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. 2014 Sep 25;3:e03251. doi: 10.7554/eLife.03251

Figure 5. Modulation of fusion activity through asymmetric distribution of PI(3)P.

(A) 1R-RPLs (with 0.5 mol% Marina-Blue-phosphatidylethanolamine [PE], containing biotinylated R-phycoerythrin) and 3Q-RPLs (with 3 mol% NBD-PE, containing Cy5-labeled streptavidin), bearing PI(3)P as indicated, were assayed for protected lumenal compartment mixing. Where indicated (columns 17–20), 3Q-RPLs bore a truncated version of the Qc-SNARE Vam7p that lacks the PI(3)P binding PX domain (Xu and Wickner, 2012). Reactions contained Sec17p (600 nM), Sec18p (200 nM), and HOPS (100 nM), as indicated. The lower panel provides a magnified view of the bottom 5% of the data (indicated by dotted box). (B) Kinetics of 1R-3Q content mixing assays with RPLs not bearing PI(3)P. Reactions contained HOPS (100 nM; red curve), Sec17p and Sec18p (600 and 200 nM; gray curve), or no added proteins (blue curve). The averages of five experiments are shown. The reactions containing Sec17p/Sec18p were normalized to zero, and these values were subtracted from all other conditions. Light-colored areas indicate standard deviations.

DOI: http://dx.doi.org/10.7554/eLife.03251.014

Figure 5.

Figure 5—figure supplement 1. Representative kinetic data for panel A in figure 5.

Figure 5—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.03251.015
Figure 5—figure supplement 2. Effect of varying the ratio of 1R to 3Q RPLs.

Figure 5—figure supplement 2.

Protected lumenal compartment mixing was measured as the decrease in fluorescence intensity of Biotin-PhycoE when it binds to Streptavidin-Cy5. Incubations either contained Sec17p (600 nM) and Sec18p (200 nM) (light gray) or no additional proteins (dark gray). All 1R-RPLs contained PI(3)P (1 mol%), while all 3Q-RPLs did not. (A) Content mixing reactions of 1R-RPLs (50 µM lipid; containing the lumenal marker Biotin-PhycoE) and 3Q-RPLs (0, 50, 150, or 450 µM lipid; containing the lumenal marker Streptavidin-Cy5). (B) Content mixing reactions of 3Q-RPLs (50 µM lipid; containing the lumenal marker Biotin-PhycoE) and 1R-RPLs (0, 50, 150, or 450 µM lipid; containing the lumenal marker Streptavidin-Cy5).
DOI: http://dx.doi.org/10.7554/eLife.03251.016