Figure 4.

Sec18p action is required on both fusion partners to activate them for docking and fusion. Separate samples of BJ3505 vacuoles (with pro-alkaline phosphatase) and DKY6281 vacuoles (carrying the maturation enzymes), each equivalent to three standard fusion reactions, were incubated at 27°C in the presence or absence of antibodies to Sec18p (70 μg/ml). After 15 min, the samples were chilled, supplemented with antibodies to Sec18p (to 70 μg/ml), and left on ice for 5 min. They were mixed pairwise to make combinations where only one of the fusion partners or neither of them had been preincubated with anti-Sec18p. The tubes were centrifuged briefly (2 min, 8,000 g, 4°C) and the vacuoles resuspended in fresh reaction mixture with 75 μM DTT. Each sample was split into aliquots which received either 1.5 μg/ml Sec18p to release the block of the Sec18p pathway or antibodies to Sec18p to maintain the block. The samples were incubated at 27°C or on ice (to indicate the background reaction which had occurred during the first incubation). After 70 min, fusion activity was determined. Control samples having both fusion partners already mixed during the first incubation were taken through the same procedure in parallel (right panel).