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An Essential Role of Hrs/Vps27 in Endosomal Cholesterol Trafficking
Ximing Du,1 Abdulla S. Kazim,1 Andrew J. Brown,1 and Hongyuan Yang1*
1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, 2052, Australia 'Correspondence: h.rob.yang@unsw.edu.au DOI 10.1016/j.celrep.2011.10.004
Cell Reports
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SUMMARY
The endosomal sorting complex required for transport (ESCRT) plays a crucial role in the degradation of ubiquitinated endosomal membrane proteins. Here, we report that Hrs, a key protein of the ESCRT-0 complex, is required for the transport of low-density lipoprotein-derived cholesterol from en-dosomes to the endoplasmic reticulum. This function of Hrs in cholesterol transport is distinct from its previously defined role in lysosomal sorting and downregulation of membrane receptors via the ESCRT pathway. In line with this, knocking down other ESCRT proteins does not cause prominent endosomal cholesterol accumulation. Importantly, the localization and biochemical properties of key cholesterol-sorting proteins, NPC1 and NPC2, appear to be unchanged upon Hrs knockdown. Our data identify Hrs as a regulator of endosomal cholesterol trafficking and provide additional insights into the budding of intralumenal vesicles.
INTRODUCTION
Mammalian cells acquire exogenous cholesterol through receptor-mediated endocytosis of low-density lipoproteins (LDLs) (Brown and Goldstein, 1986). The endocytic pathway sorts and delivers LDL from early endosomes to late endo-somes/lysosomes (LE/Ly) for the hydrolysis of cholesteryl esters, and the released free cholesterol exits LE/Ly efficiently to reach the plasma membrane and/or the endoplasmic reticulum (ER) for structural and regulatory functions (Chang et al., 2006; Ikonen, 2008; Kristiana et al., 2008; Mesmin and Maxfield, 2009). Niemann Pick type C (NPC) 1 and NPC2 are two key proteins that regulate the exit of LDL-derived cholesterol (LDL-C) from LE/Ly. Recent elegant studies suggest that NPC2 (a soluble, cholesterol binding protein that resides in the lysosomal lumen) accepts and delivers LDL-C to the N-terminal domain of NPC1 (an LE/Ly membrane protein with 13 transmembrane domains and three large lumenal loops), which then inserts LDL-C directly into the lysosomal membrane for export (Kwon et al., 2009). Putative proteins on the cytoplasmic side of the endosomal limiting membrane may be required to transport LDL-C to other membranes (Kwon et al., 2009; Yang, 2006). We have recently identified ORP5, an oxysterol binding protein (OSBP)-related
protein, as a possible key component in the post-LE/Ly transport of LDL-C (Du et al., 2011). It is likely that other yet-to-be identified proteins may also work with ORP5 to remove cholesterol from the endosomes.
We have focused on components of the endosomal sorting complex required for transport (ESCRT) (Saksena et al., 2007), which mediates the degradation of ubiquitinated endosomal membrane proteins through the formation of intralumenal vesicles (ILVs) (Hurley, 2010; Hurley and Hanson, 2010; Raiborg and Stenmark, 2009; Williams and Urbe, 2007). ESCRT consists of a highly conserved set of four hetero-oligomeric protein complexes (ESCRT-0, -I, -II, and -III) comprising more than a dozen subunits. ESCRT-0 is believed to sequester ubiquitinated cargo and recruit other ESCRT complexes. Recent elegant in vitro studies demonstrate that ESCRT-I and II can drive membrane deformation to form the ILVs whereas ESCRT-III is important for the abscission of the forming ILVs (Wollert and Hurley, 2010; Wollert et al., 2009). The AAA ATPase VPS4/SKD1 regulates the disassembly and recycling of ESCRT-III components, enabling further rounds of cargo sorting. There have been a few lines of evidence linking ESCRT function and cholesterol trafficking. We and others have shown that VPS4 can interact with yeast ORPs and regulate cholesterol sorting in yeast and mammalian cells (Bishop and Woodman, 2000; Wang et al., 2005; Yang, 2006). Overexpression of hSnf7 and hVps20 has been reported to cause endosomal cholesterol accumulation (Peck et al., 2004). A close functional relationship between ORPs and ESCRT function has been revealed in Caenorhabditis elegans (Kobuna et al., 2010). Interestingly, a recent study demonstrated that ORP5 specifically bound to the ubiquitin-interacting motif of Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate, called VPS27 in yeast for vacuolar protein sorting 27) (Pridgeon et al., 2009). Hrs interacts with STAM (signal transducing adaptor molecule) to make up ESCRT-0, which plays a crucial role in initiating the ESCRT pathway (Bache et al., 2003b; Bilodeau et al., 2002). ESCRT-0 recruits ESCRT-I possibly through the direct interaction between Hrs and Tsg101(an ESCRT-I component that also binds ubiquitinated proteins), and hands the ubiquitinated cargo to ESCRT-I (Bache et al., 2003a; Bilodeau et al., 2003; Katzmann et al., 2003; Lu et al., 2003).
Here, we report that Hrs, but not other ESCRT-0, -I, -II, or -III subunits, is required for the transport of LDL-C from endosomes to the ER. Our data indicate that Hrs regulates endosomal cholesterol transport independent of NPC1 and NPC2, and therefore identify Hrs as a regulator of cholesterol trafficking.
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Figure 1. Hrs Knockdown Causes LDL-Derived Cholesterol Accumulation in HeLa Cells
(A) HeLa cells were transfected with two siRNAs targeting different regions of Hrs (siHrs.1, siHrs.2), sINPCI or a universal control siCtrl for 72 hr. Efficiency of knockdown was analyzed by immuno-blotting using polyclonal anti-NPC1, monoclonal anti-Hrs, or anti-actin antibodies.
(B) HeLa cells were transfected with indicated siRNAs for 72 hr. Cells were then fixed and stained by filipin for free cholesterol. Representative fluorescent images are shown.
(C) Filipin intensities of cells from (B) were measured using ImageJ and the values were normalized to those of control images (mean ± SD, n > 30).
(D) HeLa cells were transfected with siCtrl or siHrs for 54 hr, followed by incubation in medium A (FBS), medium B (LPDS), or medium B supplemented with 50 mg/ml of LDL (LPDS + LDL). Cells were then fixed and stained by filipin for free cholesterol.
(E) Filipin intensities of cells from (D) were measured using ImageJ and the values were normalized to those of control cells grown in FBS medium (mean ± SD, n > 30). Bars = 10 mm.
RESULTS AND DISCUSSION
Hrs Knockdown Causes Endosomal Accumulation of LDL-Derived Cholesterol
We have recently shown that ORP5 plays an important role in endosomal cholesterol transport (Du et al., 2011). The possible link between ORP5 and Hrs (Pridgeon et al., 2009) prompted us to investigate whether Hrs and other ESCRT components may regulate intracellular cholesterol transport and homeostasis. Strikingly, an NPC-like cholesterol accumulation phenotype was observed in HeLa cells when Hrs was depleted by either of the two distinct siRNA oligos (Figures 1A-1C). Both free and total cholesterol was increased upon Hrs knockdown (Figure S1A available online). The accumulation of cholesterol in Hrs knockdown cells is dependent on the presence of LDL (Figures 1D and 1E), indicating that Hrs may regulate the transport of LDL-C in HeLa cells. To test whether Hrs silencing causes the accumulation of LDL-C in LE/Ly, we investigated the colocaliza-tion between accumulated free cholesterol and the late endosomal marker, Lamp-1. Cholesterol mainly accumulated in Lamp-1-positive compartment in Hrs knockdown cells, similar to that in NPC1 knockdown cells (Figures S1B and S1C). Cholesterol in Hrs knockdown cells colocalized mainly with GFP-Rab7 and partially with GFP-Rab5 or GFP-EEA1 (Figures S1D and S1E), further indicating that Hrs depletion resulted in late endosomal accumulation of free cholesterol. Together, these data indicate that Hrs is specifically involved in endosomal transport of LDL-C in HeLa cells.
Hrs Has a Distinct Role in Endosomal Cholesterol Transport
To investigate whether other ESCRT components may also regulate cholesterol trafficking, HeLa cells were treated with
siRNAs targeting Hrs and selected ESCRT subunits: Hrs (ESCRT-0), Tsg101 (ESCRT-I), EAP20 (ESCRT-II), and CHMP6 (ESCRT-III) (Figure 2A). Among these ESCRT subunits, only Hrs appears to be involved in cholesterol transport, since the downregulation of other ESCRT subunits in HeLa cells did not cause any apparent cholesterol accumulation (Figure 2B). We carefully examined the effect of Tsg101 knockdown because Hrs and Tsg101 are closely related, both functionally and physically (Bache et al., 2003a; Lu et al., 2003). Tsg101 was efficiently depleted after siRNA transfection as indicated by immunoblotting (Figure 2C), and by the enlargement and/ or clustering of Lamp-1-positive compartments that indicates impaired Tsg101 function as previously described (Doyotte et al., 2005; Figure 2D). To further ensure that Tsg101 is effectively knocked down, we examined the trafficking of caveolin-1 (CAV1) tagged with mEGFP and mCherry at its C terminus (Hayer et al., 2010) in Hrs and Tsg101 single or double knockdown cells. Consistent with the previous report, most of LE/Ly compartments were red in control cells (Figure S2A), whereas both red and green LE/Ly compartments were observed in cells depleted of Hrs or Tsg101 due to defects in ILV targeting of CAV1 (Figures S2B and S2C). This phenotype became more severe in HeLa cells depleted of both Hrs and Tsg101 (Figure S2D, line profile). These results indicate that the function of Tsg101 is compromised by siRNA, but cholesterol transport is not, further supporting the distinct role of Hrs in cholesterol trafficking.
A recent study revealed a close relationship between sorting nexin 3 (SNX3) and Hrs (Pons et al., 2008). Interestingly, silencing of SNX3 in HeLa cells appeared to have no effect on cholesterol transport (Figures S2E and S2F). We also knocked down VPS26 (Figure S2G), a component of the retro-mer complex that associates with endosomes and mediates
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endosomes to trans-Golgi trafficking (Bonifacino and Hurley, 2008). Consistent with a previous report (Arighi et al., 2004), VPS26 depletion caused a dispersed distribution of cation-independent mannose 6-phosphate receptor (CI-MPR) in HeLa cells (Figure S2H). However, no detectable defects in cholesterol distribution upon VPS26 knockdown were observed in HeLa cells (Figure S2H). Taken together, these data indicate that Hrs has a unique role in endosomal cholesterol trafficking.
Hrs Is Required for LDL-C Transport to the ER
To monitor the arrival of LDL-C at the ER, HeLa cells depleted for Hrs were incubated with LDL, followed by examining the conversion of cholesterol to cholesteryl ester that is catalyzed by the ER-localized enzyme: ACAT (acyl coA cholesterol acyl trans-ferase). NPC1 knockdown in HeLa cells ablated LDL-induced cholesterol esterification (Figure 3A, lane 3). Similar to NPC1 knockdown, Hrs depletion also significantly decreased LDL-induced cholesterol esterification (Figure 3A, lanes 3 and 4 versus lane 2, Figure 3B), indicating a blockage of endosomal cholesterol transport to the ER. The lack of LDL-C in the ER as indicated by the cholesterol esterification assay was further confirmed by the sterol-regulatory element binding protein (SREBP)-2 processing assay. Cholesterol-loading in the ER
Figure 2. Hrs Is Specifically Involved in Intracellular Cholesterol Trafficking
(A) HeLa cells were transfected with siRNAs targeting Hrs (ESCRT-0), Tsg101 (ESCRT-I), EAP20 (ESCRT-II), or CHMP6 (ESCRT-III) for 72 hr. Efficiency of knockdown was analyzed by qRT-PCR (means ± SD, n = 3 replicate cultures).
(B) HeLa cells were transfected with the indicated siRNAs for 72 hr. Cells were then fixed and stained by filipin for free cholesterol. Representative fluorescent images are shown.
(C) HeLa cells were transfected with siHrs, siTsg101, or a universal control siCtrl for 72 hr. Efficiency of knockdown was analyzed by immu-noblotting using monoclonal anti-Hrs, anti-Tsg101, or anti-actin antibodies.
(D) HeLa cells were transfected with the indicated siRNAs for 72 hr, followed by processing for immunofluorescence staining with the monoclonal antibody to Lamp-1 and filipin staining for free cholesterol. Representative confocal images are shown. Bars = 10 mm.
blocks the transport of SREBP-2 precursor from the ER to the Golgi complex for proteolytic cleavage, inhibiting the formation of nuclear form of SREBP-2 (nSREBP-2) and inactivating genes involved in cholesterol uptake and synthesis (Goldstein et al., 2006). SREBP-2 processing was revealed by immunoblotting using an antibody (IgG-1D2) against both precursor and nSREBP-2 (Figure 3C). As expected, NPC1 silencing impeded LDL-C transport to the ER because the nuclear form of SREBP-2 was still produced in the presence of LDL (Figure 3C, lane 4 versus lane 2, and Figure 3D). Consistent with the results from cholesterol esterification assay (Figures 3A and 3B), Hrs knockdown also blocked LDL-C transport to the ER and abolished the inhibitory effect of LDL on SREBP-2 processing (Figure 3C, lane 6 versus lane 2, and Figure 3D). This effect was further tested using qRT-PCR to examine the expression of two SREBP-dependent genes: LDL receptor (LDLR) and 3-hydroxy-3-methyglutaryl-CoA reductase (HMGR). Under control conditions, LDL treatment almost completely shut down LDLR or HMGR expression (Figure 3E); however, when NPC1 or Hrs was depleted, the inhibitory effect of LDL on LDLR or HMGR expression was significantly reduced (Figure 3E). Collectively, these data demonstrate a deficiency of LDL-C transport to the ER when Hrs is depleted, further supporting that Hrs is required for this process.
NPC1 and NPC2 Still Localize to LE/Ly upon Hrs Depletion
NPC1 and NPC2 work in concert to deliver LDL-C out of LE/Ly membranes. Hrs knockdown may interfere with the level and localization of NPC1 or NPC2, thereby indirectly causing cholesterol accumulation in LE/Ly compartments. However, the levels of NPC1 and NPC2 were not compromised upon Hrs depletion
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Figure 3. Hrs Is Required for LDL-Derived Cholesterol Transport to the Endoplasmic Reticulum
(A) HeLa cells grown In medium A were transfected with siNPCI, sIHrs, or a universal control siCtrl for 48 hr, followed by incubation in medium Bfor 18 hr. Cells were then chased with LDL (50 mg/ml) and [14C]-palmitate in medium B for 7 hr. Lipids extracted from cell lysates were standardized for total cell proteins and separated by TLC. Cholesteryl [14C]-esters were revealed by phosphor-imaging. A representative phosphorimage of three experiments with similar results is shown.
(B) Quantification of cholesteryl [14C]-esters formed in (A) by densitometry. Values of siCtrl + LDL were arbitrarily set as 100, against which experimental data were normalized. The results are expressed as means ± SD from three independent experiments.
(C) HeLa cells grown in medium A were transfected with siNPCI, siHrs, or a universal control siCtrl for 48 hr, followed by incubation in medium Bfor 18 hr. Cells were then treated with LDL (50 mg/ml) in medium B for 6 hr. Whole cell lysates were subjected to SDS-PAGE and immuno-blotting with a monoclonal antibody (IgG-1D2) against both precursor (P) and nuclear forms (N) of SREBP-2. The membrane was stripped and reblotted with antibodies to NPC1, Hrs, and actin.
(D) Relative intensity of the N band relative to the total (N+P bands) in (C) was quantified by densitometry and presented relative to none LDL-treated control in each siRNA treatment. The results are expressed as means ± SD from three independent experiments.
(E) qRT-PCR analysis of cells with the same treatment in (C). mRNA levels for LDL-receptor (LDLR) or HMG-CoA reductase (HMGR) were measured and normalized to actin mRNA levels. Data are presented relative to no LDL-treated control in each siRNA treatment and are means ± SD (n = 3 replicate cultures).
(Figure S3). In fact, whereas NPC2 appeared to be unchanged (Figures S3A and S3B), NPC1 was upregulated in Hrs knockdown cells in a time-dependent manner (Figures S3C and S3D). NPC1 or NPC2 may be mislocalized upon Hrs silencing, which could lead to cholesterol accumulation in LE/Ly. To examine this possibility, NPC1 localization was investigated by immunofluorescence in cells treated with control, Hrs or NPC1 siRNAs. In control cells, NPC1 almost completely colocalized with the LE/Ly marker, Lamp-1 (Figure 4A). Depletion of either NPC1 or Hrs by siRNA led to the accumulation of free cholesterol in Lamp-1-positive compartments (Figures 4B and 4C). In Hrs knockdown cells, NPC1 clearly overlapped with Lamp-1-posi-tive structures, which encircled the accumulated cholesterol (Figure 4C, inset). These observations demonstrate that NPC1 is not mislocalized upon Hrs depletion.
To examine NPC2 localization, HeLa cells treated with control siRNA or siRNAs against NPC1, NPC2, or Hrs were immunola-beled for endogenous NPC1 and NPC2, and stained with filipin for free cholesterol. In control cells, NPC1 and NPC2 colocalized to the same compartments (Figure 4D), which were shown to be Lamp-1-positive structures (Figures 4A-4C). Cholesterol accumulation in LE/Ly caused by NPC1 depletion overlapped with NPC2, also confirming the cellular localization of NPC2 (Figure 4E). This was also the case for NPC1, which overlapped
with the accumulated cholesterol caused by NPC2 depletion (Figure 4F). Importantly, in Hrs-depleted cells, NPC1, NPC2 and also cholesterol clearly colocalized (Figure 4G, inset). These microscopic data reveal that Hrs knockdown causes cholesterol accumulation in LE/Ly without affecting the localization of NPC1 and NPC2.
Hrs and Cholesterol Sorting
Here, we identify Hrs, but not other ESCRT components, as a regulator of endosomal cholesterol trafficking. Compared with other ESCRT components, Hrs is unique in at least two aspects: (1) Hrs functions at the very beginning of the ESCRT pathway, and therefore may have a critical role in cargo sorting and the initiation of intralumenal vesicle (ILV) budding (Babst, 2011; Hurley et al., 2010). (2) Hrs has multiple interacting partners that include many non-ESCRT components and has been implicated in a number of trafficking/sorting events. Besides STAM, clathrin and Tsg101, Hrs has also been reported to interact with SNX1 and Vps35, components of the retromer complex (Popoff et al., 2009). Moreover, Hrs, but none of the other ESCRT components, is required for the recycling of plasma membrane G protein coupled receptors (Hanyaloglu et al., 2005). Together, it seems that the ability of Hrs to interact with multiple partners forms the basis for its multiple functions. Therefore, it is not entirely surprising
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that Hrs, but not other ESCRT components, is required for the proper delivery of LDL-C.
Because of its well-established role in endosomal protein sorting, one way Hrs may regulate cholesterol transport is through controlling the level/degradation and localization of two endosomal proteins: NPC1 and NPC2. However, NPC1 and NPC2 both appear to be correctly localized upon Hrs knockdown (Figure 4).
Figure 4. NPC1 and NPC2 Localize to Lamp-1 Positive Compartments in Hrs Knockdown Cells
HeLa cells were transfected with siCtrl (A and D), siNPCI (B and E), siHrs (C and G), or siNPC2 (F) for 72 hr, followed by processing for immunofluorescence staining with antibodies to Lamp-1 or NPC1 (A-C), NPC1 or NPC2 (D-G), and filipin staining (A-G) for free cholesterol. Representative confocal images are shown. Bars = 10 mm.
The level of NPC1 is upregulated by ~50% upon Hrs depletion, whereas NPC2 is unchanged. These results indicate that Hrs may regulate the efflux of cholesterol from endosomes through other mechanisms. Hrs may recruit putative lipid carriers in the cytoplasm, and facilitate cholesterol removal from the limiting membrane. In support of such a hypothesis, a genome-wide screen of Hrs-interacting proteins identified ORP5 (Pridgeon et al., 2009), which has recently been shown to mediate endosomal cholesterol transport (Du et al., 2011). We are currently examining the detailed physical and functional relationship between ORP5, Hrs, and NPC1. Additionally, disturbed ILV formation upon Hrs knockdown may further interrupt the efflux of LDL cholesterol from LE/Ly as ILVs may play a role in the storage and trafficking of lumenal cholesterol. It is likely that the striking cholesterol accumulation phenotype upon Hrs depletion results from its combined effect on ILV formation, and on the recruitment of cytoplasmic sterol carriers. In summary, although further work is needed to elucidate the interactions between NPC1, ORP5, and Hrs, it is clear that Hrs can regulate endosomal cholesterol sorting. Hrs, Cholesterol, and Intralumenal Budding
The ESCRT pathway is characterized by the formation of ILVs through a unique mechanism, i.e., membrane budding away from the cytoplasm. An important unanswered question is what triggers the initial membrane deformation in this budding event, although both lipids and ESCRT proteins have been implicated (Babst, 2011; Hurley et al., 2010). Given the fact that most coatless budding mechanisms rely on membrane microdomains, it has been hypothesized that the ESCRT-mediated budding could involve cholesterol-rich domains, which may initiate the budding (Hurley et al., 2010). However, little is known about how ESCRTs may regulate the formation of such microdomains, which could form spontaneously as recently proposed (Babst, 2011).
Here, a function of Hrs in endosomal cholesterol transport is revealed. Hrs may initiate the ESCRT pathway by orchestrating the formation of putative cholesterol rich microdomains that
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help drive the "budding away'' from cytosol. In one hypothetical scenario, Hrs may selectively facilitate the removal of cholesterol from specific regions of the limiting membrane, thereby generating a cholesterol rich neighboring region that forms the basis of membrane deformation, which initiates the ESCRT pathway. The potential role of Hrs in generating microdomains on the endosomes may have broader implications. For instance, the recycling of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, is an active process mediated by distinct endosomal subdomains that are stabilized by a highly localized but dynamic actin machinery (Puthenveedu et al., 2010). The fact that knocking down Hrs, but none of the other ESCRT components, can significantly impair this recycling pathway suggests that cholesterol-rich microdomains generated by Hrs may also be involved in the formation of this actin-stabilized subdomain (Hanyaloglu et al., 2005).
In summary, our results uncover a role of Hrs in cholesterol sorting and indicate that cholesterol efflux from endosomal compartments and the formation of ILVs may be coregulated by the multifunctional protein: Hrs.
EXPERIMENTAL PROCEDURES Cell Culture and Transfection
HeLa cells were purchased from the American Type Culture Collection (ATCC HTB-22; Rockville, MD). Monolayers of cells were maintained in specified medium supplemented with serum (medium A: 10% FBS; medium B: 5% LPDS), 100 units/ml penicillin, and 100 mg/ml streptomycin sulfate in 5% CO2 at 37°C. DNA transfection was performed using Lipofectamine LTX and Plus Reagent (Invitrogen) according to manufacturer's instruction. For each transfection, 1-2 mg/well of plasmid cDNA were used in 6-well plates. siRNA transfection was carried out in cells grown in medium A at ~20% confluence according to standard methods using Lipofectamine RNAiMAX transfection reagent (Invitrogen). Forty picomoles of duplexes of siRNA were used for transfection of one well of cells grown in 6-well plate.
Filipin Staining and Fluorescence Microscopy
Cells grown on coverslips were fixed with 4% paraformaldehyde for 30 min at room temperature. Cells were stained with freshly prepared 50 mg/ml of filipin in PBS for 1 hr at room temperature. Stained cells were imaged using a Leica CTR5500 microscope (Wetzlar, Germany) equipped with an EL6000 fluorescent lamp and a DFC300 FX digital camera. Quantification of intracellular free cholesterol was carried out as previously described (Du et al., 2011).
Immunofluorescence and Confocal Microscopy
All immunofluorescence steps were performed at room temperature and cells were extensively rinsed with 3% BSA/PBS after each step. Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 15 min. For NPC2 staining, cells were fixed with Bouin's solution (Sigma-Aldrich) for 1 hr at room temperature. Cell permeabilization was carried out using 0.1% saponin/ PBS for 30 min, followed by blocking with 3% BSA/0.05% saponin in PBS for 1 hr. Incubation with primary antibodies and appropriate conjugated secondary antibodies were performed at room temperature for 1 hr. Cells were mounted in ProLong Gold antifade reagent (Invitrogen). Confocal images were acquired on an Olympus FV1000 laser-scanning microscope. The manufacturer's software and Adobe Photoshop CS4 were used for data acquisition. For colocalization analysis, Image-Pro Plus 6.0 software was used.
Cholesterol Esterification, SREBP-2 Processing, and Immunoblotting Analysis
Cholesterol esterification, SREBP-2 processing, and immunoblotting analysis were carried out as previously described (Du et al., 2006, 2011) and are detailed in Supplemental Experimental Procedures.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Extended Experimental Procedures and three figures and can be found with this article online at doi:10.1016/j.celrep. 2011.10.004.
LICENSING INFORMATION
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported License (CC-BY-NC-ND; http://creativecommons.org/licenses/by-nc-nd/3.0/ legalcode).
ACKNOWLEDGMENTS
This work is jointly supported by research grants from the Ara Parseghian Medical Research Foundation and the National Health and Medical Research Council of Australia (#510271). H.Y. is a Future Fellow of the Australian Research Council.
Received: August 11, 2011 Revised: September 27, 2011 Accepted: October 25, 2011 Published online: January 26, 2012
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