Imaging of Bet1p-Cy3 molecules reconstituted in a planar lipid bilayer. (A) Overview of the horizontal planar lipid bilayer formation apparatus. The apparatus consists of two chambers. On the bottom of the upper chamber, a thin plastic film with a small hole (∼100 μm) was attached and an artificial planar lipid bilayer was formed horizontally across the hole on an agarose-coated coverslip. The upper chamber could be moved vertically using a micromanipulator. (B) A typical fluorescent spot of Bet1p-Cy3 in the bilayer membrane was photobleached in a single step (red) to the background level (blue). (C–G) Distribution of the fluorescence intensity of Bet1p-Cy3 in the presence of the COPII component(s). To the bilayer membranes reconstituted with Bet1p-Cy3 (0.28 molecules μm⁻²) and Sec12Δlum (C) was added Sar1p (70 ng) (D), Sar1p (70 ng) and Sec23/24p (410 ng) (E), or Sar1p (70 ng), Sec23/24p (410 ng), and Sec13/31p (1.4 μg) (F) in molar excess of Bet1p-Cy3 in the presence of GTP, except for experiments in (G), in which GDP was added in place of GTP. Histograms of the fluorescence intensity were fit to either single or double Gaussian distributions (lines) and the percentages of fractions of each component are indicated (numbers).

Clustering of Bet1p-Cy3 molecules and downward COPII vesicle budding with the bilayer membrane formed in an aqueous environment. (A) Sequential images of the Bet1p-Cy3 clustering in the bilayer membrane (Supplementary Movie S2 online). Sar1p (70 ng), Sec23/24p (410 ng), and Sec13/31p (1.4 μg) were added to the bilayer membrane reconstituted with Bet1p-Cy3 (95.6 molecules μm⁻²) and Sec12Δlum at t=0 s in the presence of GTP or GDP as illustrated in the upper panel. Epifluorescence images were taken with exposure times of 66 ms. (B) Schematic illustration of downward COPII vesicle budding. (C) Time course of the increase in the number of fluorescent particles that appeared on the coverslip after the addition of the COPII components. Each thin trace corresponds to an individual experiment. The numbers of fluorescent particles were normalized so that the initial number of fluorescent particles on the coverslip was 100%. Thick traces with standard error deviations represent the averages of all thin traces.

COPII-induced Bet1p-Cy3 assembly in the decane bilayer membrane. (A) COPII components (70 ng Sar1p, 410 ng Sec23/24p, and 1.4 μg Sec13/31p) were added from the upper chamber to the decane bilayer membrane formed in an aqueous environment and fluorescent spots were acquired (as shown in Supplementary Movie S4 online) in the presence of GTP (upper panel) or GMP-PNP (lower panel). The bilayer membrane was then attached to the coverslip. Fluorescent images were taken under evanescent field illumination. (B) Distribution of the fluorescence intensity of the small spots observed in (A) under different concentrations of Bet1p-Cy3 in the membrane. Insets show the same histograms plotted with a narrower bin size. (C) A correlation plot of the Bet1p-Cy3 concentration versus the mean fluorescent intensity of the fractions obtained from (B).

The mutual localization of Ufe1p-ATTO647N and Bet1p-Cy3 in the presence of the COPII components. (A) Sar1p (70 ng), Sec23/24p (410 ng), and Sec13/31p (1.4 μg) were added from the upper chamber to the decane bilayer membrane (formed in an aqueous environment) reconstituted with Ufe1p-ATTO647N (41.3 molecules μm⁻²), Bet1p-Cy3 (113.1 molecules μm⁻²), and Sec12Δlum molecules in the presence of GTP or GMP-PNP. Bet1p-Cy3 was omitted in some cases. The following procedures were the same as those for Figure 3A. ATTO647N (left) and Cy3 (centre) fluorescence channels, and merged image (right) are shown. ATTO647N and Cy3 fluorescence is shown in red and green, respectively. (B) The relative density of Ufe1p-ATTO647N in the large clusters compared to that outside the cluster.