Figure 6. HOPS increases the rate of content mixing substantially more than the formation of trans-SNARE associations.

(A) Kinetics of 1R-3Q content mixing assays. RPLs (reconstituted with a protein:lipid ratio of 1:2500 for SNAREs) were incubated for 30 min at 27°C before and after the addition of HOPS (100 nM) or its buffer. (B) Additional 40 µl reactions that were performed in parallel were analyzed for trans-SNARE association by co-immunoprecipitation of Nyv1p (R-SNARE) with Vam3p (Q_a-SNARE). Lanes 3 and 4 correspond to the conditions as shown in (A). Lanes 7 and 8 are from equivalent reactions, but containing soluble Nyv1p instead of R-RPLs. For lanes 1, 2, 5, and 6, the components were mixed directly in detergent solution without prior incubation. (C) Quantification of content mixing and trans-SNARE association (as seen in panels A and B) from three independent repeat experiments. The rate of fusion was determined as the slope for the first minute of the content mixing reaction after addition of HOPS or its buffer (between minutes 30 and 31). Trans-SNARE interactions were quantified as the amount of Nyv1p that co-precipitated with Vam3p (lanes 3 and 4 in panel B) using UN-SCAN-IT gel 5.3 software (Silk Scientific, Orem, UT). (D) 1R- and 3Q-RPLs were mixed with increasing concentrations of soluble Nyv1p (sR), and incubated for 30 min at 27°C. HOPS (100 nM) was added, and the reactions were assayed for protected lumenal compartment mixing. (E) A mixture of 1R- and 3Q-RPLs was pre-incubated with or without 1 µM soluble Nyv1p for 30 min at 27°C. Sec17p (600 nM), Sec18p (200 nM), and HOPS (100 nM) or their respective buffers were added as indicated, and the reactions were assayed for protected lumenal compartment mixing.

DOI: http://dx.doi.org/10.7554/eLife.03251.017

Figure 6—figure supplement 1. Suppression of the formation of 1R-3Q SNARE complexes in detergent by increasing concentrations of GST-tagged Nyv1p.

The 40 µl fusion reactions were incubated at 27°C as indicated, and analyzed for trans-SNARE association by co-immunoprecipitation of Nyv1p (R-SNARE) with Vam3p (Q_a-SNARE). The co-immunoprecipitation was carried out in the presence of increasing concentrations of GST-Nyv1p. Lanes 1 and 2 contained only R- or 3Q-RPLs. Lanes 3-6 contained R- and 3Q-RPLs. Lanes 7 and 8 contained 3Q-RPLs and soluble Nyv1p.

DOI: http://dx.doi.org/10.7554/eLife.03251.018

Figure 6—figure supplement 2. Representative kinetic data for panels D–E in figure 6.

DOI: http://dx.doi.org/10.7554/eLife.03251.019

Figure 6—figure supplement 3. Reversible inhibition by soluble Q-SNAREs.

(A) 1R- and 3Q-RPLs were mixed with increasing concentrations of soluble Vam3p, Vti1p, and Vam7p, and incubated for 30 min at 27°C. HOPS (100 nM) was added, and the reactions were assayed for protected lumenal compartment mixing. (B) A mixture of 1R- and 3Q-RPLs was pre-incubated with or without 4 µM soluble Vam3p, Vti1p, and Vam7p for 30 min at 27°C. Sec17p (600 nM), Sec18p (200 nM), and HOPS (100 nM) or their respective buffers were added as indicated, and the reactions were assayed for protected lumenal compartment mixing. (C) Reactions as in (A), but with increasing concentrations of either soluble Vam3p, or soluble Vti1p, or Vam7p individually.

DOI: http://dx.doi.org/10.7554/eLife.03251.020