Figure 7. High cooperativity does not explain the disproportion between trans-SNARE interaction and fusion rate resulting from HOPS addition.
(A) Kinetics of content mixing reactions of 3Q-RPLs (reconstituted with a protein:lipid ratio of 1:2000 for Vam3p, Vti1p, and Vam7p) and 1R-RPLs (reconstituted with a protein:lipid ratio of either 1:2000 [blue], 1:4000 [red], 1:8000 [green], or 1:16,000 [purple] for Nyv1p). RPLs were incubated for 30 min at 27°C before and HOPS (100 nM) or its buffer were then added. (B) Equivalent 40 µl reactions were analyzed for trans-SNARE association by co-immunoprecipitation of Nyv1p (R-SNARE) with Vam3p (Qa-SNARE). Lanes 1–8 correspond to the conditions as shown in (A) with odd numbers representing conditions with no addition and even numbers representing conditions with HOPS (100 nM). For lanes a, b, c, and d, the components were mixed directly in detergent solution without prior incubation. (C) Quantification of content mixing (as seen in panel A) from three independent repeat experiments. The rate of fusion was determined as the slope for the first 10 min of the content mixing reaction (between minutes 0 and 10) for samples that did not receive HOPS or as the slope for the first minute of the content mixing reaction after addition of HOPS (between minutes 30 and 31). (D) Quantification of trans-SNARE association (as seen in panel B). Trans-SNARE interactions were quantified as the amount of Nyv1p that co-precipitated with Vam3p (lanes 1–8 in panel B) using UN-SCAN-IT gel 5.3 software (Silk Scientific, Orem, UT), and are represented as percentage of the amount of Nyv1p that co-precipitated in the reaction that contained HOPS and R-RPLs with a SNARE:lipid ratio of 1:2000 (lane 8 in panel B).