Membrane recruitment and shmoo tip localization of Ste5 do not require actin filament function or PtdIns 3-kinase Vps34 activity. (A) Exponentially growing cultures of ste5Δ cells harboring a low-copy (CEN) vector expressing from the STE5 promoter Ste5-(GFP)₃ (Left) were either not treated (−) or treated (+) with 100 μM latrunculin A (Lat A) for 15 min and then treated with 20 μM α-factor for 30 min (Right, Upper) or treated with 20 μM α-factor for 30 min and then either not treated (−) or treated (+) with 100 μM Lat A for 30 min (Right, Lower). Cells were then examined by standard epifluorescence microscopy; each panel depicts a collage of representative cells. (B) Upper, PtdIns 3-kinase Vps34 is not required to generate PtdIns(4,5)P₂ anisotropy. Expression of the GST-GFP-PH^PLCδ1 probe was induced and shut off in a vps34Δ mutant as described in Fig. 1A, and the cells then were either not treated (−) or treated (+) with 5 μM α-factor for 60 min and examined by standard epifluorescence microscopy. Lower, PtdIns 3-kinase Vps34 is not required for plasma membrane recruitment of Ste5. Exponentially growing cultures of a ste5Δ vps34Δ double mutant harboring a low-copy (CEN) vector expressing from the STE5 promoter Ste5-(GFP)₃ were either not treated (−) or treated (+) with 20 μM α-factor for 90 min and examined by epifluorescence microscopy. Each panel depicts a collage of representative cells.

Continuous PtdIns(4,5)P₂ synthesis is required for stable accumulation of Ste5 at the shmoo tip during pheromone response. (A–E) Exponentially growing cultures of ste5Δ cells (A), ste5Δ mss4^ts cells (B), ste5Δ stt4^ts cells (C), ste5Δ pik1^ts cells (D), or ste5Δ stt4^ts pik1^ts cells (E), each harboring a low-copy (CEN) vector expressing from the STE5 promoter Ste5-(GFP)₃, were treated with 20 μM α-factor for 30 min, at which point half of each culture was left at the permissive temperature (26 °C) and half was shifted to the restrictive temperature (37 °C). After 90 min, cells in each culture were examined by standard epifluorescence microscopy. Each panel depicts a collage of representative cells at each time point: arrowheads, mating projections where Ste5-(GFP)₃ accumulated; arrows, mating projections lacking Ste5-(GFP)₃.

Continuous synthesis is required for maintenance of PtdIns(4,5)P₂ anisotropy in the plasma membrane during pheromone response. (A–E) Expression of the GST-GFP-PH^PLCδ1 probe was induced and shut off, as described in Fig. 1A, in either wild-type cells (A) or otherwise isogenic mss4^ts (B), stt4^ts (C), pik1^ts (D), or stt4^ts pik1^ts (E) mutants. After 15 min, each culture was treated with 5 μM α-factor for 30 min, at which point half of each culture was left at the permissive temperature (26 °C) and half was shifted to the restrictive temperature (37 °C). After 1 h, cells in each culture were examined by standard epifluorescence microscopy. Each panel depicts a collage of representative cells at each time point: arrowheads, mating projections with a marked asymmetric distribution of the PtdIns(4,5)P₂ probe; arrows, mating projections lacking a marked asymmetric distribution of the probe.

Plasma membrane PtdIns(4,5)P₂ is necessary for MAPK activation and signaling. (A) Exponentially growing cultures of wild-type cells (strain YPH499) and otherwise isogenic mss4^ts, stt4^ts, pik1^ts, stt4^ts pik1^ts, and vps34Δ mutants, as indicated, were each divided into two equal portions, which were incubated for 45 min at either permissive (26 °C) or restrictive (37 °C) temperature, as indicated. Cells then were treated with 5 μM α-factor for 30 min, harvested, and ruptured by alkaline lysis and TCA precipitation (Materials and Methods). Samples of the resulting extracts were resolved by SDS/PAGE and immunoblotted with anti-phospho-p44/p42 and anti-Fus3 antisera. A representative experiment is shown. (B) Plots show the extent of Fus3 phosphorylation, relative to the level of total Fus3 in the same samples, normalized to the amount of Fus3 phosphorylated in the wild-type control cells at 26 °C (set at 100%). Values represent the average of three independent experiments (error bars represent the SE of those means). (C) Continuous phosphoinositide production at the plasma membrane and in the nucleus is required for pheromone-induced reporter gene expression. Independent cultures (n = 6) of wild-type cells or of the otherwise isogenic mutant strains indicated, each carrying a copy of the FUS1_prom-lacZ reporter gene integrated into their genome (strains YLG70, YLG71, YLG72, YLG73, and YLG74), were grown at 26 °C, divided into two portions, one of which was maintained at 26 °C (shaded bars) and the other of which was shifted to 37 °C for 45 min (solid bars), and then treated with 5 μM α-factor for 60 min. The cells in each culture were then harvested and the amount of β-galactosidase produced by each was measured (value given represents the mean and error bars represent ±SD).

Fluorescent reporters reveal highly polarized PtdIns(4,5)P₂ distribution during pheromone response and do not perturb signaling. (A) Exponentially-growing wild-type cells (strain BY4741) carrying a 2-μm DNA vector expressing from the GAL1 promoter the PtdIns(4,5)P₂-binding GST-GFP-PH^PLCδ1 probe were induced by addition of galactose for 30 min and then harvested by brief centrifugation, washed, and resuspended in glucose-containing medium to shut off any further probe expression. After 15 min, the cells were treated with 5 μM α-factor, and samples were withdrawn every 15 min, as indicated, and examined by standard epifluorescence microscopy. Each panel depicts a collage of representative cells at each time point; arrows denote the tips of mating projections that exhibited marked asymmetric distribution of the probe. (B) Strain YCS234 carrying a 2-μm DNA vector (pRS426) expressing from the constitutive PRC1 promoter an alternative PtdIns(4,5)P₂-binding probe (GFP-2XPH^PLCδ1) was treated with 0.1 μM α-factor for 1 h, spread on an agar pad, and examined by deconvolution microscopy. False color image represents the indicated scale of measured pixel intensity. (C) Upper, strain BY4741 was transformed with an empty 2-μm DNA vector or the same plasmid expressing GST-GFP-PH^PLCδ1 from the GAL1 promoter (pPP1872), as indicated and tested for sensitivity to pheromone-induced growth arrest by halo bioassay on galactose-containing medium. Lower, strain BYB88 (ste4Δ ste5Δ) was cotransformed with a URA3-marked 2-μm DNA vector expressing from the GAL1 promoter the hyperactive Ste5(P44L) allele and a LEU2-marked CEN vector expressing GST-GFP-PH^PLCδ1 from the GAL1 promoter (pLG99) and tested for mating proficiency by patch mating assay on galactose-containing medium. (D) Strain YLG36 carrying a FUS1_prom-lacZ reporter gene was transformed with an empty 2-μm DNA vector or the same plasmid expressing GST-GFP-PH^PLCδ1, as indicated. Representative transformants (n = 3) of each type were grown to early exponential phase, induced with galactose for 2 h, and treated with 3 μM α-factor for 90 min, and expression of β-galactosidase was measured (mean ± SD).