JCS02754 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A) Halo assay of a-factor pheromone production. 0.6% top agar was mixed with 10⁵ indicator cells (MATa sst1). Aliquots (approximately 4310⁶ cells) of wild-type W303-1B (MATa) and W303-1A (MATa) wild-type, and mdy2 mutant cells were spotted directly onto the top agar. The plate was incubated at 30°C for 2 days and photographed. 1. MATa mdy2 mutant cells (HZH683). 2. MATa wild-type cells (W303-1B). 3. MATa wild-type cells (W303-1A). (B) Halo assay of a-factor pheromone production. 0.6% top agar was mixed with 10⁵ indicator cells (MATa sst2). Aliquots (approximately 4x10⁶ cells) of wild-type HZH175 (MATa) and HZH176 (MATa) wild-type, and mdy2 mutant cells were spotted directly onto the top agar. The plate was incubated at 30°C for 2 days and photographed. 1. MATa wild-type cells (HZH175). 2. MATa control (HZH111). 3. MATa control (HZH176). 4. MATa mdy2 mutant cells (HZH119). (C) Time course of FUS1-lacZ induction after the addition of a-factor to haploid strains. Log-phase cultures of wild-type cells (W303-1A) and the mdy2 mutant (HZH686) harbouring plasmid pSB234 were shifted to fresh medium and treated with 5 mM a-factor for induction of FUS1-lacZ. Samples were harvested at the indicated times for the measurement of b-galactosidase activity.

• Supplemental Figure 2 -

  Fig. S2. Tests of MDY2 overexpression. Pheromone-induced FUS1-lacZ expression. Wild-type W303-1A and mdy2 (HZH686) carrying the empty vector pRS416 and mdy2 mutant harbouring pRS416-GAL-MDY2 were grown to log phase in SRG medium, induced with different concentrations of a-factor (0.05, 0.2, 1, 5 and 10 mM) for 90 minutes, and the b-galactosidase activity was measured. The data are the means of three independent assays.

• Supplemental Figure 3 -

  Fig. S3. Stability of Mdy2 during pheromone induction. Cultures of mdy2 mutant (HZH686) harbouring pGREG546-GST-MDY2 plasmid were grown to log phase and incubated in 3% raffinose and 1% galactose medium for 3 hours, and then shifted to 2% glucose medium and treated with 5 mM a-factor for different times as indicated. The cell lysates prepared from aliquots of cultures at the indicated times after shift (from galactose to glucose) were separated by SDS-PAGE and immunoblotted with anti-GST antibody. Immunoblots probed with anti-a-tublin antibody were treated with peroxidase-conjugated anti-mouse secondary antibody. Bound antibody was visualized by incubation of the immunoblots with Lumi light western blotting substrate and luminescent bands were visualized using Fuji film LAS-1000 combined with the image reader LAS-1000 V1 program.

• Supplemental Figure 4 -

  Fig. S4. Microscopic analysis of mating mixture using DIC optics in combination with soluble fluorescent marker GFP. MATa and MATa strains of wild-type (W303-1A and W303-1B) and mdy2 mutants (HZH686 and HZH683) were transformed with pRS424MET-GFP plasmid. The log-phase cells were mixed and incubated for 4 hours. The unbudded zygotes were scored for GFP distribution. Four mating tests were carried out as follows: MATa wild-type strain (w303-1A) harbouring pRS424MET-GFP plasmids was crossed with MATa wild-type strain (W303-1B); wild-type strain (W303-1A) was crossed with wild-type strain (W303-1B) harbouring pRS424MET-GFP; MATa mdy2 mutant (HZH686) harbouring pRS424MET-GFP plasmids was crossed with MATa mdy2 mutant (HZH683); mdy2 mutant (HZH686) was crossed with mdy2 mutant (HZH683) harbouring pRS424MET-GFP plasmids. The cells were grown to log-phase and the same numbers of cells of each mating type were mixed. After incubation for 4 hours, the unbudded zygotes were scored for the GFP distribution.

• Supplemental Figure 5 -

  Fig. S5. Microscopic analysis of mating mixtures using DIC optics in combination with the fluorescent DNA marker DAPI. Cultures of wild-type strains (W303-1A and W303-1B) and mdy2 mutant (HZH686 and HZH683) were grown to log phase. The same number of cells of each mating type were mixed and incubated at 30°C for 5.5 hours. The samples were harvested and cells were stained with DAPI. Zygotes were scored as having a cell fusion defect if a septum separating two unfused nuclei was present after 5.5 hours of mating (n>150).