Figure 4.

Reconstitution of ceramide and subsequent IPC synthesis with ER- and Golgi-enriched membranes. (A and B) Purification of ER- and Golgi-enriched membranes was performed as described in Materials and methods. (A) Microsomal membranes from RH5256 were subjected to sequential centrifugation to obtain pellet (P27), supernatant (S27) fraction, and from the S27 fraction, pellet (P45), supernatant (S45) fraction. (B) The P27 fraction was subjected to sucrose density step gradient, and the resulting gradient fractions were collected from the top. Equal fractions were analyzed for the distribution of Aur1p-HA, Emp47p, and Wbp1p by Western blotting. (C) In vitro labeling using [³H]DHS with either ER- (40/50%-I fraction, 5–10 μg) or Golgi- (S45 fraction, 25–50 μg) enriched membranes or both (40/50%-I fraction, 5–10 μg and S45 fraction, 25–50 μg), and NaOH treatment of the labeled lipids were performed as described in the legend to Fig. 2 A. Control experiments were done without membranes or with both membranes in the presence of AbA (50 nM). The lipids were applied to TLC plates using solvent I. Images of portions of individual lanes were shown, and the maximal signal of [³H]ceramide in the experiment, which was done with both membranes, was set to 100 as the relative signal. In these diagrams, the total amount of incorporation of [³H]DHS into each lipid was expressed as the percentage of that of [³H]DHS with both membranes.