Figure 4.
The formation of quaternary SNARE complexes occurs sequentially and only in the presence of Sed5p. In A–C, the relative amount of a single SNARE was sequentially increased (lanes 2–5), whereas the amount of the other three SNAREs remained constant. SNARE complex formation was monitored following affinity purification of GST-Sft1p on glutathione Sepharose beads (Precipitate) and immunostaining with an anti-(His)6 antibody. Aliquots of supernatants, removed following incubation of the mixtures with reduced glutathione beads (∼5% of total volume), were also analyzed by immunostaining (Supernatant). (A) Effect of increasing concentrations of Ykt6p. Note that (His)6-Yktp stimulates (weakly) the formation of the ternary complex of [Sed5p + Gos1p + Sft1p] (lane 2). (B) Gos1p. Progressively increasing (His)6-Gos1p results in the eventual reduction of the amount of GST-Sft1p quaternary complex (compare lanes 2–5). (C) Sed5p. Neither binary nor ternary Sft1p-SNARE complexes (with Gos1p or Ykt6p) form in the absence of Sed5p (lane 1).