Strains used in this study are summarized in Appendix 1-table 1. All cell growth, starvation, and pairing experiments were conducted at 30°C. Cells were grown in Super Proteose Peptone (SPP) medium (1% Proteose Peptone, 0.1% yeast extract, 0.2% glucose, 0.003% Sequestrene) or Neff medium (0.25% Proteose Peptone, 0.25% yeast extract, 0.5% glucose, 0.003% Sequestrene). Cells were starved in 10 mM Tris-Cl (pH 7.4) for 16 h before all pairing experiments. For normal pairing assays, equal numbers of starved cells of different mating types (at 2×10 6 cells/ml) were mixed. To obtain costimulated (pre-incubated) cells, two starved strains were mixed at a 9 : 1 ratio for 30 min (unless otherwise stated). Before mixing costimulated cells, any potentially pairing cells were separated by shaking. Figure 2A shows the setup of costimulation experiments. For all mating experiments (whether or not they involved mutant cells), the starting WT cell density was 2×10 6 cells/ml. The following formula was used to calculate pairing ratios and correct for the presence of mutant cells: