A. Samples were taken at 6 h for microscope analysis. Bars, 5 μm. (C) Percentage of cells from the same cultures used in B, showing nuclear Cdc24. (D) Western blotting analysis of GFP-Cdc24 from the same cultures used in B, using anti-GFP and anti-GST antibodies. (E) RAY1773 cells expressing HA-tagged Cdc24 were cotransformed with p414-3xmycCdc24Fl plasmid and pEG(KG)H (GST), pEG(KG)H-SteC (GST-SteC), or pEG(KG)H-SteC^1-229 (GST-SteC^1-229) and cultured as in B. HA-tagged Cdc24 was immunoprecipitated from cell extracts, and bound Myc-Cdc24 was assayed by Western blotting. (F) Fluorescence microscopy photographs showing the in vivo localization of YKT725 cells cultured as in B but with samples taken at 2 h after galactose addition. Bars, 5 μm. (G) Percentage of cells showing nuclear (gray bars) or growth sites (bud tips or septa, white bars) localization of GFP-Cdc24 at different stages of the cell cycle. The indicated transformants were cultured as in F. n > 100 cells for each strain and for each cell cycle stage. (H) Western blotting analysis of YKT725 cell extracts of cells showed in G.