Figure 2.

Sec18p action is a prerequisite for dilution resistance. (A) Sec18p dependence. Two 3× fusion reactions were started at 27°C in the presence or absence of F_ab-fragments (150 μg/ml) prepared from the total immunoglobulin fraction from antiserum directed against Sec18p. After 20 min, the samples were chilled and made chemically equivalent by supplementation with antibodies to Sec18p. The samples were then split into three standard reactions. These were either left concentrated (Undil.) or diluted 20fold (Dil.) with reaction mixture. Each received 15 μg/ml purified Sec18p and 75 μM DTT and all were incubated at 27°C. An aliquot kept on ice serves as a measure of the background reaction which had occurred during the first incubation. After 70 min, all samples were chilled on ice and adjusted to the same volume with reaction mixture. The vacuoles were reisolated by centrifugation, resuspended in 30 μl ice-cold reaction mixture and assayed for fusion. The same results were obtained if Sec18p was added to the diluted sample to 0.75 μg/ml to satisfy only the stoichiometric requirement for saturation of the antibodies (not shown). (B) ATP dependence. Two 3× fusion reactions were incubated for 20 min at 27°C in the presence or absence of the ATP regenerating system. The samples were then chilled and the regenerating system was added to the sample lacking it. The samples were split into aliquots which were diluted with reaction mixture or left concentrated. Further incubation and analysis was as in A. (C) Temperature dependence. Fusion reactions equivalent to three standard reactions were incubated for 20 min at 0°C or 27°C. The samples were split into aliquots, diluted with reaction mixture where indicated, and further processed as in A.