Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

Abstract

Osh/Orp proteins transport sterols between organelles and are involved in phosphoinositide metabolism. The link between these two aspects remains elusive. Using novel assays, we address the influence of membrane composition on the ability of Osh4p/Kes1p to extract, deliver, or transport dehydroergosterol (DHE). Surprisingly, phosphatidylinositol 4-phosphate (PI(4)P) specifically inhibited DHE extraction because PI(4)P was itself efficiently extracted by Osh4p. We solve the structure of the Osh4p-PI(4)P complex and reveal how Osh4p selectively substitutes PI(4)P for sterol. Last, we show that Osh4p quickly exchanges DHE for PI(4)P and, thereby, can transport these two lipids between membranes along opposite routes. These results suggest a model in which Osh4p transports sterol from the ER to late compartments pinpointed by PI(4)P and, in turn, transports PI(4)P backward. Coupled to PI(4)P metabolism, this transport cycle would create sterol gradients. Because the residues that recognize PI(4)P are conserved in Osh4p homologues, other Osh/Orp are potential sterol/phosphoinositol phosphate exchangers.

Figure 1.

Real-time measurement of DHE loading and extraction by Osh4p from large DOPC liposomes. (A) Intrinsic fluorescence at 340 nm of Osh4p (0.5 µM) upon addition to DOPC/DHE (99.5:0.5 mol/mol) liposomes (blue trace) or to DOPC liposomes (black trace). The final lipid concentration is 0.5 mM. (B) Fluorescence emission spectra (λ_ex = 285 nm) of the samples shown in A at the end of the kinetics. (C) Close-up view of the Osh4p structure (PDB accession no. 1ZHZ) showing the lid (segment 1–29, black) covering ergosterol (bright green). Tryptophans located at ≤15 Å, between 15 and 20 Å, or at ≥20 Å from ergosterol are colored in cyan, blue, and purple, respectively. Cystein C98 is indicated. (D) Fluorescence at 510 nm (λ_ex = 310 nm) of 0.5 µM DNS-Osh4p added to buffer alone (gray trace) or to DOPC/DHE (99.5:0.5 mol/mol, blue trace), DOPC (black trace), DOPC/ergosterol (95:5, light green trace), or DOPC/ergosterol/DHE (94.5:5:0.5, dark green trace) liposomes. Bottom trace, unlabeled Osh4p added to DOPC/DHE liposomes. (E) 0.5 µM Osh4p was incubated with DOPC or DOPC/DHE (99.5:0.5) liposomes (0.5 mM lipids) filled with sucrose. A reference experiment (100%) was done without liposomes. After centrifugation, the percentage of Osh4p in the pellet (P) and supernatant (S) was determined by SDS-PAGE. The fluorescence spectrum of each sample was recorded (λ_ex = 285 nm). Control spectra corresponding to buffer or liposome alone (0.5 mM lipids) were subtracted from the spectra of the supernatant and pellet, respectively. Data in E are from a single representative experiment out of two independent experiments.

Figure 2.

Influence of membrane composition on the solubilization of DHE by Osh4p. (A) Solubilization of DHE by Osh4p from DOPC liposomes doped with the indicated anionic lipid using the same protocol as in Fig. 1 E. When the fluorescence intensity was high enough, the I₃₇₂/I₃₄₀ ratio, which reflects the amount of DHE in complex with Osh4p in the supernatant (S) or in the pellet (P), was determined. (B) 0.5 µM NBD-Osh4p was added to buffer (−lip) or to liposomes (0.5 mM lipids) of various compositions. Trp fluorescence was monitored at 340 nm (λ_ex = 285 nm; top), whereas NBD fluorescence was followed at 525 nm (λ_ex = 495 nm; bottom). The contribution of the liposomes to the fluorescence signal was subtracted for each curve, and the fluorescence in buffer at t = 0 was used as a reference for normalization. Data shown in A are from a single representative experiment out of two independent experiments.

Figure 3.

DHE and PI(4)P compete for extraction by Osh4p. (A) 0.5 µM NBD-Osh4p was added to buffer alone or to DOPC liposome (0.5 mM lipids) containing 0.5 mol% DHE and increasing amounts of PI(4)P or PI(4,5)P₂. The changes in Trp and NBD fluorescence, which reflect DHE extraction and liposome binding, respectively, were monitored as in Fig. 2 B. The plots show the fluorescence levels 9 min after the addition of Osh4p. The amount of DHE-free Osh4p reported on the right axis was determined from the Trp intensity. (B) Lipid extraction assay. Sucrose-loaded DOPC liposomes (0.5 mM lipids) containing 2% PI(4)P (red curve), 2% PI(4,5)P₂ (green curve), 2% PI (purple curve), or 2% cholesterol (black curve) and doped, respectively, with [³²P]PI(4)P, [³H]PI(4,5)P₂, [³H]PI, or [³H]cholesterol were incubated with Osh4p (0–5 µM) for 20 min (room temperature). After centrifugation, radioactivity in the supernatant and in the pellet was counted to calculate the fraction of extracted lipid. Data are represented as mean ± SEM (error bars; n = 2–3). (C) Two-stage flotation assay. DOPC liposomes containing the indicated amounts of PI(4)P and DHE were mixed with 0, 5, or 10 µM Osh4p, collected by flotation, and then incubated with 0.75 µM PH_FAPP (GST-tagged). The final amount of liposome-bound Osh4p and PH_FAPP after the second stage was determined by SDS-PAGE. (D) Solubilization of DHE by Osh4p from DOPC liposomes containing 2% PI(4)P and increasing amounts of DHE (0–10%). After centrifugation, the amount of protein in the supernatant (top) was assessed by SDS-PAGE. The bottom shows Osh4p fluorescence at 340 nm (λ_ex = 285 nm). Data are represented as mean ± SEM (error bars; n = 2). For comparison, an experiment was performed with liposomes containing 0.5% DHE but no PI(4)P (black curve).

Figure 4.

Crystal structure of Osh4p in complex with PI(4)P. (A) Overall structure. The lid region is shown in red (residues 13–29), the N-terminal domain in orange (30–116), the β-barrel in green (117–307), and the C-terminal domain in cyan (308–434). PI(4)P is shown as spheres with carbon atoms colored in white, oxygen in red, and phosphorus in orange. The missing loops are represented by dashed lines. (B) Structure superposition of Osh4p in complex with PI(4)P (in orange) or ergosterol (in purple; PDB accession no. 1ZHZ). The black circle indicates the region that differs significantly in the two structures. (C) Close-up view of the PI(4)P binding site. PI(4)P is shown in black with oxygen in red and phosphorus in orange. (D) Superposition of PI(4)P (colored in orange) and ergosterol (purple) molecules in Osh4p. The backbone of Osh4p is shown in light gray. (E) Stereo view of the interaction network of the PI(4)P polar head. The electron density colored in blue represents the Fo-Fc–simulated annealing omit map contoured at 2.5 σ. Nitrogen, oxygen, and phosphorus atoms are shown in blue, red, and orange, respectively. Key water molecules contacting PI(4)P and the protein are displayed as cyan spheres. (F) Effect of mutations on the ability of Osh4p to extract DHE from DOPC liposomes doped or not doped with 2% PI(4)P. The experimental conditions were the same as in Fig. 2 B.

Figure 5.

Osh4p delivers DHE to PI(4)P-containing liposomes. (A) Osh4p loaded with DHE (∼0.5 µM Osh4p[DHE]) was incubated at 30°C in buffer. At the indicated time, 30 µl of a stock suspension of liposomes (5 mM lipids) was injected (final concentration = 240 µM). The release of DHE was followed by measuring the increase in Osh4p fluorescence at 340 nm (λ_ex = 285 nm). The liposomes contained DOPC and, when indicated, 2 mol% of DOPS, PI, PI(3)P, PI(4)P, PI(5)P, or PI(4,5)P₂. (B) DNS-Osh4p loaded with DHE (∼0.5 µM) was mixed with 240 µM liposomes with (left) or without (right) 5 mol% ergosterol at 30°C. The release of DHE was followed by measuring the diminution in FRET between DHE and DNS (λ_ex = 310 nm; λ_em = 510 nm). In all assays, the recordings were corrected for the light-scattering signal and the dilution effect was due to liposome addition.

Figure 6.

Osh4p optimally transports DHE from neutral to PI(4)P-containing membranes. (A) Effect of anionic lipids in the acceptor liposomes. DOPC/DNS-PE/DHE liposomes (87.5:2.5:10 mol/mol, 100 µM lipids) were mixed with DOPC liposomes (900 µM lipids) containing 2 mol% of the indicated lipid. After 3 min, 0.5 µM Osh4p was added. FRET between DHE and DNS-PE in the donor liposomes diminishes as DHE is transported to the acceptor liposomes. The initial fluorescence signal was set at 100%. The slow decay observed without Osh4p is caused by spontaneous DHE transfer between the liposomes. (A, right) Initial transport rates. (B) Osh4p WT or mutants (0.5 µM) were added to DOPC/DNS-PE/DHE liposomes (87.5:2.5:10 mol/mol, 100 µM lipids) mixed with DOPC liposomes (900 µM lipids) containing 0, 1, or 2% PI(4)P. The initial transport rates are indicated on a linear scale for each mutant (black bars, 0% PI(4)P; dark red bars, 1% PI(4)P; red bars, 2% PI(4)P). The broken lines correspond to the value of the initial transport rate measured for the wild-type Osh4p with acceptor liposomes containing 1% or 2% PI(4)P. Data are represented as mean ± SEM (error bars; n = 2). (C) Transport was measured by mixing 0.5 µM Osh4p with DOPC/DNS-PE/DHE liposomes (87.5:2.5:10 mol/mol, 100 µM lipids) and acceptor liposomes (900 µM lipids) of different compositions (DOPC, DOPC/DOPS or POPC/POPS 70:30 mol/mol) with 2% PI(4)P (red traces) or without PI(4)P (black traces). (C, right) Initial transport rate. Black bars, without PI(4)P; red bars, with 2% PI(4)P. Experiments in A and C were completed once but include common conditions.

Figure 7.

Osh4p transfers PI(4)P between membranes. (A) 0.5 µM Osh4p was mixed at 30°C with DOPC liposomes containing 6% PI(4)P (liposome A1, 15 µM) and DOPC liposomes doped with 2.5% DNS-PE (A2, 75 µM). In a second step, DOPC liposomes containing 18% DHE were added (donor liposome, 90 µM). Control experiments were done with A1 and A2 liposomes containing either 0 or 1% PI(4)P (black and dark red trace, respectively) or without Osh4p (gray trace). (B) Sucrose-loaded DOPC/DHE liposomes (98:2 mol/mol, 0.5 mM lipids, labeled with 0.1% mol NBD-PE) were incubated with DOPC/PI(4)P liposomes doped with [³²P]PI(4)P (98:2 mol/mol, 0.5 mM lipids, labeled with 0.1 mol% Rho-PE). After centrifugation on a sucrose gradient, a bottom and top fraction were collected. The fluorescence of NBD-PE, Rho-PE, and DHE was measured and PI(4)P radioactivity was counted for each fraction. (B, bottom) The relative amount of donor and acceptor liposomes in each fraction. (B, top) The gain or loss of DHE and PI(4)P (in percentages) for each liposome population. Data are represented as mean ± SEM (n = 2).

Figure 8.

Model for the coupling between sterol and PI(4)P exchange by Osh4p. Osh4p solubilizes sterols efficiently from neutral and loosely packed membranes. The sterol is locked by the lid. The interaction of Osh4p with anionic membranes promotes the opening of the lid and sterol release. Only PI(4)P promotes net sterol delivery because PI(4)P is extracted by Osh4p at the expense of sterol re-extraction. Tight lipid packing in the acceptor membrane limits the retention of Osh4p through its ALPS motif. These observation suggest that Osh4p is suited to transport sterols preferentially from ER to the trans Golgi marked by PI(4)P. Conversely, Osh4p transports PI(4)P from the trans Golgi to the ER, and another round of transport can resume. Although each step of the cycle is reversible by itself, the presence of a PI-4 kinase at the TGN (Pik1p) and of a PI(4)P phosphatase at the ER (Sac1p) favors directionality of lipid transport. The sterol/PI(4)P exchange activity of Osh4p is also coupled to that of Sec14p, a PC/PI exchanger.