The initiation of Cla4 signaling at the vacuole occurs at the bud cortex. (A and B) cla4Δ vac17Δ mutant transformed with Cla4-tdTomato and either Vac17-GFP or vac17-S222A-GFP plasmids. Cells synchronized with α-factor were imaged every 10 min after release. Representative images from >25 cells per strain per time point per n. n = 3. DIC, differential interference contrast. (A) Cla4-tdTomato colocalized with Vac17-GFP at the bud cortex (arrowheads). After Vac17-GFP was no longer detected, Cla4-tdTomato was localized away from the cortex (yellow arrows). (B) Cla4-tdTomato and vac17-S222A-GFP colocalized at the bud cortex (arrowheads). vac17-S222A-GFP persisted at the bud tip (white arrows) and later at the mother-bud neck (blue arrow) after Cla4-tdTomato localized to a different location on the vacuole (yellow arrows). (C) Model showing how Myo2 brings a portion of the vacuole and Vac17 to the bud cortex where Cla4 phosphorylates Vac17. A punctum of Cla4 then moves onto the vacuole. Cla4-dependent phosphorylation of Vac17 may either recruit a downstream binding partner or change the conformation of Vac17 to facilitate Dma1-dependent ubiquitylation of Vac17 and thus the termination of vacuole transport.