Myo2p dissociates from the vesicle ∼4 s before fusion while the GEF Sec2p and exocyst components dissociate at the fusion event. (A) Kymographs of GFP-Sec4 and Myo2-3×mCherry puncta during the fusion event. Note the gradual decrease in intensity of Myo2-3×mCherry before its complete disappearance. (B) Line scans of three different kymograph traces showing Myo2-3×mCherry leaving a vesicle gradually before the fusion event at t = 0 s. Intensity was normalized. (C) Kymographs of GFP-Sec4 and Sec2-3×mCherry puncta during the fusion event. (D) Kymographs of GFP-Sec4 and Sec15-3×mCherry puncta during the fusion event. (E) Column scatter plot of the time of disappearance (in seconds) for the protein of interest compared with the fusion event (GFP-Sec4 disappearance). Sec2-3×mCherry, n = 23; Myo2-3×mCherry, n = 22; Sec15-3×mCherry, n = 30. Lines show mean and 95% confidence intervals. (F) Kymographs of Sec3-3×GFP and Sec15-3×mCherry puncta during the fusion event. (G) Kymographs of Sec5-3×GFP and Sec15-3×mCherry puncta during the fusion event. (H) Column scatter plot of the time of disappearance (in seconds) for Sec3p or Sec5p compared with the fusion event (Sec15p disappearance). n = 25 for both datasets. Lines show mean and 95% confidence intervals. (I) FLIP experiments showing the normalized intensity in the bud for tagged exocyst components. Best-fit curves were single-exponential decay with one rate of loss from the bud. Half times of loss from the bud were 71.0 ± 9.4 s (Sec3-GFP, n = 15), 58.1 ± 9.4 s (Sec5-GFP; n = 13), and 78.2 ± 17.1 s (Sec15-GFP; n = 10). Error bars indicate standard deviation.