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

Figure 3. Discrepancies between lipid mixing and content mixing assays in 1R-3Q RPL fusion reactions.

Incubations contained Sec17p (600 nM), Sec18p (200 nM), and HOPS (100 nM), as indicated. (A and B) Incubations contained 1R-RPLs (containing Biotin-PhycoE) and 3Q-RPLs (containing Streptavidin-Cy5) (250 µM lipid each), with the two fluorescent lipids Marina-Blue-phosphatidylethanolamine (MB-PE) and NBD-PE (1.5 mol% each) being together on either the 1R-RPLs or the 3Q-RPLs. Lipid dequenching (A) and protected lumenal content mixing (B) were recorded from the same reactions. (C and D) Incubations bore 1R-RPLs (containing Biotin-PhycoE) and 3Q-RPLs (containing Streptavidin-Cy5) (250 µM lipid each), with each RPL bearing a single fluorescent lipid, either Marina-Blue-phosphatidylethanolamine (MB-PE) (1 mol%) or NBD-PE (3 mol%). Lipid quenching (C) and protected lumenal content mixing (D) were recorded from the same reactions.

DOI: http://dx.doi.org/10.7554/eLife.03251.008

Figure 3.

Figure 3—figure supplement 1. Representative kinetic data for panels A–D in figure 3.

Figure 3—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.03251.009
Figure 3—figure supplement 2. Characterization of liposome size by dynamic light scattering.

Figure 3—figure supplement 2.

(A–H) The size distribution of various proteoliposome preparations was analyzed by dynamic light scattering with a Zetasizer nano ZS (Malvern Instruments, Westborough, MA) through non-invasive back-scatter at 173°. Samples of 400 μl at a lipid concentration of 20 μM were measured in low volume disposable sizing cuvettes at 25°C. The Z averages (r.nm) were: 101.5 for 1R(MB/NBD) (A), 90.63 for 1R() (B), 99.18 for 1R(MB) (C), 91.07 for 1R(NBD) (D), 89.78 for 3Q() (E), 95.36 for 3Q(MB/NBD) (F), 86.93 for 3Q(NBD) (G), and 89.40 for 3Q(MB) (H).
DOI: http://dx.doi.org/10.7554/eLife.03251.010
Figure 3—figure supplement 3. Characterization of dye and protein incorporation.

Figure 3—figure supplement 3.

(A) Samples (20 µl) of 1R(MB/NBD) and 3Q(MB/NBD) RPLs (25 µM lipid) were incubated for 30 min at 27°C in the presence or absence of Thesit (0.2% [wt/vol]) in 384-well plates in a SpectraMax Gemini XPS, and the fluorescence of Marina-Blue (Ex: 370 nm, Em: 465 nm, cutoff: 420 nm) and NBD (Ex: 460 nm, Em: 538 nm, cutoff: 515 nm) was recorded. The average of three repeats ± standard deviations is shown. (B) RPLs of various composition were analyzed for their protein composition by SDS-PAGE and Colloidal Coomassie staining.
DOI: http://dx.doi.org/10.7554/eLife.03251.011