PMC

Fluorescence microscopic visualization of the lipid microdomains in cells during vegetative growth and α-factor treatment. The various lipid microdomains indicated in are represented. Arrows indicate location of membrane strands.

Ste4 overexpression does not rescue arv1 signaling defect. pGAL1-Ste4-GFP was expressed in MATa ste4∷ADE2 FUS1∷FUS1-lacZ∷LEU2 or MATa ste4∷ADE2 arv1∷KANr FUS1∷FUS1-lacZ∷LEU2 cells. FUS1-lacZ induction was measured 4 hr after galactose treatment (without α-factor) (mean ± SEM, n = 4, *P < 0.02, **P < 0.01).

Arv1 protein constructs. (A) A schematic of the various Arv1 protein constructs studied. Constructs are driven by the ARV1 promoter and are C-terminally tagged with 3HA in the vector pRS416. (B) Western analysis of cells expressing constructs described in A. Whole cell protein lysate was probed with anti-HA and anti-Pgk1.

Activated Ste11 mutants do not rescue signaling defect in arv1 cells. (A) Expression of Ste11-Cpr does not rescue signaling defect in arv1, AHD, or ΔAHD cells. pGAL1-Ste11-Cpr was expressed in MATa ste11∷ADE2 FUS1∷FUS1-lacZ∷LEU2 or MATa ste11∷ADE2 arv1∷KANr FUS1∷FUS1-lacZ∷LEU2 cells. FUS1-lacZ induction was measured 4 hr after galactose treatment (without α-factor) (mean ± SEM, n = 3, *P < 0.02, **P < 0.001). (B) Ste11ΔN expression does not rescue signaling defect in arv1 or ΔAHD cells. pGAL1-Ste11ΔN was expressed in strains described in . FUS1-lacZ induction was measured 4 hr after galactose treatment (without α-factor) (mean ± SEM, n = 4, *P < 0.01, **P < 0.001).

arv1 cells harbor bilateral mating defects. Cells to be tested for mating efficiency were patched onto YEPD plates and grown for 1 day (WT and arv1). A total of 10⁶ cells of the opposite mating partner that were grown to exponential phase in liquid YEPD media were spread onto YEPD plates and allowed to dry (MATα WT; MATa WT; MATα arv1; MATa arv1). Mating was performed at 30° for 3 hr. Diploid progeny were selected by replica plating onto the appropriate media and allowed to grow for 1 day at 30°.

arv1 cells harbor defects in shmoo formation. A total of 2 × 10⁷ cells/ml were incubated with 20 μg/ml of α-factor and cell aliquots were collected at 0, 1.5, 3, 4.5, and 7.5 hr. arv1 bar1 cells were transformed with the indicated Arv1 constructs which were C-terminally tagged with 3HA and on a pRS416 plasmid. Cells were collected at indicated time points and the numbers of shmoos/300 cells were counted for each sample. Arv1 full-length (solid circles), AHD (solid squares), ΔAHD (solid diamonds), empty vector (solid triangles) (mean ± SEM, n = 3, *P < 0.01, **P < 0.001).

Ste5 recruitment to plasma membrane is altered in arv1 cells. (A) Pheromone signaling is rescued by overexpression of Ste5^Q59L. pGAL1-Ste5^Q59L-GFP was expressed in strains described in . FUS1-lacZ induction was measured 4 hr after galactose treatment (without α-factor) (mean ± SEM, n = 3). (B) Expression of Ste5ΔN-CTM does not rescue signaling defect in arv1 or ΔAHD cells. pGAL1-Ste5ΔN-CTM-GFP was expressed in strains described in . FUS1-lacZ induction was measured 4 hr after galactose treatment (without α-factor) (mean ± SEM, n = 4, *P < 0.01). (C) Ste5-GFP was expressed in cells. Cells with polarized Ste5-GFP were counted after 1 hr of α-factor treatment (mean ± SEM, n = 4, *P < 0.001). (D) Cells with pGAL1-GST-GFP-PH^PLCδ were treated with galactose for 3 hr. α-Factor was added during the last 1 hr of galactose treatment. Cells with polarized PI(4,5)P₂ were counted (mean ± SEM, n = 3, *P < 0.005, **P < 0.001).

arv1 cells harbor defects in pheromone response pathway signaling. (A) Pheromone signaling is defective in arv1 cells treated with α-factor. MATa FUS1∷FUS1-lacZ∷LEU2 and arv1 FUS1∷FUS1-lacZ∷LEU2 cells were transformed with plasmids containing full-length Arv1, AHD, and ΔAHD; the constructs are under the control of the ARV1 promoter and are C-terminally tagged with 3HA in vector pRS416. FUS1-lacZ induction was measured after 2 hr of α-factor treatment (mean ± SEM, n = 3, *P < 0.02, **P < 0.006). (B) Ste12 overexpression rescues signaling defect in arv1 cells. pGAL1-STE12 was expressed in the strains described in A. FUS1-lacZ induction was measured 4 hr after galactose treatment (without α-factor) (mean ± SEM, n = 3).

arv1 cells harbor defects in pheromone-induced G1 cell cycle arrest. A total of 2 × 10⁷ cells/ml of cells were treated with 20 μg/ml of α-factor for the indicated times. MATa bar1 and arv1bar1 cells were transformed with plasmids containing full-length Arv1, AHD, and ΔAHD, as indicated; the constructs are under the control of the ARV1 promoter and are C-terminally tagged with 3HA in vector pRS416. Data are shown as the quantification of FACS profiles measuring propidium iodide-stained DNA. G1 phase (light shading), S phase (solid), G2 phase, (dark shading).