Traffic 2011; 12: 218–231 © 2010 John Wiley & Sons A/S

doi:10.1111/j.1600-0854.2010.01142.x

Role of ORPs in Sterol Transport from PlasmaMembrane to ER and Lipid Droplets in MammalianCells

Maurice Jansen1, Yuki Ohsaki1, Laura Rita

Rega2,3, Robert Bittman4, Vesa M. Olkkonen5

and Elina Ikonen1,∗

1Institute of Biomedicine, University of Helsinki, Finland2Telethon Institute of Genetics and Medicine (TIGEM),Naples, Italy3Department of Cell Biology and Oncology, ConsorzioMario Negri Sud, Santa Maria Imbaro, Chieti, Italy4Department of Chemistry and Biochemistry, QueensCollege of The City University of New York, Flushing, NY11367-1597, USA5Minerva Foundation Institute for Medical Research,Helsinki, Finland*Corresponding author: Elina Ikonen,elina.ikonen@helsinki.fi

In this study, we investigated the mechanisms of sterol

transport from the plasma membrane (PM) to the

endoplasmic reticulum (ER) and lipid droplets (LDs) in

HeLa cells. By overexpressing all mammalian oxysterol-

binding protein-related proteins (ORPs), we found that

especially ORP1S and ORP2 enhanced PM-to-LD sterol

transport. This reflected the stimulation of transport

from the PM to the ER, rather than from the ER to LDs.

Double knockdown of ORP1S and ORP2 inhibited sterol

transport from the PM to the ER and LDs, suggesting a

physiological role for these ORPs in the process. A two

phenylalanines in an acidic tract (FFAT) motif in ORPs

that mediates interaction with VAMP-associated proteins

(VAPs) in the ER was not necessary for the enhancement

of sterol transport by ORPs. However, VAP-A and VAP-B

silencing slowed down PM-to-LD sterol transport. This

was accompanied by enhanced degradation of ORP2

and decreased levels of several FFAT motif-containing

ORPs, suggesting a role for VAPs in sterol transport by

stabilization of ORPs.

Key words: BODIPY-cholesterol, cholesterol, FFAT motif,

lipid droplet, oxysterol-binding protein, sterol traffic,

VAMP-associated protein

Received 11 August 2010, revised and accepted for

publication 8 November 2010, uncorrected manuscript

published online 9 November 2010, published online 7

December 2010

Cholesterol is a key constituent in the plasma membrane(PM) of mammalian cells (1), where it is involved indiverse processes, such as regulation of membranepermeability, signal transduction and endocytosis. For the

proper functioning of these processes, tight control of themembrane cholesterol content is necessary. An importantmechanism by which cells regulate their membranecholesterol abundance is the deposition of excess sterolin lipid droplets (LDs). Cholesterol storage in LDs mainlyoccurs in the form of fatty acyl esters generated by acylcoenzyme A: cholesterol acyltransferase (ACAT) in theendoplasmic reticulum (ER) (2), and cholesterol targetedfor esterification moves from the PM to the ER (3). Inaddition, there is some free, i.e. unesterified cholesterolin LDs. This represents a major fraction of the storedsterol in adipocytes where LDs are abundant (4). On theother hand, the ER, where LDs are formed, is a sterolpoor organelle (1). Hence, the transfer of sterols betweenthe PM, ER and LDs is critical for controlling membranecholesterol content and cellular sterol homeostasis.

The mechanisms controlling the transport of cholesterolbetween the PM, ER and LDs in mammalian cells are notwell understood. Newly synthesized cholesterol can movefrom the ER to the PM largely via Golgi bypass route(s), asjudged by the minor inhibition of transport by brefeldin A(BFA) (5,6). Non-vesicular cholesterol transport pathwayscould be facilitated by cytosolic lipid-binding proteins (7,8).Moreover, the membrane lipid composition is important.Neutral sphingomyelinase (SMase) treatment induces PMcholesterol esterification, apparently by shunting sterolout of the PM (9,10). In lipid-loaded macrophages, thefluorescent sterol dehydroergosterol (DHE) was efficientlytransported from the PM to LDs in an ATP-independentmanner (11).

Studies in yeast have provided evidence that steroltransfer from the ER to the PM is non-vesicular (12) andfacilitated by the Osh family of lipid transport proteins (13).Sterol movement from the PM to the ER was also reportedto be non-vesicular (14) and requires Osh proteins (15).In a yeast mutant capable of aerobic sterol uptake andlacking all seven Osh proteins, esterification of PM-derivedcholesterol was significantly slowed down. The crystalstructure of Osh-4 revealed that a single sterol moleculebinds within a hydrophobic tunnel in a manner consistentwith a transport function for Osh4 (16). Indeed, all yeastORPs can transport sterols in vitro (17).

The mammalian homologs of Osh proteins are oxysterol-binding protein-related proteins (ORPs). Humans have12 ORP/OSBPL genes (18,19). Besides an OSBP-relateddomain (ORD) present in all ORPs and involved in lipidbinding, most members of the family contain a plextrinhomology (PH) domain and a two phenylalanines in anacidic tract (FFAT) motif. The FFAT motif interacts with

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VAMP-associated proteins (VAPs) in the ER (20) and wasshown to be required for efficient lipid trafficking, e.g.by ceramide transfer protein (CERT) (21). Whether ORPproteins are involved in sterol movement from the PM tothe ER or LDs in mammalian cells is not known.

In this study, we screened for the potential involve-ment of human ORPs in PM-to-LD sterol delivery using aBODIPY-labeled cholesterol analog (BODIPY-chol) (22) inORP-overexpressing cells. We compared the kinetics andORP dependence of the transfer route(s) and dissectedthe functionally relevant ORP domains involved. The phys-iological relevance of ORPs and VAPs in PM to ER andLD sterol delivery was studied by acute gene silencingand the key conclusions were confirmed by using radioac-tive cholesterol. The results provide the first evidence fora role of mammalian ORPs in sterol transport from thePM to the ER and LDs, and uncover a role for VAPs inmaintaining ORP levels.

Results

Kinetics of sterol trafficking from the PM to LDs

To analyze sterol trafficking from the PM to LDs, we devel-oped a live-cell microscopy assay employing the fluores-cent sterol BODIPY-chol. First, HeLa cells were prelabeledwith the fluorescent fatty acid analog BODIPY-C12 andchased for 2–6 h to visualize LDs. BODIPY-C12 is mostlyincorporated in triacyl- and diacylglycerides during thistime (23). Accordingly, we found that BODIPY-C12 colocal-izes extensively with the neutral lipid dye BODIPY-493/503(Figure S1). Next, the PM was pulse labeled with BODIPY-chol from a methyl-β-cyclodextrin (MβCD) complex. Label-ing of cells for 1 min at 37◦C resulted in the incorpo-ration of roughly 1 molecule of BODIPY-chol for every1000 molecules of unlabeled cholesterol, as assessed bythin-layer chromatography (TLC) analysis of cellular lipidextracts. After labeling, the cells were chased at 37◦Cwhile imaging with a confocal microscope. Within the firstfew minutes of after labeling, BODIPY-chol showed a clearPM localization (Figure 1A). After 10 min of chase, someof the BODIPY-chol colocalized with LDs. The LD BODIPY-chol content continued to increase until ∼1 h postlabelingafter which a steady state appeared to be reached.

To quantify the kinetics of BODIPY-chol transport fromthe PM to LDs, we determined the time-dependentcolocalization between BODIPY-chol and BODIPY-C12by calculating the Pearson’s correlation coefficient ofimages acquired during the chase. This shows thatBODIPY-chol reached LDs with a half-time value ofapproximately 30 min (Figure 1B). To study if BODIPY-chol could be detected in LDs biochemically, we labeledthe cells with BODIPY-chol, chased for 3 h and isolatedLDs using sucrose density gradient fractionation. Theobtained LD fraction was highly enriched in cholesterolesters, triacylglycerols and adipocyte differentiation-related protein (ADRP), as expected, and displayed no

calnexin or Na+K+-ATPase immunoreactivity, indicatinglow ER and PM contamination (Figure 1C). The LD fractionwas found to contain both free cholesterol and BODIPY-chol (Figures 1C and S2), in agreement with the presenceof free sterol in LDs. Of note, the fraction of BODIPY-cholin LDs was higher than that of free cholesterol (Figure S2),probably because of the affinity of the BODIPY group forLDs. No BODIPY-chol esters were detectable with thischase time (not shown).

To compare PM–LD transport of BODIPY-chol to that ofradiolabeled cholesterol, we pulse labeled the PM of cellswith [3H]cholesterol ([3H]chol) for 30 min at 14◦C. Afterdifferent chase times at 37◦C, LDs were isolated usingsucrose density fractionation as above. We found that thefraction of free [3H]chol in LDs increased with increasingchase time at 37◦C, reaching a steady state in ∼90 min.During this time, cholesterol esterification still continuedin a linear manner (Figure 1D). Thus, a fraction of free[3H]chol was rapidly transported from the PM to LDs,followed by a slower conversion of the sterol to esters fordeposition in LDs. The kinetics of free [3H]chol transportto LDs resembled that of BODIPY-chol (Figures 1D andS2). Together, these data indicate that BODIPY-chol canbe used as a tool to analyze the transport of unesterifiedsterol to LDs.

Effects of pharmacological treatments on sterol

transport from the PM to LDs

The transport of DHE from the PM to LDs was reported tobe ATP independent (11). We therefore analyzed the ATPdependence of BODIPY-chol PM–LD transport and foundthat ATP depletion did not alter the kinetics (Figure 2). Tofurther study the characteristics of BODIPY-chol PM–LDtransport, we tested its sensitivity to SMase or BFAtreatments. We found that SMase treatment significantlyincreased the colocalization of BODIPY-chol with the LDsmarker after 15–25 min of chase, but had no effect onthe degree of colocalization at steady state (180 min)(Figure 2). This suggests that SMase treatment does notincrease the overall amount of BODIPY-chol transportedto LDs but facilitates its transfer to LDs. Instead, BFA hadno significant effect on BODIPY-chol PM–LD transport(Figure 2). Thus, BODIPY-chol transport from the PM toLDs resembles the transport of DHE to LDs, taking placeunder ATP-depleting conditions. It also shares similaritieswith the movement of cholesterol between the PM andER, being sensitive to SMase (9,10) but rather insensitiveto BFA (6).

Screening of ORPs in sterol transport from the

PM to LDs

We next analyzed the effect of ORP overexpressionon cholesterol transport from the PM to LDs usingBODIPY-chol. All human ORP splice variants (exceptC-terminally truncated ORP3, devoid of most of the ORDdomain) (Figure 3A) containing an N-terminal Xpress tagwere transiently overexpressed in HeLa cells. Western

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Figure 1: Cholesterol trafficking from the PM to LDs. A) HeLa cells, prelabeled with BODIPY-C12 to stain LDs, were pulse labeledwith BODIPY-chol for 1 min. Confocal images were taken during the chase. White circles indicate colocalization. Scale bar: 15 μm.B) Pearson’s correlation of BODIPY-chol and BODIPY-C12 as a function of time. Combined data from three pulse-chase experiments,with each data point containing ∼15 cells. Gray areas indicate time windows used for quantification in later experiments. C) LDs wereisolated by sucrose density gradient fractionation (top fraction). Lipids were extracted and analyzed by TLC and proteins were analyzedby western blotting. D) Cells were pulse labeled with [3H]chol and chased for 0, 30, 90 or 270 min. LDs were isolated and [3H]chol LDcontent and [3H]chol esterification were determined (mean ± SEM; n = 2 or 3).

blotting showed that all of the proteins were expressedalbeit at varying levels (Figure 3B). To identify theORP-overexpressing cells by live-cell microscopy, theXpress-ORP constructs were co-overexpressed with anHcRed-encoding plasmid and confocal images were takenof fields containing at least 50% HcRed-expressingcells. Roughly 90% of the cells with detectable HcRedfluorescence also expressed the ORP construct, as judgedby immunofluorescence staining with α-Xpress antibodies

(Figure S3). Images were acquired at 15–25 min afterBODIPY-chol labeling of the cells, during which there wasa net transport of BODIPY-chol to LDs (Figure 1B). Wefound that overexpression of especially Xpress-ORP1Sand Xpress-ORP2 enhanced the transport of BODIPY-cholto LDs during this time (Figure 3C; p < 0.01 for bothORP1S and ORP2, see also Figure 4A). At 3 h of chase,there was no difference in the degree of colocalization ofBODIPY-chol and the LD marker (Figure 3C), suggesting

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Figure 2: Effects of chemical treatments on sterol transport

from the PM to LDs. A) Cells were prelabeled with BODIPY-C12

to stain LDs, and pulse labeled with BODIPY-chol. Cells weretreated with 5 μg/mL BFA or ATP depletion mix before and afterBODIPY-chol labeling, or with 25 mU/mL SMase only duringchase, as detailed in Materials and Methods. Confocal imagestaken after 15 min chase are shown. Scale bar: 15 μm. B) Barsindicate Pearson’s correlation of BODIPY-chol and BODIPY-C12

from images taken after 15–25 min or 180 min chase (mean ±SEM; n = 4 for 15–25 min and n > 2 for 180 min).

that Xpress-ORP1S and Xpress-ORP2 increased the rateat which BODIPY-chol was transported to LDs, not thetotal amount transported. Interestingly, although OSBP,ORP1L, ORP3, ORP5, ORP7 and ORP9S were expressedat levels roughly comparable to ORP1S and ORP2, theywere not as effective in stimulating BODIPY-chol transport(Figure 3C), suggesting selectivity between ORPs in theircapacity to facilitate PM–LD sterol delivery.

To analyze whether ORP1S and ORP2 are physiologicallyimportant for PM–LD sterol transfer, we silenced bothproteins using siRNAs and analyzed the effect on BODIPY-chol transport from the PM to LDs. We found that bothproteins were depleted by at least 80% (Figure S4) andthat this resulted in an ∼20% decrease in the colocalizationof BODIPY-chol with LDs after 15–25 min of chase(Figure 4B). Single knockdown of either ORP1S or ORP2did not significantly affect the transport (not shown).

Transport of fluorescent and radiolabeled sterols by

ORP1S and ORP2

To investigate whether the ORP2-dependent stimulationof PM–LD sterol delivery is limited to BODIPY-chol,we analyzed the effect of ORP2 overexpression on thePM–LD transport of DHE. For this purpose, we used Huh7cells because their large LD facilitated the visualization ofDHE in LDs. We found that also DHE transport fromthe PM to LDs was stimulated by ORP2 overexpression(Figure S5), indicating that the ORP2-mediated stimulationof PM–LD transport applies to this natural fluorescentsterol as well.

Furthermore, we analyzed the effect of ORP silencingor overexpression on the esterification of PM-derived[3H]chol, which was used as a measure for PM–ER steroldelivery. [3H]chol was inserted into the PM from a BSAcomplex at 15◦C and then chased at 37◦C for increasingtimes, after which the fraction of [3H]chol esterifiedwas analyzed. We found that ORP1 and ORP2 doubleknockdown reduced the fraction of [3H]chol esterifiedduring 15–30 min of chase (Figure 4C), i.e. the timewindow at which we observed a net fluorescent steroltransfer from the PM to the ER (Figure 6B). This resultsuggests that silencing of ORP1S and ORP2 inhibitsthe PM–ER transfer of cholesterol. By contrast, Xpress-ORP2 overexpression significantly increased the fractionof [3H]chol esters using the same assay (Figure 4D).One possible explanation for altered esterification is thatORP overexpression/knockdown affects ACAT activity.However, this was not the case, as the treatments didnot significantly alter ACAT activity (177 ± 7 pmol/mgprotein/min in control versus 183 ± 8 in ORP1 + ORP2siRNA-treated cells and 208 ± 16 in control plasmidtransfected versus 213 ± 4 in Xpress-ORP2-transfectedcells; n = 3 for each condition). Together, these resultssuggest that the transfer of [3H]chol from the PM to theER can be facilitated by ORP2 and that this process is inpart dependent on ORPs, as it can be inhibited by reducingORP1 and ORP2 levels.

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Figure 3: Screening of ORPs in sterol transport from the PM to LDs. A) Schematic drawing of ORP proteins used in this study.B) Cells, transiently overexpressing Xpress-ORPs, were analyzed by western blotting using α-Xpress antibodies. C) Cells, transientlyoverexpressing Xpress-ORPs and HcRed, were prelabeled with BODIPY-C12 and pulse labeled with BODIPY-chol. Confocal imageswere taken during the chase of BODIPY-chol to LDs. Bars indicate Pearson’s correlation of BODIPY-chol and BODIPY-C12 from imagestaken after 15–25 min or 180 min chase (mean ± SEM; n > 4).

To assess more directly whether ORP1S and ORP2 canfunction as cholesterol transporters, we studied choles-terol transfer in vitro using recombinant fusion proteins.We found that ORP1S significantly increased sterol trans-

fer between liposomes (Figure 4E). A similar tendencywas observed for ORP2. However, the transfer activity ofboth ORPs was substantially lower than that of MLN64START domain, which was used as a positive control.

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Figure 4: Legend on next page.

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Figure 5: Recovery of BODIPY-chol fluorescence in LDs after photobleaching. A) HeLa cells, prelabeled with BODIPY-C12 to stainLDs, were pulse labeled with BODIPY-chol and chased for more than 3 h. BODIPY-chol in LDs was photobleached and recovery wasmonitored. Scale bar: 5 μm. B) Quantification of BODIPY-chol FRAP in LDs with and without Xpress-ORP2 overexpression (mean ±SEM; n = 7).

Contribution of ORP2 domains in PM-to-LD sterol

transfer

We next analyzed the importance of two conserveddomains present in ORP2, the sterol-binding domain andthat FFAT motif, in PM–LD BODIPY-chol transport. Wefound that overexpression of an Xpress-ORP2 I249Wmutant (ORP2mORD) defective in sterol binding (24)was unable to stimulate BODIPY-chol transport toLDs (Figure 4F). On the other hand, overexpression ofan Xpress-ORP2 mutant with a defective FFAT motif(ORP2mFFAT) increased BODIPY-chol transport to LDssimilarly as wild-type Xpress-ORP2. Western blot analysisindicated that the expression level of ORP2mORD wassomewhat lower than that of wild-type Xpress-ORP2(Figure 4G). However, it seems unlikely that this relativelysmall reduction explains the total lack of BODIPY-choltransport activity by this mutant. Therefore, these resultsprovide evidence that an intact ORD domain capable ofsterol binding is essential for the ORP2 sterol transferactivity. Instead, an intact FFAT motif appears not to berequired.

Transport route of sterols moving from the PM to LDs

To obtain more insight into the exchange of BODIPY-chol between LDs and their surroundings, we usedfluorescence recovery after photobleaching (FRAP). Wefound that the half-time of BODIPY-chol fluorescencerecovery in LDs was very rapid, ≤1 min, and that thisspeed was unaffected by ORP2 overexpression (Figure 5).This implies that the majority of BODIPY-chol replenishedin LDs does not come directly from the PM, as the half-time for PM–LD transport is ∼30 min and the process isstimulated by ORP2. We considered that a likely candidatefor an intermediate station in PM–LD sterol delivery isthe ER. Indeed, we had observed that ORP2 stimulatedthe transfer of [3H]chol to the ER for esterification(Figure 4D).

We therefore set up a PM–ER sterol transport assay basedon imaging, where the PM is pulse labeled with BODIPY-chol, after which the time-dependent colocalization ofthe fluorescent sterol with an ER-tracker is determined,analogously to the PM–LD transport assay. Evidently, theextensive area occupied by the ER makes the detectionless reliable than in the case of LDs, and it is likely

Figure 4: ORP2 is involved in cholesterol transport from the PM to ER and LDs. A) Cells coexpressing ORP2 and HcRed wereprelabeled with BODIPY-C12 and pulse labeled with BODIPY-chol. Confocal images were taken after 10 min chase. HcRed (ORP2)-expressing cells display increased BODIPY-chol LD labeling. Scale bar: 15 μm. B) Cells, in which ORP1 and ORP2 were silenced, werepulse labeled with BODIPY-chol. Bars indicate Pearson’s correlation of BODIPY-chol and BODIPY-C12 after 15–25 min chase. (mean ±SEM; n = 8; *p < 0.02). C) Cells, in which ORP1 and ORP2 were silenced, were pulse labeled with [3H]chol. After chasing for differenttimes, the amount of esterified [3H]chol was determined. D) Cells overexpressing Xpress-ORP2 were pulse labeled with [3H]chol.After chasing for 30 min, the amount of esterified [3H]chol was determined (mean ± SEM; n = 4 from two independent experiments;*p < 0.01). E) Cholesterol transfer between liposomes using recombinant GST-MLN64-START domain, GST-ORP1S or GST-ORP2. Dataare expressed as percentage of [3H]chol transferred to acceptor vesicles after 15 min of incubation (mean ± SEM; n = 3–5; *p < 0.01).F) Pearson’s correlation of BODIPY-chol and BODIPY-C12 after 15–25 min chase in cells overexpressing Xpress-ORP2 wild type (WT)or mutants (mean ± SEM; n = 7; *p < 0.01). G) Western blot analysis of cells overexpressing Xpress-ORP2 WT or mutants (mean ±SEM; n = 5).

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Figure 6: BODIPY-chol transport from the PM to the ER. A) HeLa cells, prelabeled with ER-tracker, were pulse labeled withBODIPY-chol. Confocal images were taken during chase. White circles indicate colocalization. Scale bar: 10 μm. B) Pearson’s correlationof BODIPY-chol and ER-tracker as a function of time in a representative pulse-chase experiment, with each data point containing ∼15cells. C) Cells overexpressing Xpress-ORP2 were prelabeled with ER-tracker and pulse labeled with BODIPY-chol. Confocal images weretaken after 10 min chase. Intensity profiles of areas indicated by the vertical white band. Scale bar: 10 μm. D) Pearson’s correlation ofBODIPY-chol and ER-tracker from images taken after 15–20 min or 180 min chase (mean ± SEM; n = 3).

that some of the colocalization recorded is coincidentaland not because of true ER localization of BODIPY-chol. Nevertheless, we observed that the colocalizationof BODIPY-chol with the ER marker started to be visibleafter 10–15 min of chase, and that this was easiest tojudge in the vicinity of the nuclear envelope (Figure 6A).By recording the Pearson’s correlation between BODIPY-chol and the ER marker, BODIPY-chol appeared to moveto the ER with a half-time of ∼30 min (Figure 6B).Interestingly, this transfer was stimulated by ORP2overexpression (Figure 6C,D). Taken together, theseresults are compatible with a model where BODIPY-chol moves first from the PM to the ER in a processfacilitated by ORPs and then to LDs independently ofORPs.

Effect of VAPs on sterol transport and ORPs

VAPs have been implicated in lipid transport (21,25)and interact with the FFAT domain present in severalORPs. We therefore analyzed the effect of VAP-A and/orVAP-B knockdown on BODIPY-chol transport from thePM to LDs. Knockdown of either VAP-A or VAP-Balone did not affect this transport (not shown), butsimultaneous knockdown of both proteins (VAP-A/B)by > 95% (Figure S4) resulted in slower BODIPY-choltrafficking from the PM to LDs, as suggested by reducedcolocalization of BODIPY-chol and the LD marker at15–25 min of chase (Figure 7A).

To assess whether ORP2 can facilitate PM–LD steroltransfer in VAP-silenced cells, we overexpressed ORP2in control and VAP-A/B knockdown cells and analyzed

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Figure 7: VAPs stabilize ORP proteins. A) In HeLa cells, VAP-A/B was silenced with and without Xpress-ORP2 overexpression. Afterprelabeling LDs with BODIPY-C12, the cells were pulse labeled with BODIPY-chol. Bars indicate Pearson’s correlation of BODIPY-choland BODIPY-C12 from images taken after 15–25 min chase (mean ± SEM; n > 5; *p < 0.05). B) Western blot of endogenous ORP2after VAP-A/B knockdown (mean ± SEM; n = 4; *p < 0.001). C) VAP-A/B was silenced in cells expressing Xpress-ORP2. Cells werelabeled with [35S]methionine and chased for the indicated times. Xpress-ORP2 was immunoprecipitated using α-Xpress antibody.Shown are autoradiographs after SDS–PAGE and densitometric quantifications (mean ± SEM; n = 3 or 4 from three independentexperiments). D) Western blot of endogenous ORP2 after Myc-VAP-A overexpression (mean ± SEM; n = 4; *p < 0.002). E) Westernblot of overexpressed Xpress-ORP2 and Xpress-ORP2mFFAT with and without VAP-A/B knockdown (mean ± SEM; n = 4; *p < 0.01).F) Western blot of endogenous ORPs after VAP-A/B knockdown (mean ± SEM; n = 4, except for ORP3, n = 3).

BODIPY-chol PM–LD delivery. We found that ORP2 over-expression approximately doubled BODIPY-chol transportkinetics in both control and VAP-A/B knockdown cells(Figure 7A). This indicates that ORP2 can facilitate steroltransport independently of VAPs.

To investigate if VAP knockdown affects endogenousORPs in HeLa cells, we analyzed ORP2 levels by westernblotting. A significant reduction in the amount of ORP2was observed in the VAP-A/B-silenced cells (Figure 7B).We addressed the mechanism of ORP2 lowering by study-ing the stability of the ORP2 protein using [35S]methioninepulse-chase experiments followed by immunoprecipita-tion. This showed that ORP2 was more rapidly degradedin VAP-A/B-silenced cells (Figure 7C), indicating thatdecreased protein stability contributes to the decreasedORP2 content in VAP knockdown cells. Conversely, Myc-VAP-A overexpression was accompanied by increasedlevels of the endogenous ORP2 protein (Figure 7D).

The FFAT motif appeared to be important for the VAP-dependent ORP2 stability, as judged by the finding that theXpress-ORP2mFFAT protein was not downregulated afterVAP-A/B knockdown (while overexpressed Xpress-ORP2was) (Figure 7E). Consistent with this, we found that theprotein levels of the FFAT motif containing ORPs, ORP1Land ORP3 were downregulated by VAP-A/B silencing,whereas ORP1S and ORP11 that lack an FFAT motif werenot affected by the knockdown (Figure 7F). However, notall ORPs containing an FFAT motif were downregulatedupon VAP-A/B silencing, as indicated by ORP4 and ORP9(Figure 7F).

Discussion

Analysis of PM-to-LD sterol transport

In this study, we took advantage of the recentlycharacterized fluorescent cholesterol analog BODIPY-chol (22), along with more conventional techniques, to

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analyze sterol transport between the PM, ER and LDs.The transport kinetics of BODIPY-chol was found toclosely resemble that of natural sterols. BODIPY-cholmoved from the PM to LDs with a t1/2

of ∼30 min, whichis roughly similar to the kinetics observed for [3H]cholPM–LD delivery using LD isolation (Figure 1D) and for thedelivery of DHE to LDs (11). The transport of BODIPY-chol from the PM to ER also occurred with a t1/2

of∼30 min, which is similar to the t1/2

∼40 min estimated byLange et al. for the PM–ER movement of cholesterol (26).Furthermore, the recovery of BODIPY-chol fluorescencein LDs occurred very rapidly (with a t1/2

≤ 1 min), as alsoobserved for DHE (t1/2

∼1.5 min) (11). BODIPY-chol wasnot esterified during the time window analyzed, but wefound LDs to contain substantial amounts of unesterifiedcholesterol. Moreover, in the professional LD-storing cells,adipocytes, the vast majority (90%) of the LD cholesterolis unesterified (4,27). We conclude that BODIPY-chol canbe considered as a valid tool for monitoring the transportof unesterified sterol from the PM to ER and LDs.

Role of ORPs in sterol transport from the PM to the

ER and LDs

Accumulating evidence implicates mammalian ORPs insterol signaling and/or sterol transport functions (28).Recent work with mammalian ORPs showed that OSBPand ORP9L have the ability to transfer cholesterolin vitro (29), and we previously reported that overex-pressed ORP2 is capable of enhancing the transport ofnewly synthesized cholesterol to the PM (7). In this study,we visualized sterol delivery to two acceptor compart-ments, the ER and LDs. Both processes were found tobe sensitive to ORP levels. Importantly, LDs are knownto interact with the ER (30). This together with our findingthat the rate at which BODIPY-chol fluorescence recov-ered in LDs was not ORP sensitive argue that the ORPeffect occurred at the PM–ER, not the ER–LD transportstep. It is also possible that sterol moves to LDs directlyfrom the PM or via routes other than the ER. However,because of the vast surface area of the ER and the closeproximities between the PM and ER, as well as ER andLDs, movement via the ER seems very likely.

Several OSBP homologs (Osh) in yeast have, usinggene disruptant strains and in vitro transport assays,been shown to display cholesterol transport activity.Raychaudhuri et al. provided evidence that Osh-3, -4 and-5 have a function in sterol transfer from the PM to theER, as measured using an assay that monitors sterolesterification (15). Furthermore, they showed that Osh4ptransfers cholesterol and ergosterol in vitro betweenliposomes. In a recent study, the same group reportedthat Osh4 has the capacity to bridge between inside-out PM vesicles and ER microsomes, in a processthat enhances cholesterol transport from one vesiclepopulation to another (17).

In the present study, we provide evidence that mammalianORPs also have the capacity to stimulate cholesterol

transfer between vesicles. Although this would beconsistent with a sterol carrier function of ORPs, ourdata do not exclude other modes of action, such as sterol-regulated control of membrane contact site formation.Interestingly, Sullivan et al. suggested that the yeast Oshproteins function in sterol transport not by acting as steroltransporters, but rather by affecting the ability of the PM tosequester sterols (13). Our results with the sterol binding-deficient ORP2 mutant (I249W) show that sterol bindingis required for the observed transport enhancement byORP2. However, they do not allow us to draw conclusionsof whether this binding contributes to a sterol carrier or asterol-regulatory function.

Structure–function relationships of ORPs

Of the yeast ORPs, Osh-4, -5, -6 and -7 represent the’short’ subtype (31), lacking the N-terminal sequencesthat include a PH domain. Olkkonen and Levine (32)originally proposed that the long subtype ORPs with anER-targeting FFAT motif and a PH domain, interactingwith phosphoinositides at other subcellular membraneorganelles, could play a role at the contact sites of ER withother organelles. The work on yeast Osh proteins has,however, clearly shown that sterol transfer activity is notrestricted to the long ORPs but is a prominent property ofthe short protein subtype.

In an overexpression screen, we identified the shortORPs, ORP1S and ORP2, as the most efficient onesin enhancing BODIPY-chol transport from the PM to LDs.Interestingly, ORP1S and ORP2 belong to the same ORPsubfamily (subfamily II) (18) and show a high degree ofsequence identity. The long isoform of ORP1, ORP1L,which targets to late endosomes and the ER (33–35)failed to enhance PM–LD sterol transport, as predictedby its subcellular localization. ORP1S has been reportedto be largely cytosolic (33) and ORP2 localizes, in additionto a cytosolic fraction, on the surface of LDs and also thePM (36,37). It thus seems that, in the case of ORP1S,a predominantly cytosolic distribution allows it to attachand detach from membranes at a rate that allows for asterol transport function. The same may also hold true forORP2. It is possible that this function is facilitated by aclose apposition of PM and ER at contact sites (38,39).

ORP–sterol interactions and stoichiometric

considerations

Six mammalian ORPs are known to bind cholesterol oroxysterols in vitro (OSBP, ORP1, ORP2, ORP4, ORP8and ORP9). Photocrosslinking indicates that even allfamily members may be capable of sterol binding (24).Analysis of ORP2 mutants revealed that a functionalsterol-binding pocket is necessary for the enhancementof sterol transfer. Quantification of the endogenousORP1S and ORP2 by western blotting using purifiedrecombinant proteins as calibrators yielded the numberof approximately 1 million copies of each protein per cell(our unpublished data). We approximated that 40 000

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cholesterol molecules move per second from the PMto the ER. This value was obtained by assuming that2% of cellular cholesterol is in the ER (40), one HeLacell contains 5 billion cholesterol molecules, the half-time of cholesterol transport to the ER is 1800 secondsand that the transport occurs by first-order kinetics[0.02 × 5·109 × Ln(2)/1800 = ∼40000]. This means that,assuming that the cellular ORP1S and ORP2 were toact as sterol carriers, they are present at an abundancethat would allow them to significantly contribute to thePM-to-ER cholesterol transport at the kinetics observed.

Functional redundancy and VAP interaction of ORPs

The yeast Osh proteins are functionally redundant, asillustrated by the fact that expression of any singlefamily member is sufficient to maintain viability (41).Furthermore, disrupting any single osh gene onlymoderately affects the transport of cholesterol from thePM to the esterification compartment (15). Therefore,we do not find it surprising that the ORP1/ORP2double RNA interference only mildly impaired BODIPY-chol transport from the PM to the ER and LDs. Moreover,in mammalian cells other lipid transporters, e.g. membersof the START family, function in intracellular cholesteroltransport. Although most of the ORPs tested did notenhance the transport process in the overexpressionscreen, the endogenous functions of several of themmay involve cholesterol trafficking from (or to) the PM.The inhibitory effect of the VAP-A/B double knockdown,which destabilized several ORPs, on the BODIPY-choltransport, supports this notion. Importantly, we foundthat the VAP-A/B double knockdown enhanced ORP2degradation, suggesting that VAPs affect sterol transportvia stabilization of ORPs. However, the VAP interactionpartners include a number of other lipid-binding/transportproteins, such as CERT/GPBP and Nir1-3 (20), some ofwhich may contribute to the transport process monitored.

Of the short human ORPs identified as putative PMto ER sterol transporters, ORP2 carries an FFAT motif,while ORP1S lacks it. The fact that ORP1S enhancedsterol transport thus suggests that the FFAT motif isdispensable for this activity. This was further corroboratedby showing that an ORP2mFFAT mutant, defective inVAP-A binding (37), is nevertheless capable of stimulatingsterol transport. However, we find it possible that theORP–VAP interactions may, under physiologic conditions,be instrumental for PM–ER sterol transport. Upon proteinoverproduction, the necessity of the specific interactiondeterminants might be bypassed and the lipid transfermight become uncoupled from them, as shown forCERT (21).

Concluding remarks

The present study introduces a live-cell imaging assayemploying BODIPY-chol to monitor sterol transport fromthe PM to the ER and LDs, a process of central importancefor the maintenance of sterol homeostatic control. By

using this assay and by following radiolabeled cholesterolpools, we provide the first evidence for a function ofmammalian ORPs and VAPs in this transport process. Toreach a comprehensive understanding of the role playedby ORPs in intracellular sterol trafficking, one should,however, be able to simultaneously disrupt the functionof multiple – or all – ORPs in mammalian cells. Futureefforts should be aimed at reaching this goal, for instancevia the development of pharmacologic ORP inhibitors.

Materials and Methods

Cells and antibodiesHeLa and Huh7 cells were grown in DMEM supplemented with 10%fetal bovine calf serum and passaged twice per week. For all experiments(except the PM to ER pulse chase), the cells were incubated overnightin growth medium containing 350 or 500 μM oleate/BSA complex toincrease the cellular LD content for better visualization. The followingantibodies were used in this study: ORP1L (34), ORP1S (33), ORP2 (36),ORP3 (42) ORP11 (43) and anti-Xpress™ (Invitrogen). Polyclonal antibodiesdirected against human VAP-A and VAP-B were raised in rabbit inMaria Antonietta De Matteis’ laboratory (Department of Cell Biology andOncology, Consorzio Mario Negri Sud, Santa Maria Imbaro) using thefull-length His-tagged proteins. The anti-VAP-A and anti-VAP-B antibodieswere affinity purified from the sera using glutathione S-transferase (GST)full-length proteins coupled to beads. Antibodies against ORP4 (44) andORP9 (45) were kindly provided by Neale Ridgway, Dalhousie University.

Plasmid and siRNA transfectionsThe following ORP open reading frames were subcloned in thepcDNA4HisMaxC expression vector (Invitrogen): rabbit OSBP (J05056),human ORP1L (AF323726), ORP2 (BC000296), ORP3 (NM 015550), ORP4(NM 030758), ORP5 (NM 020896), ORP6 (AF323728), ORP7 (AF323729),ORP8 (NM 001003712), ORP9 (NM 024586), ORP10 (NM 017784) andORP11 (NM 022776). The short ORP sequences expressed using the samevector were ORP1S (AF274714), ORP4S (aa 413-916 of NM 030758) andORP9S (aa 268-736 of NM 024586; this construct is shorter than the naturalshort variant NM 148905). The ORP2 sterol binding-deficient mutantI249W (ORP2mORD) has been described in Ref. (24), and the mutant withthe FFAT motif sequence EFFDA replaced with EVVVA (ORP2mFFAT) hasbeen described in Ref. (46). The Myc-VAP-A expression construct was agift from Dr W. Trimble (Hospital for Sick Children). The ORP constructswere transfected together with the pHcRed1-N1 vector (Clontech)in a 3:1 mass ratio using Lipofectamine™ LTX transfection reagent(Invitrogen). For control samples, we transfected an empty expressionvector. Cells were used for experimentation 2 days after transfection.For knockdown experiments, siRNA duplexes were transfected usingHiPerFect transfection reagent (Qiagen) and used for experiments2 (ORP) or 3 days (VAP) later. For simultaneous ORP2/ORP2mFFAToverexpression and VAP-A/B knockdown, Lipofectamine 2000 (Invitrogen)was used. VAP-A (NM 003574; M-021382-00) and VAP-B (NM 004738;M-017795-00) duplexes were siGENOME SMART pool siRNAs purchasedfrom Dharmacon. SiRNAs for ORP1 (sense: GCCAGUGCCGGAUUCUGA-dTdT)(47) and ORP2 (sense: GGGAAGAUUUAGGAUUCAGtt) were fromSigma. For control samples, we used Allstar Negative Control siRNA(Qiagen).

Preparation of BODIPY-chol/methyl-β-cyclodextrin

complexBODIPY-chol was synthesized as described (48). A 370 mM MβCD (Sigma)solution was added to BODIPY-chol in a 100:1 molar ratio. The BODIPY-chol was solubilized by sonication while cooled with ice-water. UndissolvedBODIPY-chol was removed by centrifugation (60 min, 16 000 × g). This

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procedure solubilized ∼50% of the BODIPY-chol. The complex wasaliquoted and stored at – 20◦C. After thawing, the aliquots were keptat 4◦C for up to 1 month.

BODIPY-chol labeling and chaseDuring the entire procedure, the cells and incubation media were kept at37◦C. Cells grown in 35-mm dishes were prelabeled with either BODIPY558/568 C12 (BODIPY-C12; Invitrogen) or ER-Tracker Red (Invitrogen).For labeling LDs with BODIPY-C12, cells were incubated with 5 μM

BODIPY-C12 in serum-free medium for 1 h at 37◦C and chased in 1 mLof CO2-independent medium (Gibco) for 2–6 h at 37◦C. Next 0.5 μLof MβCD/BODIPY-chol complex was added to cells in 1:2000 dilution,resulting in a final concentration of 0.185 mM MβCD and ∼1 μM BODIPY-chol. After labeling for 1 min at 37◦C, the cells were washed 3× with PBS.Using this procedure, ∼3% of BODIPY-chol was transferred from MβCD tocells, resulting in roughly 1 out of 1000 sterol molecules being labeled in thecell. The cells were chased and imaged in a CO2-independent medium at37◦C. Images were taken with a Leica TCS SP2 AOBS confocal microscope(Leica Microsystems AG) equipped with an HCX PL APO LU-V-I 63×/0.9numerical aperture (NA) water immersion objective. Three fluorophoreswere imaged sequentially in the same sample using the following laserlines and emission settings: BODIPY-chol: 488 nm excitation, 506–536 nmemission; ER-tracker and BODIPY-C12: 561 nm excitation, 575–585 nmemission; HcRed: 633 nm excitation, 640–800 nm emission. For everydish, six to nine triple channel images (each containing ∼15 cells) weretaken at 15–25 min and at 180 min. The average Pearson’s correlationcoefficient was determined in all the six to nine images and used as asingle data item. Colocalization of BODIPY-chol and the red organellarmarker, as expressed by the Pearson’s correlation coefficient (calculatedusing IMARIS software, Bitplane), was used as a measure of the relativeamount of BODIPY-chol in the organelle. When pooling data from severalexperiments, the Pearson’s correlation coefficients were normalized tocontrol.

Drug treatmentsCells were treated with the following drugs during the BODIPY-chol chase:5 μg/mL BFA in CO2-independent medium, 25 mU/mL SMase in CO2-independent medium containing 0.2% BSA, and for ATP depletion, cellswere incubated in HEPES buffer [150 mM NaCl, 5 mM KCl, 1 mM CaCl2,1 mM MgCl2 and 20 mM HEPES (pH 7.4)] containing 100 mM sodiumazide and 100 mM 2-deoxyglucose. For both the BFA treatment and ATPdepletion, cells were pretreated for 30 and 5 min, respectively, beforeBODIPY-chol labeling.

Dehydroergosterol labeling and chaseHuh7 cells were labeled for LDs with BODIPY-C12 as described for theBODIPY-chol pulse-chase assay. Then, the cells were incubated with a1:8 DHE/MβCD complex for 3 min at 37◦C. The DHE/MβCD complex wasprepared as described in Ref. (49). The cells were imaged with a TILLPhotonics wide-field microscopy system equipped with a filter cube forDHE imaging with 335-nm (20-nm bandpass) excitation filter, 365-nm longpass dichromatic filter and 405-nm (40-nm bandpass) emission filter fromChroma Technology Corp., as described in Ref. (49).

In vitro cholesterol transport assayGST-fusion proteins of ORP1S, ORP2, and the MLN64 START domainexpressed using the pGEX1λT (GE Healthcare) vector were producedin Escherichia coli and purified using glutathione-Sepharose 4B (GEHealthcare). Protein concentrations were determined by the DC ProteinAssay™ (BioRad) and amounts of the full-length fusion proteins inthe preparations equalized with the help of Coomassie Blue stainedSDS-PAGE. The ability of 50 pmol of proteins to stimulate [3H]chol(54.2 Ci/mmol; PerkinElmer) transfer from large unilamellar vesicles(generated by extrusion through a 400-nm filter) containing 89 mol%egg phosphatidylcholine (PC), 10 mol% heart cardiolipin and 1 mol%

cholesterol (Sigma-Aldrich) to a 50-fold molar excess of acceptor eggPC vesicles (generated by sonication) was assayed essentially asdescribed (50). The reaction time used was 15 min, after which the donorsand acceptors were separated by using diethylaminoethyl (DEAE)-Sephacel(GE Healthcare), and cholesterol transfer to acceptors was quantified byliquid scintillation counting. By measuring 0-min control samples, we foundthat 5% of the donor vesicles did not bind to the column and contaminatedthe acceptor vesicle fraction. The data were corrected for this accordingly.

Fluorescence recovery after photobleachingFRAP was performed at 37◦C with a Leica TCS SP2 confocal microscopewith argon excitation line at 488 nm and an HCX PL APO LU-V-I 63×/0.9 NAwater immersion objective, and a zoom factor of 6. With 256 × 256 pixelresolution and 1400 Hz scanning speed, bleaching was carried out withfive high-intensity short pulses (1.5 seconds in total) and zoom-in functionto increase the bleaching power. Fluorescence recovery was monitoredfor a total duration >3 min under low-intensity illumination.

[3H]Cholesterol esterificationPM was selectively labeled by incubating cells with 2 μCi/mL [3H]cholin DMEM containing 0.2% BSA for 30 min at 15◦C (51). The cells werewashed with PBS and incubated in a medium without serum at 37◦C for10–30 min. The cells were collected by scraping in an ice-cold PBS. Lipidswere extracted (52) and free and esterified [3H]chol were separated byTLC and quantified by scintillation counting.

LD isolationLDs were isolated as described previously (53). Briefly, cells were labeledwith [3H]chol and incubated in a medium without serum as describedfor [3H]chol esterification. After scraping in PBS, cells were lysedin 3 mL of hypotonic lysis medium [20 mM Tris–HCl, pH 7.4; 1 mMethylenediaminetetraacetic acid (EDTA)] including a protease inhibitormixture (chymostatin, leupeptin, antipain and pepstatin A; 25 μg/mL each;CLAP, Sigma) on ice. The samples were kept at 0–4◦C until lipid extraction.The lysate was spun for 10 min at 1000 × g. The supernatant wascollected and mixed with 1.5 mL of 60% sucrose solution to reach a finalconcentration of 20% sucrose. The samples (4.5 mL) were transferredto SW40Ti rotor tubes, overlaid with 4 mL of hypotonic lysis mediumcontaining 5% sucrose and 4 mL containing 0% sucrose. The sampleswere centrifuged for 2 h at 28 000 × g, after which LDs were recoveredfrom the top of the gradient. The fractions were collected using a tubeslicer and stored at −20◦C.

Lipid extraction and thin-layer chromatographyLipids were extracted (52) and the neutral lipids were analyzed byTLC. A running buffer of hexane/diethyl ether/acetic acid (80:20:1) wasused except for samples containing BODIPY-chol, for which petroleumether/diethyl ether/acetic acid (60:40:1) was used. Radiolabeled lipidswere scraped from the TLC plate, extracted and quantified by scintillationcounting. BODIPY-labeled lipids were visualized using a fluorescence platereader and unlabeled lipids were detected by charring.

Western blot analysisCells were lysed in buffer containing 1% SDS and 5 mM EDTA, includingCLAP. Equal amounts of cellular protein were subjected to SDS–PAGE(10% gels) and western blotting. The blots were developed using theenhanced chemiluminiscence kit (Amersham). Western blot quantificationswere performed using IMAGEJ (NIH).

ACAT activityACAT activity was analyzed as described (54), except that lipid extractionand TLC were carried out as above.

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Metabolic labeling and immunoprecipitationCells were incubated in methionine-free medium containing 70 μCi/mLof [35S]methionine (PerkinElmer) for 2 h. Cells were washed with PBSand chased in a complete medium for the indicated periods. Cells werewashed with an ice-cold PBS and collected in lysis buffer (0.5% Triton-X-100, 20 mM HEPES pH 7.6, 150 mM NaCl, 2 mM MgCl2, 10% glycerol,1 mM DTT, CLAP). After incubating for 30 min at 4◦C, cell lysates werecentrifuged at 16 000 × g for 10 min. After preclearing with protein A-Sepharose (GE Healthcare) for 60 min, lysates were incubated sequentiallywith anti-Xpress antibody (0.25 μg per sample, overnight; Invitrogen) andprotein A-Sepharose beads (4 h) at 4◦C. The beads were washed withlysis buffer and boiled in SDS sample buffer. The samples were subjectedto SDS–PAGE, and the gels were exposed to film (Amersham HyperfilmMP, GE Healthcare).

Statistical analysisData are reported as mean ± standard error of the mean (SEM). Statisticalanalysis was performed using Microsoft Excel ’t-test: two samplesassuming unequal variances’.

Acknowledgments

We thank Anna Uro, Pipsa Kaipainen and Diyu Wang for expert technicalassistance, and Giuseppe Di Tullio and Michele Santoro for preparing theVAP antibodies. Pentti Somerharju and Martin Hermansson are thankedfor help with the in vitro cholesterol transfer assay. Professor DaoguangYan (Jinan University) is thanked for kindly providing the full-length ORP4and ORP5 cDNA constructs. This study was financially supported by theSigrid Juselius Foundation (E. I. and V. M. O.), the Academy of Finland(grants 131429 and 218066 to E. I. and grant 121457 to V. M. O.),the Alfred Kordelin Foundation (M. J.), the Instrumentarium Foundation(M. J.), the Helsinki Biomedical Graduate School (M. J.), the Ida MontinFoundation (M. J.), the Telethon Foundation (grants numbers GSP08002and GGP06166 to L. R. R.), the Associazione Italiana per la Ricerca sulCancro (AIRC, grant IG 8623 to L. R. R.) NIH Grant HL-083187 (R. B.), the Livoch Halsa Foundation (V. M. O.), the Finnish Foundation for CardiovascularResearch (V. M. O., E. I.), the Magnus Ehrnrooth Foundation (V. M. O.) andthe European Union FP7 (LipidomicNet, agreement no. 202272).

Supporting Information

Additional Supporting Information may be found in the online version ofthis article:

Figure S1: LD labeling. Cells were labeled with BODIPY-C12 for 1 h andchased for 2 or 8 h. After fixation, the cells were labeled with BODIPY-493/503 and confocal images were acquired. Scale bar: 10 μm.

Figure S2: Time-dependent localization of BODIPY-chol in isolated

LDs. A) Cells were pulse labeled with BODIPY-chol and chased for 0, 30,90 or 270 min. LDs were isolated as detailed in Materials and Methodsand BODIPY-chol in the fractions was analyzed by TLC (shown are thedata from 0 and 270 min chase). Please note that two third of fractions1 and 2 and one fourth of fractions 3 and 4 were analyzed by TLC, forbetter visualization of BODIPY-chol in the LD fraction. B) The percentageof BODIPY-chol in the LD fraction was plotted as a function of time.

Figure S3: Cotransfection efficiency. Xpress-ORP2 was cotransfectedwith an HcRed construct to identify cells overexpressing ORP2 during live-cell microscopy. The cotransfection efficiency was determined by fixingand staining the cells with α-Xpress antibody; ∼90% of the HcRed-positivecells were also positive for ORP2 and ∼20% of the cells negative forHcRed were positive for ORP2. Scale bar: 100 μm.

Figure S4: Knockdown efficiency using siRNAs. Cells were simultane-ously transfected with siRNA duplexes against ORP1 and ORP2 or VAP-Aand VAP-B. Protein levels were analyzed by western blotting.

Figure S5: ORP2 overexpression increases the transport of DHE from

the PM to LDs. Huh7 cells, transiently overexpressing Xpress-ORP2and GFP, were prelabeled with BODIPY-C12 and pulse labeled with DHE.Shown are exemplary images, both taken after 45 min chase. Scale bar:15 μm.

Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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