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. 2002 Apr 1;157(1):79–90. doi: 10.1083/jcb.200112098

Figure 2.

Figure 2.

Released Vam7p is functional. (A) Complete Vam7p release during consecutive ice incubations. BJ3505 vacuoles (12 μg) were incubated in the presence of ATP on ice. After 30 min, the reaction was centrifuged (5 min, 20,000 g, 4°C), supernatant was removed, and a fresh reaction mix was added to the vacuoles. This procedure was repeated four times. The supernatant and the pellet were analyzed by immunoblotting with antibodies to Vam7p. Loss of Vam7p in the supernatant in the second reaction is due to the instability of released Vam7p (see text). (B) Removal of the supernatant coincides with loss of fusion activity. A fusion reaction containing the two tester strains was incubated as in A. After each incubation, vacuoles were reisolated (5 min, 20,000 g, 4°C) and resuspended either in new reaction mix containing Sec18p and CoA (white bars), the same supernatant (gray bars) to monitor loss of fusion due to centrifugation, or in the Vam7-containing supernatant removed in the first incubation (black bars). All samples were then incubated for 60 min at 26°C before assaying for fusion activity. (C) Fusion of vacuoles poisoned with anti-Vam7p. Fusion reactions containing both tester strains were incubated in a first incubation on ice or at 26°C for 30 min. Vacuoles were then reisolated (20,000 g, 5 min, 4°C), incubated for 5 min with or without anti-Vam7p on ice, and reisolated. Supernatant of a parallel reaction (30 min on ice in the presence of ATP) containing wild-type vacuoles or vam7Δ vacuoles was added to the pretreated vacuoles, incubated for 60 min at 26°C, and assayed for alkaline phosphatase activity. (D) Depletion of Vam7p from the supernatant blocks fusion. Priming on ice was done two times as described in A. The supernatant containing released Vam7p was incubated for 30 min at 4°C with protein A–Sepharose containing nonimmune IgG, anti-Vam7 (1–122) (α-PX), or anti-Vam7 (165–316) (α-cc). Then, the cleared supernatant was added to primed vacuoles as in A. (E) Soluble Vam7p can rescue fusion at a stage that is insensitive to docking inhibitors. Inhibitors were added to a fusion reaction either from the beginning (white bars) or after pretreatment with anti-Vam7p (black bars) as described in C. Fusion reactions were then incubated for 60 min at 26°C, and fusion activity was determined.