Figure 6. Effect of large protein tags on lipid and content mixing.

1. Vacuoles were isolated from the indicated yeast strains and 30 μg of proteins analyzed by SDS–PAGE and Western blotting.
2. Vacuole morphology was assessed for the indicated cells as in Fig 3C. Scale bar: 5 μm.
3. Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that the presence of Nyv1 on only one fusion partner still permits efficient fusion 64 but ensures that trans‐SNARE complexes can only form in one orientation, between the Q‐SNARE in DKY6281 and the R‐SNARE in BJ3505. The vacuoles were used in standard fusion reactions, and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either on ice, which prevents fusion, or in the presence of 0.5% Triton X‐100, which allows fusion‐independent access of the maturase Pep4 to pro‐ALP and controls for the levels of these two reporter enzymes in the samples. Means ± s.d. are shown from three independent experiments.
4. Using strains from (C), lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti‐Vam3 antibody (3 μg) was added to some reactions in order to inhibit trans‐SNARE pairing and fusion. Means ± s.d. are shown from three independent experiments.