Local negative regulation of Ras1 couples cell fusion with cell contact. (A) Localization of Gap1 GAP domain on its own (GAP^Gap1), fused to a single or triple RBD (RBD-GAP^Gap1, RasAct-GAP^Gap1), and full-length Gap1 fused to 3GFP expressed in gap1Δ cells. These constructs are detailed in Fig. S5 A. Myo52-tdTomato (red) foci indicate cell pairs in fusion (arrowheads). (B) Percentage of cell lysis and cell pair formation of h90 gap1Δ cells expressing or not the fusion constructs shown in A 3 h (left) and 12 h (right) after nitrogen (N) starvation (see also Fig. 4 E; n > 300 for three independent experiments). (C) GAP^Gap1-3GFP strains expressing Myo52-GBP-mCherry 8 h after nitrogen starvation. GAP^Gap1-3GFP is recruited to the fusion focus via interaction with the GBP moiety. (D) Percentage of cell lysis and fusion efficiency of WT, gap1Δ, GAP^Gap1-3GFP, and gap1Δ GAP^Gap1-3GFP strains expressing Myo52-GBP-mCherry 12 h after nitrogen starvation. Note that GAP^Gap1-3GFP recruitment to the fusion focus suppresses gap1Δ cell lysis. (E) Duration of Myo52-GBP-mCherry focalization in strains of indicated genotypes; n = 25. ***, 1.2 × 10⁻¹⁶ ≤ P ≤ 7.2 × 10⁻⁷. Error bars, SD. Bars, 2 µm.