Opy2 regulates the filamentous growth pathway. (A) The MAPK pathway that regulates filamentous growth and biofilm/mat formation. It is not clear how glucose limitation leads to pathway activation. (B) The plate-washing assay. Wild-type, msb2Δ, opy2Δ, ste50Δ, and ste12Δ strains were spotted onto YEPD medium and incubated for 2 days at 30°. The plate was photographed (left panel, YEPD), washed under a stream of water, and photographed again to reveal invaded cells (right panel, Washed). (C) Microscope-based assays and colony morphology assays to evaluate the role of Opy2 in the filamentous growth pathway. (Far left column) The single-cell invasive growth assay. Wild-type, opy2Δ, and ste12Δ cells were spread at low concentrations onto synthetic complete medium lacking glucose (SC-GLU). Plates were incubated for 24 hr and examined by DIC microscopy at ×100 magnification. More than 100 microcolonies were examined, and representative images are shown. Arrows point to axial (nondistal) budding patterns commonly seen in the opy2Δ and ste12Δ mutants. Bar, 5 µm. (Second column from left) Biofilm/mat assay. Strains were grown for 16 hr in YEPD and spotted onto YEPD media with 0.3% agar (YEPD + 0.3% agar). Plates were incubated at 30° for 4 days and photographed. The opy2 and ste12 mats were smaller and less ruffled. Bar, 1 cm. (Third column from left) Strains were spotted onto YEPD + 4% agar atop a nitrocellulose filter and incubated at 30° for 7 days and photographed. Bar, 1 cm. (Far right column) Cells were spotted onto YEP + 0.3% agar for 21 days. Microscopic images (×20 magnification) of mat perimeters are shown. Dense mats containing pseudohyphae at borders can be seen for wild type but not for the opy2Δ and ste12Δ mutant. Bar, 200 µm. (D) Phosphorylation of the MAP kinase Kss1 in wild-type cells and the indicated mutants. Immunoblots are shown to detect phosphorylated Kss1 (P∼Kss1, 42 kDa) and Fus3 (P∼Fus3, 40.7 kDa) with p44/42 antibodies. Blots using antibodies to Pgk1 (44.7 kDa) are shown as a loading control for protein levels. Blots using antibodies to the Kss1 protein (Kss1, 42 kDa) are shown. The asterisk refers to a nonspecific band. (E) β-Galactosidase assays to measure expression of the pFRE-LacZ and pYLR042c-LacZ reporters in wild-type and opy2Δ strains. Enzymatic activity is expressed in Miller units. Two independent experiments were performed, with error bars showing differences between trials. (F) Immunoblots showing the level of the Flo11-HA protein (>220 kDa) in culture supernatant (S) and pellet (P) fractions. The level of Flo11 in supernatants probably reflects the levels seen in pellets.

Opy2 is required for plasma-membrane recruitment of Ste50. (A) Genetic suppression analysis showing phosphorylated Kss1 in an opy2Δ mutant with a control plasmid (CTL) or plasmids containing hyperactive alleles of MSB2, SHO1, and STE11. For rga1Δ, an rga1Δ opy2Δ strain was examined. Blots were performed as in . (B) Levels of phosphorylated Kss1 in wild-type and opy2Δ mutant strains that expressed Myr-Ste50 under the control of its own promoter or the GAL1 promoter (P_GAL-Myr-Ste50). (C) Strains in B were spotted onto YEPGAL media for 48 hr and examined by the plate-washing assay to measure agar invasion. (D) Phosphorylation of Kss1 in cells overexpressing a wild-type (GAL-OPY2) or membrane-anchored version of the cytosolic domain of Opy2 (P_GAL-Myr-Opy2^CT). (E) Phosphorylation of Kss1 in an opy2Δ mutant containing a control plasmid (Ctl) and wild-type (WT) Opy2, Opy2^C30Y, or Opy2^G207D. (F) Two-hybrid analysis of the interaction between pGADC1-STE50 (Ste50), pGBDUC1-OPY2 (Opy2), or pGBDUC1-OPY2^G207D (G207D). Interactions are represented by growth on synthetic medium lacking adenine (SD-Ura-Leu-Ade, bottom panel). Growth of strains on control media is also shown (SD-Ura-Leu, top panel).

Opy2 interacts with the transcriptional repressor Mig1. (A) The Opy2 ORF and deletions used in the study. Yellow vertical lines, conserved cysteines; red bars, Ste50-binding sites; green bars, Mig1-binding sites. (B) Opy2 associates with Ste50, Mtc1, Mig1, and Mig2 by two-hybrid analysis. (Left) Growth of strains on control media (SD-URA-LEU). (Right) Growth of strains represents protein interactions (SD-URA-LEU-HIS+3-ATA). (C) Two-hybrid deletion mapping. Two-hybrid analysis of versions of Opy2 lacking specified deletions in its cytoplasmic tail with Mig1. (D) Pull-down assay. GST (∼26 kDa) or GST-Mig1 (∼81.5 kDa) were purified (Input, anti-GST) and bound to glutathione-sepharose beads (Pull Down, anti-GST). Beads were incubated with purified MBP-Opy2^CT (Input, anti-MBP, ∼69.4 kDa). Coprecipitated MBP-Opy2^CT is shown (Pull Down, anti-MBP).

The Mig1 and Mig2 proteins regulate starvation-dependent induction of the filamentous growth pathway. (A) Phospho-Kss1 blots of wild type, opy2Δ, mig1Δ, mig2Δ, and mig1Δ mig2Δ strains grown to mid-log phase in YEPD or YEPGAL media. (B) Strains in A were grown on YEPD media for 48 hr and assessed by the plate-washing assay. (C) Two-hybrid analysis of Mig1 and Mig2 with Msb2^CT.

Snf1 regulates the filamentous growth pathway independently of the HOG pathway. (A) Phospho-Kss1 levels for wild type, opy2Δ, and snf1Δ strains grown in YEPD (GLU) and YEPGAL (GAL) media. (B) Wild-type cells were grown in YEPD (GLU) or YEPGAL (GAL) for 6 hr. After 6 hr in galactose, cells harvested by centrifugation and transferred to YEPD media (GLU). Equal numbers of cells were withdrawn at 5, 15, and 30 min, and cell extracts were prepared and probed by immunoblot analysis. (C) Hog1 phosphorylation analysis. Phospho-blot of the MAP kinase Hog1 in response to 0.5 M KCl for 5 min was performed in the indicated strains in YPD media.

Different roles for Opy2 and Mig1/Mig2 in regulating the mating and filamentous growth pathways. (A) Halo assay. Wild type, P_GAL-OPY2, and P_GAL-MIG1 were spread on YEPGAL media, onto which was spotted 2, 10, and 20 µl of 1 mg/ml α-factor. Plates were incubated for 48 hr and photographed. Bar, 1 cm. Inset portraying the loss in pheromone sensitivity is also shown. (B) Overexpression of OPY2, MIG1, and STE50 suppresses expression of the FUS1-HIS3 reporter. Strains were spotted onto synthetic medium with galactose (SC-GAL) or SC-GAL lacking histidine (SC-GAL-HIS), grown for 24 hr, and photographed. In the bottom panels, Myr-Ste50 was expressed in wild-type cells or in an opy2Δ mutant, as indicated. (C) Overexpression of OPY2 and MIG1 induces hyper-filamentous growth. Cells were grown in YEPGAL and examined by DIC microscopy at ×100; representative images are shown. Bar, 5 µm. (D) Mig1 and Mig2 associate with Ste7 and Kss1 but not with Fus3 by two-hybrid analysis.

The filamentous growth pathway does not regulate the glucose repression function of the Mig proteins. Immunoblots with antibodies to HA-tagged Act1, expressed from a GAL promoter (P_GAL), P_GAL-ACT1 was obtained from . Wild-type, mig1Δ mig2Δ, opy2Δ, and ste12Δ strains were grown in YEP media supplemented with glucose and galactose (GLU + GAL) or galactose (GAL). Pgk1, loading control.