Fig. 6. Cdc42p acts before trans-SNARE pairing. Standard fusion reactions (5-fold volume) with cytosol containing vacuoles from strains BJ3505Δvam3 and BJ3505Δnyv1 were incubated without ATP on ice or with ATP at 27°C. One of the samples at 27°C received buffer only (None) and another one 6 µM affinity-purified antibodies to Cdc42p (Ab). After 45 min, trans-SNAREs were assayed as described in Materials and methods by the amount of Nyv1p that co-immuno precipitates with Vam3p. As can be seen by comparison of co-precipitated Nyv1p with the corresponding input, formation of trans-SNARE pairs in the control reaction is in the range of literature values (Ungermann et al., 1998a).