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. 2013 Aug;54(8):2174-2184.
doi: 10.1194/jlr.M037713. Epub 2013 Jun 3.

The LXR-IDOL axis defines a clathrin-, caveolae-, and dynamin-independent endocytic route for LDLR internalization and lysosomal degradation

Vincenzo Sorrentino  1 , Jessica K Nelson  1 , Elena Maspero  2 , André R A Marques  1 , Lilith Scheer  1 , Simona Polo  3 , Noam Zelcer  4
Affiliations

The LXR-IDOL axis defines a clathrin-, caveolae-, and dynamin-independent endocytic route for LDLR internalization and lysosomal degradation

Vincenzo Sorrentino et al. J Lipid Res. 2013 Aug.

Abstract

Low density lipoprotein (LDL) cholesterol is taken up into cells via clathrin-mediated endocytosis of the LDL receptor (LDLR). Following dissociation of the LDLR-LDL complex, LDL is directed to lysosomes whereas the LDLR recycles to the plasma membrane. Activation of the sterol-sensing nuclear receptors liver X receptors (LXRs) enhances degradation of the LDLR. This depends on the LXR target gene inducible degrader of the LDLR (IDOL), an E3-ubiquitin ligase that promotes ubiquitylation and lysosomal degradation of the LDLR. How ubiquitylation of the LDLR by IDOL controls its endocytic trafficking is currently unknown. Using genetic- and pharmacological-based approaches coupled to functional assessment of LDL uptake, we show that the LXR-IDOL axis targets a LDLR pool present in lipid rafts. IDOL-dependent internalization of the LDLR is independent of clathrin, caveolin, macroautophagy, and dynamin. Rather, it depends on the endocytic protein epsin. Consistent with LDLR ubiquitylation acting as a sorting signal, degradation of the receptor can be blocked by perturbing the endosomal sorting complex required for transport (ESCRT) or by USP8, a deubiquitylase implicated in sorting ubiquitylated cargo to multivesicular bodies. In summary, we provide evidence for the existence of an LXR-IDOL-mediated internalization pathway for the LDLR that is distinct from that used for lipoprotein uptake.

Keywords: E3-ubiquitin ligase; endocytosis; epsins; inducible degrader of low density lipoprotein receptor; lipoprotein receptors; liver X receptor; low density lipoprotein receptor.

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Figures

Fig. 1.
Fig. 1.
The LXR-IDOL axis targets the LDLR membrane pool for lysosomal degradation. A: A431 cells were cultured as indicated in the presence (+) or absence (−) of 2 μM of the synthetic LXR agonist GW3965 for 4 h and 100 nM bafilomycin A1 or 10 mM NH4Cl. LDLR at the cell surface was determined by FACS analysis. Cell surface LDLR in sterol-depletion medium was set to 100% (n = 6; *P < 0.01; n.s, not significant). B, C: Total cell lysates were immunoblotted as indicated. Immunoblots are representative of three independent experiments.
Fig. 2.
Fig. 2.
IDOL-mediated degradation of the LDLR is clathrin independent. A: Inducible CHC knockdown HeLa cells were cultured in the presence (+) or absence (−) of doxycycline for 6 days to induce silencing of the CHC. Subsequently, cells were infected with a GFP- or IDOL-expressing adenovirus for 10 h at an MOI of 5. Total cell lysates were immunoblotted as indicated. B: Inducible CHC knockdown cells were cultured as in (A). Subsequently, cells were shifted to sterol-depletion medium for 16 h and then incubated for 30 min with 5 μg/ml DyLight 488-labeled LDL. After extensive washing, internalized LDL was quantified by measuring fluorescence in total cell lysates. LDL uptake in −Dox cells was set to 100%. Each bar and error represent the average ± SD (n = 8; *P < 0.01; DOX/dox, doxycycline). C: Inducible CHC knockdown cells were cultured as in (A) and then treated with rhodamine-transferrin (red) for 30 min. After fixation, cells were stained for clathrin (green), and counterstained with DAPI (blue). Note that in clathrin knockdown cells transferrin internalization is blocked, while in control cells transferrin is internalized and colocalized with clathrin. Immunoblots are representative of three independent experiments.
Fig. 3.
Fig. 3.
IDOL-mediated degradation of the LDLR does not require ARH. A: Human fibroblasts (parental) and their engineered derivatives in which WT LDLR or Y828C LDLR were stably introduced and cultured in the absence (−) or presence (+) of 1 μM GW3965 for 24 h. Total cell lysates were immunoblotted as indicated. B: A431 cells were transfected with 20 nM control or ARH siRNA. Subsequently, cells were shifted to sterol-depletion medium for 16 h and then treated for an additional 6 h with 2 μM GW3965. Total cell lysates were immunoblotted as indicated. Immunoblots are representative of three independent experiments.
Fig. 4.
Fig. 4.
Degradation of the LDLR by IDOL is independent of caveolin and macroautophagy. Cav1(−/−) (A) and Atg5(−/−) (B) mouse embryonic fibroblasts and their corresponding WT cells were incubated for 16 h in sterol-deficient medium with vehicle (−) or 1 μM GW3965 (+). Total cell lysates were immunoblotted as shown. Immunoblots are representative of three independent experiments.
Fig. 5.
Fig. 5.
IDOL-mediated LDLR degradation is dynamin independent. A, B: HepG2 cells were cultured for 16 h in sterol-depletion medium to stimulate LDLR expression. Subsequently, cells were treated with 1 μM GW3965 or vehicle in the presence (+) or absence (−) of 80 μM dynasore for 4 h. A: Cells were incubated for 30 min with 5 μg/ml DyLight 488-labeled LDL. LDL uptake in vehicle-treated cells was set to 100% (n = 4, *P < 0.01 from vehicle treated cells). B: Total cell lysates were immunoblotted as indicated. C: HeLa cells were transfected with 20 nM control or DNM1/2 siRNA and subsequently incubated for 16 h with sterol-depletion medium to induce LDLR expression. Cells were then treated for 6 h with vehicle or 1 μM GW3965. Immunoblots are representative of three independent experiments.
Fig. 6.
Fig. 6.
The LXR-IDOL degradation pathway targets a lipid-raft resident LDLR pool. HepG2 cells were cultured in FBS-containing medium and treated with vehicle (A) or 1 μM GW3965 for 4 h (B). Membrane fractions were isolated and an equal amount of protein per fraction was separated by SDS-PAGE and immunoblotted as indicated. Immunoblots are representative of two independent experiments with similar results. C: HepG2 and A431 cells were incubated for 16 h with sterol-depletion medium and subsequently pretreated with 1 μM GW3965 (GW; +) or vehicle (−) for 30 min followed by addition of 5 mg/ml MβCD for an additional 4 h. D: HepG2 cells were cultured as in (C) except that 10 μg/ml filipin was added for an additional 3 h. Total cell lysates were immunoblotted as indicated.
Fig. 7.
Fig. 7.
Epsin1 over-expression blocks IDOL-mediated degradation of the LDLR and leads to accumulation of ubiquitylated receptor. A: HEK293T cells were transfected with expression plasmids for LDLR, FLAG-IDOL, increasing amounts of Myc-rEpn1, and GFP to monitor transfection efficiency. Total cell lysates were immunoblotted as indicated. B: HEK293T cells were transfected with expression plasmids for LDLR, FLAG-IDOL, and Myc-rEpn1. Samples were immunoprecipitated and analyzed by immunoblotting as indicated. Immunoblots are representative of three independent experiments.
Fig. 8.
Fig. 8.
The ESCRT system is required for sorting of ubiquitylated LDLR. A: A431 cells were transfected with 20 nM of control or TSG101 siRNA and subsequently incubated for 16 h with sterol-depletion medium followed by treatment with vehicle (−) or 1 μM GW3965 (+) for 5 h. B: HEK293T cells were transfected with expression plasmids for LDLR, FLAG-IDOL, GFP-VPS4WT, GFP-VPS4E228Q, or GFP. C: HEK293T cells were transfected with expression plasmids for LDLR, Myc-IDOL, FLAG-AMSH, FLAG-USP8, and GFP to monitor for transfection efficiency. In (A–C) total cell lysates were analyzed by immunoblotting as indicated. Immunoblots are representative of three independent experiments.
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