Figure 2. Asymmetric lipid dequenching.
(A) Lipid dequenching reactions of 1R- and 3Q-RPLs (250 µM lipid each), with both fluorescent lipids (Marina-Blue-phosphatidylethanolamine [MB-PE] and NBD-PE, 1.5 mol% each) on either the 1R-RPLs or the 3Q-RPLs. Incubations contained either Sec17p (600 nM) and Sec18p (200 nM) (light gray) or no additional proteins (dark gray). (B) Lipid dequenching reactions of MB/NBD-labeled 1R- (light gray) or 3Q-RPLs (dark gray) with increasing amounts of non-labeled complementary RPLs (3Q or 1R; 1:1 corresponds to 50 µM lipid of each). No additional proteins were added. (C) Sensitivity of lipid dequenching reactions of 1R(MB/NBD)-RPLs and 3Q-RPLs to various inhibitors that might prevent trans-SNARE complex formation. Incubations received: (1) no addition, (2) Sec17p/Sec18p (400 nM each), (3) antibodies directed against Vam3p (1 µM), or (4) soluble Nyv1p(1-231) (1 µM). Column 5: 1R(MB/NBD)-RPLs were incubated with non-fluorescent RPLs bearing various combinations of SNAREs other than 3Q: QabcR, QbcR, QacR, QabR, QcR, QbR, Qbc, QaR, Qac, Qab, R, Qc, Qb, Qa. The extent of these reactions was comparably low for all conditions, and is shown as average for these 14 conditions with standard deviations.
Figure 2—figure supplement 1. Representative kinetic data for panels A–C in figure 2.
