Sey1p-independent ER–ER fusion requires the ER SNARE Ufe1p. (a) Serial 10-fold dilutions of cells with the indicated genotypes were spotted onto YPD plates and incubated at 23°C for 5 d. (b) Cells with the indicated genotypes expressing Sec63-GFP were grown at 23°C and visualized live, focusing on either the center plane or the periphery of the cells. (c) A box and whiskers plot of the amount of time between cell fusion and ER fusion when cells with identical genotypes were mated with each other. Top and bottom ends of the boxes represent the 75th and 25th percentiles, and whiskers indicate the maximum and minimum values. The median is depicted with a solid line (n = 12–16 from at least three independent experiments). Cells were grown at 23°C and either mated at this temperature or shifted to 32°C immediately before mating. Where indicated (+ SEY1), the strains contained a plasmid that overexpressed Sey1p. (d) Strains expressing Sec63-GFP were visualized live in growth medium, and images were taken at 2-s intervals. Beginning at time 0, the area in the red rectangle was bleached every 2 s. Selected images from the time-lapse video are shown. All strains except sey1Δ ufe1-1 were grown at 30°C. The sey1Δ ufe1-1 cells were grown at 23°C and then shifted to 37°C for 30 min. Bars, 1 µm.