Induction of the FUS1-lacZ reporter gene by glycosylation defects. The indicated yeast mutant cells carrying the pFUS1-lacZ plasmid were grown exponentially and were treated with (+tun) or without (−tun) tunicamycin (final conc. 25 μg/ml) for the indicated times. Activity of β-galactosidase in cell extracts was normalized using cell densities and expressed as Miller units (Miller, 1972). The strains used, and their full genotypes, are listed in Supplementary Tables S1 and S2, respectively.

Signalling elements required for induction of FUS1-lacZ expression by glycosylation defects. (A) Induction of the FUS1-lacZ reporter gene by glycosylation defects. The indicated yeast mutant cells carrying the pFUS1-lacZ plasmid were grown exponentially, treated with (+tun) or without (−tun) tunicamycin for 8 h, and assayed for β-galactosidase activity as in Figure 1. (B) The indicated mutants were assayed as in (A). (C) Exponentially growing cells of the indicated genotypes were treated with 25 μg/ml tunicamycin for the indicated times. Cell extracts were probed for phosphorylated (activated) forms of Fus3 (P-Fus3), Kss1 (P-Kss1) and Mpk1 (P-Mpk1) by immunoblotting using anti-phospho-p44/42 antibody.

Glycosylation of Opy2 by Pmt4, and its role in the FG response. (A) Schematic diagrams of the Opy2 constructs used in (B–D). FL, full-length; SR, Ser-rich; TM, transmembrane segment. (B) C-terminally GFP-tagged Opy2 constructs were generated using a single-copy vector with the GAL1 promoter (p414GAL1). Expression of Opy2-FL-GFP, or the indicated deletion constructs, was induced by 2% galactose for 4 h in PMT4⁺ or pmt4Δ host cells. Total cell extracts were probed with an anti-GFP antibody by immunoblotting. The horizontal arrow indicates cleavage products. The estimated cleavage site is shown in (A) by an upright arrow. (C) The full-length OPY2 gene (with the OPY2 promoter) carried on a single-copy vector (pRS414) or its deletion constructs were individually expressed in pmt4Δ ste4Δ opy2Δ host cells that also carried the pFUS1-lacZ reporter plasmid. Cells were treated with (+tun) or without (−tun) tunicamycin (final conc. 25 μg/ml) for 8 h, before extracts were prepared for β-galactosidase assays. (D) GST-tagged Ste50 (GST-Ste50), or GST alone, was constitutively expressed using pFP122 or the vector p426TEG1, and expression of GFP-tagged Opy2-FL (Opy2-FL-GFP), or Opy2ΔC-GFP, was induced by 2% galactose for 4 h using the expression constructs based on the p414GAL1 vector, in pmt4Δ ste50Δ opy2Δ host cells. GST-Ste50 (or GST) was immunoprecipitated by glutathione-Sepharose beads (bottom panel), and co-precipitated Opy2-GFP protein was detected by immunoblotting (top panel). Expression levels of the Opy2-GFP proteins are shown in the middle panel.

Pmt4 glycosylation of Msb2. (A) Schematic diagrams of the deletion constructs of Msb2 used in (B–F). Each construct contains two HA epitopes, one following amino acid position 48 and another at the C terminus. STR, Ser/Thr rich; HMH, Hkr1-Msb2 Homology domain; TM, transmembrane segment; FL, full-length. (B–E) Indicated Msb2-HA constructs, under the control of the GAL1 promoter in the single-copy vector p416GAL1, were induced by 2% galactose for 3 h in PMT⁺ or the indicated pmt mutant cells. Cell extracts were probed by immunoblotting with an anti-HA antibody. (F) Endo H treatment of Msb2. The Msb2-ΔSTR2-HA construct, under the control of the GAL1 promoter in the single-copy vector p416GAL1, was induced by 2% galactose for 3 h in PMT ⁺ cells and in the indicated pmt mutant cells. Cell extracts were prepared in Buffer N, immunoprecipitated with anti-HA antibody (3F10), treated with (+) or without (−) 0.4 U/ml endoglycosidase H (Endo H) at 37°C for 15 h, applied to SDS–PAGE, and probed with another anti-HA antibody (12CA5).

Domains of Msb2 essential for the FG response. (A) Schematic diagrams of the Msb2 deletion constructs used in (B) and (C). STR, Ser/Thr rich; HMH, Hkr1-Msb2 Homology domain; TM, transmembrane segment; FL, full-length. (B) Induction of the FG reporter gene FUS1-lacZ by glycosylation defects. The full-length MSB2 gene, or its deletion constructs, carried on the single-copy vector pRS416 were individually expressed in pmt4Δ ste4Δ msb2Δ host cells in which the FUS1-lacZ reporter gene has been integrated. Cells were treated with (+tun) or without (−tun) tunicamycin (final conc. 25 μg/ml) for 8 h, before extracts were prepared for β-galactosidase assays. In HMH(Hkr1), the HMH domain of Msb2 was replaced by the homologous HMH domain from Hkr1. (C) Expression of the indicated Msb2 or Opy2 construct, placed under the GAL1 promoter, was induced by galactose for 2 h. Induction of the FG reporter gene FUS1-lacZ was assayed.

Cross-inhibition of the Hog1 MAPK by the FG MAPK pathway. (A–C) Induction of the HOG reporter gene 8xCRE-lacZ by glycosylation defects. Yeast strains of the indicated genotypes, carrying the reporter plasmid, were stimulated with (+) or without (−) 25 μg/ml tunicamycin for 8 h, before cell extracts were prepared for β-galactosidase assay. [KSS1] and [FUS3] in (B) indicate that the strain contains a single-copy plasmid carrying the KSS1 or FUS3 gene with its own promoter. (D) Activation of the Hog1 MAPK by glycosylation defects. Exponentially growing cells of the indicated genotypes were treated with (+) or without (−) 25 μg/ml tunicamycin for 2 h. Cell extracts were probed for phosphorylated Hog1 (P-Hog1) and total Hog1 by immunoblotting. (E) Induction of the FUS1-lacZ reporter gene by glycosylation defects. The indicated yeast mutant cells carrying the pFUS1-lacZ plasmid were grown exponentially, treated with tunicamycin for 8 h (+tun) or untreated (−tun), and assayed for β-galactosidase.

Schematic diagrams of signal flow in the Kss1 and Hog1 MAPK pathways in three yeast mutants: (A) pmt4Δ, (B) pmt4Δ ptp2Δ, and (C) pmt4Δ ptp2Δ hog1Δ. Black arrows and T-shaped bars indicate, respectively, activation and inhibition. Gray arrows and bars represent lack of activation or inhibition. Crosses (X) signify disrupted genes. The precise molecular mechanisms by which Kss1 inhibits Hog1, or Hog1 inhibits Kss1, are currently unknown.