Figure 1.

Osh4p and other Osh proteins are required for efficient nonvesicular PM to ER sterol transport. (A–I) Cells were grown at either 23°C (A–C) or shifted to 37°C for 1 h (D–I) and ¹⁴C-cholesterol or ¹⁴C-ergosterol was added to medium. To ensure that the strains took up similar amounts of ¹⁴C-cholesterol, the OSH ⁺ strain (A and D) was given 1.0 μM ¹⁴C-cholesterol and the oshΔ strains (B,C,E,F) were given 4.0 μM ¹⁴C-cholesterol. All three strains were given¹⁴C-ergosterol at 2.5 μM. At the indicated times, cells were harvested, lipids were extracted, and the amount of free and esterified radiolabeled sterol in the cells was quantitated. (J) ACAT activity of the OSH ⁺ and oshΔ strains. Cells were grown at 25°C and shifted to 37°C for 1 h, and the ACAT activity of protein extracts from each strain was determined. (K) To estimate the amount of endogenous sterol in the ER, lysates from the OSH ⁺ and oshΔ cells were reacted to completion with ¹⁴C-oleoyl CoA. The amount of sterol in the ER was calculated from the amount of ¹⁴C-steryl ester formed. (L) To determine if ¹⁴C-cholesterol esters are hydrolyzed after they are formed, oshΔ osh4-1 and OSH ⁺ cells were grown at 23°C, shifted to 37°C for 30 min, and 2.0 μM ¹⁴C-cholesterol was added to the medium. After 30 min, the 250-μM unlabeled cholesterol was added to the medium and cells grown for an additional 40 min. The amount of ¹⁴C-cholesteryl esters per OD₆₀₀ of cells before (pulse) and after addition of unlabeled cholesterol (chase) was determined. In all graphs, values are the mean of two determinations. Error bars indicate the SEM. n = 2.