ORDs can simultaneously bind ISO-PM and ER membranes. Sucrose-loaded ISO-PM vesicles were incubated with light microsomes (that do not sediment at 16,000 g) and 0.4 µM of the indicated proteins for 30 min at 30°C. Mixtures were centrifuged for 10 min at 16,000 g, and the percentage of light microsomes pelleting was determined by quantitative immunoblotting. Error bars indicate mean ± SEM (n = 3).

Four Osh proteins are enriched on ER near the PM. Cells expressing the indicated GFP fusions and the ER marker ss-RFP-HDEL were visualized live, focusing on either the center plane or the periphery of the cells. Images taken in the GFP and RFP channels were merged. (A and B) GFP-Osh2p (A) and GFP-Osh3p (B) were expressed in a strain that overexpressed Scs2p. (C–E) Osh6-GFP (C), Osh7-GFP (D), and Rtn1-GFP (E) were expressed in wild-type cells. Arrowheads indicate ER tubules that are not closely apposed to the PM. Bar,1 µm.

ORDs can bind two membranes simultaneously. (A) Dense, sucrose-loaded liposomes with the indicated lipid composition were incubated with light liposomes of identical lipid composition plus a trace amount of [³H]triolein (0.5 mM each) with and without purified ORD or BSA (0.4 µM) for 30 min at 30°C. Radioactivity in the supernatant after centrifugation was measured by scintillation counting. (B) Liposome pull-down assay performed as in A but with varying concentrations of Osh6p or Osh7p. (C) Turbidity assay (absorbance at 350 nm after 10 min) using 90 µM lipid and varying concentrations of Osh6p or BSA. (D) Time course of change in turbidity using 900 µM lipid and 0.4 µM Osh6p. Error bars indicate mean ± SEM (n = 3).

Cholesterol extraction by Osh4p is regulated by PIPs in a second membrane. (A) 200 pmol Osh4p was incubated at 30°C with 500 µM 99:1 PC/[¹⁴C]cholesterol liposomes together with 400 µM auxiliary liposomes containing 100% PC (red squares), 99.5:0.5 PC/PI(4,5)P₂ (blue triangles), or 99:1 PS/PI(4)P (gray triangles). All liposomes were sucrose filled. At the indicated times, the samples were placed on ice, and the liposomes were pelleted. The amount of [¹⁴C]cholesterol in the supernatant was determined by scintillation counting. (B) 2 µM Osh4p was incubated at 30°C with 500 µM 99:1 PC/[¹⁴C]cholesterol liposomes. After 15 min (arrow), auxiliary liposomes were added, and the amount of [¹⁴C]cholesterol extracted was determined as in A (n = 3). (C) Performed as in B except that auxiliary liposomes were added to a final concentration of 400 µM (1×), 800 µM (2×), or 1.6 mM (4×). The total incubation was 30 min at 30°C (n = 4). Error bars indicate mean ± SEM.

Sterol transfer by Osh4p requires close contact of membranes. (A) DHE transfer from donor to acceptor liposomes was determined fluorimetrically as described in Materials and methods. Reaction components were separated by a barrier (1-µm–pore size Nuclepore membrane) where indicated. The distribution of components across the barrier at the start of the reaction is indicated. The amount of transfer with and without the indicated amount of Osh4p is shown (n = 2). (B) For the assays in A, the amount of Osh4p on the side of the barrier with the donor and acceptor liposomes at the end of the transfer assay was determined by SDS-PAGE. (C) Osh4p lacking endogenous cysteines and with the indicated mutations was covalently attached to sucrose-filled liposomes (containing 1% [¹⁴C]cholesterol) as described in Materials and methods. After a washing to remove unbound proteins, the amount of protein attached to the liposomes (picomole × linked) was determined (n = 3). These liposomes were incubated with acceptor liposomes for 1 h at 30°C, and the amount of [¹⁴C]cholesterol transferred to the acceptors was determined. (bottom) In a control reaction, the amount of transfer by free (not covalently attached) Osh4p and the same liposomes was determined. Error bars indicate mean ± SEM.

Osh4p has more than one membrane-binding surface. (A) The indicated mutations were introduced into Osh4p lacking endogenous cysteines (cys-less). The proteins were incubated with liposomes with the indicated lipid composition (N-MCC-PE contains a sulfhydryl-reactive headgroup) for 2 h at RT. The amount of protein pelleting with the membranes after washing (P) and the total amount of input protein (T) were determined by SDS-PAGE. The percentage of protein in P is shown (n = 3). Black lines indicate that intervening lanes have been spliced out. (B) Single-cysteine mutants were allowed to react with a sulfhydryl-reactive NBD derivative. The ratio of the fluorescence of the proteins before (Fo) and after (F) the addition of liposomes (99:1 PS/PI(4,5)P2) was measured (n = 6–8). (C, left) The structure of Osh4p (blue) bound to cholesterol (yellow) is shown. The flexible lid domain is shown in orange. (middle) A surface rendering of Osh4p, indicating the positively (blue) and negatively (red) charged surface. (right) The opposite side of the protein is shown. The positions of the residues changed to cysteine are indicated, and those that may interact with liposomes are shown in green. Error bars indicate mean ± SEM.

The distal membrane–binding surface of Osh4p is required for function. (A, left) Lysine residues changed to glutamate in the Osh4p mutant M4 are indicated in red, and the bound cholesterol are shown in yellow. (right) A surface rendering of Osh4p indicating the positively (blue) and negatively (red) charged surface. (B) Cross-linking experiments as in Fig. 3 A were performed with Osh4p mutant E284C or M4 proteins. The percentage of protein pelleting with the liposomes is shown. (C) Pull-down assay as described in Fig. 2 A using wild-type Osh4p or the M4 mutant and liposomes containing the indicated amount of PS. (D) DHE transport assays using 20 pmol wild-type Osh4p and liposomes containing the indicated amount of PS. The amount of DHE transferred in 1 h (30°C) was calculated by subtracting the amount of transfer in control reactions lacking protein. (E) DHE transport assays performed as in D but with the indicated liposomes. (F) [¹⁴C]cholesterol was added to cultures of strains with the indicated genotypes that contained plasmids that encoded no protein, wild-type Osh4p, or the M4 mutant. Samples were taken every 10 min for 40 min, and the rate at which the total amount of [¹⁴C]cholesterol was converted to cholesteryl ester per minute was calculated. (G) Plasmids encoding wild-type, cys-less, cys-less E284C, M4, or L111D Osh4p proteins were introduced into either oshΔ osh4-1 (left) or sec14-3 osh4Δ cells (right). Serial dilutions of the strains were incubated for 4 d at 37°C. Error bars indicate mean ± SEM (n = 3).