ORP5 localizes to ER-lipid droplet contacts and regulates the level of PI(4)P on lipid droplets

Abstract

Lipid droplets (LDs) are evolutionarily conserved organelles that play important roles in cellular metabolism. Each LD is enclosed by a monolayer of phospholipids, distinct from bilayer membranes. During LD biogenesis and growth, this monolayer of lipids expands by acquiring phospholipids from the endoplasmic reticulum (ER) through nonvesicular mechanisms. Here, in a mini-screen, we find that ORP5, an integral membrane protein of the ER, can localize to ER-LD contact sites upon oleate loading. ORP5 interacts with LDs through its ligand-binding domain, and ORP5 deficiency enhances neutral lipid synthesis and increases the size of LDs. Importantly, there is significantly more phosphatidylinositol-4-phosphate (PI(4)P) and less phosphatidylserine (PS) on LDs in ORP5-deficient cells than in normal cells. The increased presence of PI(4)P on LDs in ORP5-deficient cells requires phosphatidylinositol 4-kinase 2-α. Our results thus demonstrate the existence of PI(4)P on LDs and suggest that LD-associated PI(4)P may be primarily used by ORP5 to deliver PS to LDs.

Figure S1.

Localization of mCherry-ORPs in HeLa cells. (A) HeLa cells were transfected with mCherry-tagged ORPs (ORP1L-ORP11) for 24 h, then treated with oleate for 16 h. Bars = 10 µm (insets, 2.5 µm). (B) Percentage of mCherry- or GFP-ORPs transfected cells that show LD localizations (n = 10–27).

Figure S2.

GFP-ORP5 targets to LDs in different mammalian cell lines. HEK-293, Cos-7, Huh7, PH5CH8, SHEP, and HeLa cells were transfected with GFP-ORP5 or GFP-ORPB for 24 h, then treated with oleate for 16 h. Bars = 10 µm.

Figure S3.

Characterization of LD targeting of ORP5 and the role of the AH. (A) Huh7 cells were transfected with GFP-ORP2 and mCherry-ORP5 for 24 h and treated with oleate in the absence or presence of 22R-hydroxycholesterol (5 µM) for 16 h. Bar = 10 µm (inlay, 2.5 µm). (B) HeLa cells were transfected with GFP-ORP5 for 24 h and treated with oleate in the absence or presence of 25-hydroxycholesterol (1 µM) or itraconazole (ITZ, 10 µM) for 16 h. Bars = 10 µm (inlay, 2.5 µm). (C) Helical wheel representation of the AH within ORP8ORD generated at HeliQuest. (D) HeLa cells were transfected with GFP-ORP8 for 24 h and treated with oleate in the for 16 h. Bar = 10 µm (inset, 2.5 µm). (E) Helical wheel representation of ORP8AH substituted with ORP5AH generated at HeliQuest. (F) HeLa cells were transfected with GFP-ORP8 containing ORP5AH for 24 h and treated with oleate for 16 h. Bar = 10 µm (inset, 2.5 µm).

Figure 1.

ORP5 localizes to LDs. (A) Diagrams of domain structures for ORP5 and ORP8. (B) Confocal images of mCherry-ORP5/8 localizations in HeLa cells treated with oleate for 16 h. LDs were stained with BODIPY. Bars = 10 µm (insets, 2.5 µm). (C and D) Confocal images of mCherry-ORP5 or mCherryORP8 localizations in oleate-treated cells transfected with either the ER–PM junction marker MAPPER or the ER marker GFP-Sec61β. LDs were stained with LipiTox DeepRed. Bars = 10 µm (insets, 2.5 µm). (E) Percentage of the cells expressing mCherry-tagged ORP5 or ORP8 that show LD localization. (F) GFP signal recovery after photobleaching in GFP-ORP5–expressing HeLa cells treated with oleate for 16 h. LDs were stained with LipidTox DeepRed. Bar = 10 µm (inlay, 1 µm). (G) Time-dependent recovery of GFP signal in F (n = 2). Mean ± SD. (H) 3D reconstruction of LDs enveloped by GFP-ORP5. Bar = 1 µm. All data are representative of at least three independent experiments with similar results.

Figure 2.

ORP5 localizes to ER–LD contacts. (A) Principle of APEX-EM. (B) APEX-EM images of oleate-treated cells expressing GFP-ORP5 or GFP-ORP8 together with APEX2-GBP. Arrowheads indicate GFP-ORP5 patch at ER–PM contact sites. N, nucleus. Bars = 10 µm (magnifications, 2.5 µm). (C) Cartoon of ORP5 localization at ER–PM and ER–LD contact sites. (D) AMEX-EM images of GFP-ORP5 and GFP-ORP5B localization at ER–LD contact sites. Arrows indicate ER. Bars = 0.5 µm (magnifications, 0.25 µm). (E) Airyscan confocal image of GFP-ORP5B/DsRed-ER–expressing HeLa cells treated with oleate for 16 h. LDs were stained with LipidTox DeepRed. Arrowheads indicate GFP-ORP5B association with ER and LDs. Bar = 10 µm (inlay, 2.5 µm). (F) Western blot analysis of ORP5 in HeLa cells treated with control or ORP5 specific siRNAs. (G) Immunofluorescence of endogenous ORP5 in siRNA transfected cells treated with oleate for 16 h. LDs were stained with BODIPY. Arrowheads indicate ORP5 association with LDs. Bars = 10 µm (inlay, 2.5 µm). All data are representative of at least three independent experiments with similar results.

Figure 3.

The ORD of ORP5 is required for its LD targeting. (A) Constructs of GFP-ORP5 and its variants with domain deletions. (B) Confocal images showing the localizations of GFP-ORP5 and variants with domain deletion in cells cotransfected with the ER marker DsRed-ER and treated with oleate for 16 h. LDs were stained with LipidTox DeepRed. Bars = 10 µm (insets, 2.5 µm). (C) Percentage of the cells expressing GFP-ORP5 and variants with domain deletion that show LD localizations. (D) LD fractionation in cells expressing GFP-ADRP, GFP-ORP5, GFP-ORP5B, and GFP-ORP5ΔORD. IB, immunoblot. (E) Relative fractions of LD-associated GFP-ADRP, ORP5, ORP5B, and ORP5ΔPHΔORD (LD/Pellet) in D. Mean ± SD. All data are representative of at least three independent experiments with similar results.

Figure 4.

The AH of ORP5-ORD and LD targeting of ORP5. (A) Helical wheel representation of one of the amphipathic helices (aa 422–439) within ORP5-ORD generated at HeliQuest (http://heliquest.ipmc.cnrs.fr/). (B) Point mutations disrupting the amphipathic character of the helix within ORP5-ORD. (C and D) Confocal images showing the localizations of GFP-ORP5 or GFP-ORP5B with the AH deleted or disrupted in cells cotransfected with the ER marker DsRed-ER and treated with oleate for 16 h. LDs were stained with LipidTox DeepRed. Bars = 10 µm (insets, 2.5 µm). (E) Percentage of the cells expressing GFP-ORP5 or GFP-ORP5B with the AH deleted or disrupted that show LD localizations. (F) Construct of GFP-tagged ORP5AH fused with the transmembrane helix (TM) of ORP5. (G) Confocal images showing the localizations of GFP empty vector (EV) and GFP-ORP5AH shown in F in cells treated with oleate for 16 h. LDs were stained with LipidTox DeepRed. Bars = 10 µm (insets, 2.5 µm). (H) Percentage of cells expressing GFP EV or GFP-ORP5AH that show LD localizations. All data are representative of at least three independent experiments with similar results.

Figure 5.

ORP5 regulates LD size. (A) Western blot analysis of ORP5 in cells treated with control or two different ORP5 siRNAs. (B) Confocal images of oleate-treated cells transfected with siRNAs as in A and stained with BODIPY fluorescent dye. Bars = 10 µm (insets, 5 µm). (C) Relative population of LD sizes from the results in B (∼2,000 LDs from 10–20 cells). (D) Percentage of LDs with different diameters (<1, 1 to ∼2, and >2 µm) in control and ORP5 knockdown cells shown in B. Mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Each dot represents one cell (n = 13–25). (E) Western blot analysis of ORP5 in HeLa WT and ORP5 KO cells generated by the CRISPR/Cas9 system. (F) Confocal images of oleate-treated HeLa WT and ORP5 KO cells stained with BODIPY. Bars = 10 µm (insets, 5 µm). (G) Relative population of LD sizes from the results in E (∼2,000 LDs from 10–20 cells). (H) Percentage of LDs with different diameters (<1, 1 to ∼2, and >2 µm) in HeLa WT and ORP5 KO cells. Mean ± SD. ****, P < 0.0001. Each dot represents one cell (n = 21–23). (I) Confocal images of oleate-treated HeLa WT and ORP5 KO cells expressing empty vector (EV) or GFP-ORP5. LDs were stained with LipidTox DeepRed. Bars = 10 µm (insets, 5 µm). (J) Sizes of the largest LDs in transfected cells in G. Mean ± SD. ****, P < 0.0001, n = 15–20. (K) Percentage of LDs with different diameters (<1, 1 to ∼2, and >2 µm) in HeLa WT and ORP5 KO cells. Mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Each dot represents one cell (n = 13–14). (L) Confocal images of oleate-treated ORP5KO cells expressing GFP-ORP5 or the mutants deficient for PI4P (H478A/H479A) or PS (L389D) transport. LDs were stained with LipidTox DeepRed. Bars = 10 µm (insets, 5 µm). (M) Sizes of the largest LDs in transfected cells in L. Mean ± SD. ****, P < 0.0001, n = 15–20. (N) Percentage of LDs with different diameters (<1, 1 to ∼2, and >2 µm) in ORP5 KO cells expressing GFP-ORP5 WT or mutants. Mean ± SD. *, P < 0.05; ****, P < 0.0001. Each dot represents one cell (n = 13–15). (O) Percentage of transfected ORP5 KO cells expressing GFP-ORP5 WT or mutants that show LD localizations. All data are representative of at least three independent experiments with similar results.

Figure S4.

Role of ORP5AH in LD size regulation and the effect of ORP5 deficiency on early LD formation. (A) Confocal images of oleate-treated ORP5KO cells expressing GFP-ORP5 or the AH mutant, GFP-ORP5 (MVL/RRR). Arrowheads indicate transfected cells. Bars = 10 µm (insets, 2.5 µm). (B) Sizes of the largest LDs in transfected cells in A. Mean ± SD. ****, P < 0.0001; n = 15–20. (C) Percentage of LDs with different diameters (<1, 1 to ∼2, and >2 µm) in oleate-treated ORP5KO cells expressing GFP-ORP5 or the AH mutant, GFP-ORP5 (MVL/RRR). Mean ± SD. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001; n = 20–23. (D) WT HeLa or ORP5 KO cells were transfected with GFP-Hpos for 24 h, starved in serum-free medium overnight, then chased with oleate for 1, 5, 10, 15, 30, and 60 min. LDs were stained with LipidTox DeepRed. Arrowheads indicate LDs. Bars = 10 µm.

Figure S5.

Effect of ORP5 deficiency on TAG synthesis, localizations of DGAT1/2 and PI4K2A, and cholesterol distribution. (A) Relative TAG content in HeLa WT and ORP5 KO cells. Cells were treated with oleate for 4 h, and neutral lipids were extracted with hexane and isopropanol, resolved by TLC, and stained with iodine. Densitometry was used to determine the relative TAG content. Mean ± SD. **, P < 0.01, n = 4. Data are representative of three independent experiments with similar results. (B) TAG content in HeLa WT and ORP5 KO cells treated as in A was quantified using a fluorometric assay kit. Mean ± SD. ***, P < 0.001, n = 6. Data are representative of two independent experiments with similar results. (C) HeLa cells were transfected with GFP-DGAT1 for 24 h and treated with oleate for 4 h. Bars = 10 µm (inlay, 2.5 µm). (D) Percentage of transfected HeLa WT and ORP5 KO cells expressing GFP-DGAT1 that show LD localizations. (E) HeLa cells were transfected with GFP-DGAT2 for 24 h and treated with oleate for 4 h. Bars = 10 µm (inlay, 2.5 µm). (F) Percentage of transfected HeLa WT and ORP5 KO cells expressing GFP-DGAT2 that show LD localizations. (G) WT HeLa or ORP5 KO cells were transfected with GFP-PI4K2A and DsRed-ER for 24 h. Bars = 10 µm (insets, 2.5 µm). (H) WT HeLa or ORP5 KO cells were treated with oleate for 16 h, followed by filipin staining for free cholesterol and BODIPY staining for LDs. Bars = 10 µm (insets, 2.5 µm).

Figure 6.

ORP5 controls the amount of PI(4)P and PS on LDs. (A) Immunofluorescence of PI(4)P and PI(4,5)P₂ in oleate-treated HeLa WT and ORP5 KO cells. LDs were stained with BODIPY. Bars = 10 µm (inlay, 2.5 µm). (B) Quantitation of LDs per cell associated with PI(4)P or PI(4,5)P₂ puncta. Mean ± SD. ****, P < 0.0001, n = 14–15. (C) Confocal images of oleate-treated HeLa WT and ORP5 KO cells expressing the PI(4)P sensor GFP-P4M and the PI(4,5)P₂ sensor GFP-PLCPH. LDs were stained with LipidTox DeepRed. Bars = 10 µm (inlay, 2.5 µm). (D) Quantitation of GFP-P4M or GFP-PLCPH intensities on the surface of LDs in C. ****, P < 0.0001, n = 37–38. (E) Confocal images of oleate treated WT HeLa and ORP5KO cells expressing the PS sensor GFP-evt2PH. LDs were stained with LipidTox DeepRed. Bars = 10 µm (inlay, 2.5 µm). Mean ± SD. (F) Quantitation of LDs associated with the PS sensor in G. Mean ± SD. ****, P < 0.0001, n = 15–20. All data are representative of at least three independent experiments with similar results.

Figure 7.

PI4K2A controls PI(4)P association with LDs. (A) Overexpression of mCherry-PI4K2A or -PI4K2A(K152A) together with the PI(4)P probe Sidc-GFP in oleate-treated cells. LDs were stained with LipidTox DeepRed. Bars = 10 µm (inlay, 2 µm). (B) Percentage of transfected cells with PI(4)P on LDs in A. (C) Airyscan confocal images of HeLa WT and ORP5 KO cells expressing GFP-PI4K2A and treated with oleate for 16 h. LDs were stained with LipidTox DeepRed. Arrowheads indicate LD-associated PI4K2A. Bars = 10 µm. (D) RT-PCR analysis of PI4K2A expression in HeLa and ORP5 KO cells treated with PI4K2A siRNAs for 48 h (n = 3). Mean ± SD. (E) Western blot analysis of HeLa WT and ORP5 KO cells treated with control or PI4K2A siRNAs for 48 h. (F) Confocal images of oleate-treated HeLa and ORP5KO cells expressing the PI4P sensor GFP-P4M. Cells were treated with siRNAs for 72 h. LDs were stained with LipidTox DeepRed. Bars = 10 µm (inlay, 2.5 µm). (G) Quantitation of GFP-P4M intensities surrounding LDs in F. Mean ± SD. ****, P < 0.0001, n > 51–52. (H) Confocal images of oleate-treated HeLa and ORP5KO cells transfected with siRNAs for 72 h. LDs were stained with BODIPY. Bars = 10 µm (inlay, 2.5 µm). (I) Percentage of LDs with diameters >2 µm in oleate-treated HeLa and ORP5KO cells transfected with siRNAs for 72 h. Mean ± SD. All data are representative of at least three independent experiments with similar results.