Fig. 6. Disassembly of the cis-SNARE complex by NSF/Sec18 and stimulation of trans-SNARE complex assembly by Sly1. The cell-free assay system was carried out with or without adding ATP or its unhydrolyzable analog AMPPNP. Yeast lysate and a recombinant protein were mixed as described in Fig. 4. The mixture was supplemented with 1 mM ATP (lanes 2 and 6) or 1 mM AMPPNP (lanes 3 and 7) or had nothing added (lanes 1 and 5). The reactions were started by shifting the temperature to 35°C and continued for up to 30 minutes. In the experiments shown in lanes 4 and 8, the mixture with exogenous Sly1/Sly1^ts-Strep was incubated in the presence of 1 mM ATP at 35°C for 30 minutes. Then 1 mM AMPPNP was added and the incubation was continued for another 30 minutes before addition of Triton X-100. The bands of Sly1/Sly1^ts-Strep, 6myc-Sed5 and 3HA-Bet1 are indicated by arrowheads. ^*indicates the endogenous Sly1^ts protein in lysate I and II.