Fig. 5.
SNARE-complex formation is stimulated by both deletion of the Habc domain from Tlg2p, and by Vps45p. (A) The ability of Tlg2p-GST, or a version lacking the Habc domain (Tlg2pΔHabc-GST), to form SNARE complexes in vitro was assessed by incubating the GST fusions (purified from E. coli and immobilised on glutathione-Sepharose) with tenfold molar excesses of His6-tagged versions of Snc2p and Vti1p, and the untagged Sx8-Tlg1p chimera [all purified from E. coli as described (Paumet et al., 2005)]. After incubation for 20 or 120 minutes at 4°C with continuous mixing, Sepharose beads were harvested by centrifugation at 1000 g for 1 minute and washed extensively with binding buffer. Immunoblot analysis was used to assess the amount of Snc2p bound to the Tlg2p-GST fusions (lower panel; a sample of the input cognate SNAREs was included in this analysis). Upper panel shows a Coomassie-stained gel of the Tlg2p-GST fusions that were incubated either with (+) or without (–) the cognate SNARE-binding proteins. (B) The effect of adding purified His6-tagged Vps45p on the ability of Tlg2p-GST and Tlg2pΔHabc-GST to form SNARE complexes in vitro during a 20-minute incubation was assessed by performing the experiment presented in A in the absence or presence of a tenfold molar excess of His6-tagged Vps45p (over Tlg2p-GST and Tlg2pΔHabc-GST).