3640 Research Article

IntroductionNiemann-Pick disease Type C (NP-C) is a fatal autosomalrecessive disorder characterized by the lysosomalaccumulation of lipids. Specifically, exogenously derivedcholesterol is aberrantly accrued in late endosomes andlysosomes (Liscum et al., 1989; Pentchev et al., 1985), and itis thought that this defect is due to an impairment in theretrograde movement of lipids from these organelles (Davieset al., 2000; Neufeld et al., 1999) to the plasma membrane orendoplasmic reticulum (ER) (Cruz et al., 2000). Although thetwo mutated genes responsible for the disorder have beenidentified as human NPC1 (hNPC1) and human NPC2(hNPC2) (Carstea et al., 1997; Loftus et al., 1997; Naureckieneet al., 2000), the precise functions of the gene products remainunknown. hNPC1 is a highly conserved integral membraneprotein of 1278 amino acids composed of 13 transmembrane-spanning domains, a conserved sterol-sensing domain and a C-terminal di-leucine motif, which has been proposed to bedispensable for proper function (Carstea et al., 1997; Daviesand Ioannou, 2000; Scott et al., 2004). Examination of thesubcellular distribution of hNPC1 reveals that the proteinlocalizes to vesicles containing LAMPI (Garver et al., 2000;Higgins et al., 1999), LAMPII (Neufeld et al., 1999) and RAB9(Higgins et al., 1999), indicating that the protein resides in late

endosomes. The proper intracellular localization of hNPC1seems to be important for its function, because a mutant formof the protein that accumulates in the ER is causative of NP-Cdisease (Blom et al., 2003).

hNPC2 is the second protein associated with NP-C disease(Naureckiene et al., 2000). hNPC2 is a secreted 151 amino acidprotein containing an N-terminal ER signal peptide and anMD-2-related lipid-recognition domain (Inohara and Nunez,2002). The protein is bound by the mannose-6-phosphatereceptor (Naureckiene et al., 2000) and co-localizes withcathepsin D (Zhang et al., 2003), indicating that hNPC2 islocated, in part, in the lysosome.

The mechanisms that target the NP-C proteins to the lateendosome/lysosome have not been fully investigated; however,intrinsic signals and modifications found within the NP-Cproteins suggest how these proteins could be targeted to theirrespective organelles. hNPC1 contains a C-terminal di-leucinemotif (Carstea et al., 1997; Loftus et al., 1997; Scott et al.,2004), which – in other membrane proteins – is necessary andsufficient to confer endocytic targeting (Bonifacino and Traub,2003; Johnson and Kornfeld, 1992a; Johnson and Kornfeld,1992b; Letourneur and Klausner, 1992). However, in the caseof NPC1, its C-terminal di-leucine motif is not sufficient forits late endosomal localization (Scott et al., 2004). As

Niemann-Pick Type C (NP-C) disease, caused by mutationsin either human NPC1 (hNPC1) or human NPC2 (hNPC2),is characterized by the accumulation of unesterifiedcholesterol in late endosomes. Although it is known that theNP-C proteins are targeted to late endosomal/lysosomalcompartments, their delivery mechanisms have not beenfully elucidated. To identify mechanisms regulating NP-Cprotein localization, we used Saccharomyces cerevisiae,which expresses functional homologs of both NP-C proteins– scNcr1p and scNpc2p. Targeting of scNcr1p to thevacuole was perturbed in AP-3-deficient yeast cells,whereas the delivery of scNpc2p was affected bydeficiencies in either AP-3 or GGA. We focused on the roleof the AP-3 pathway in the targeting of the mammalian NP-C proteins. We found that, although mouse NPC1 (mNPC1)and hNPC2 co-localize with AP-3 to a similar extent in

fibroblasts, hNPC2 preferentially co-localizes with AP-1.Importantly, the targeting of both mammalian NPC1 andNPC2 is dependent on AP-3. Moreover, and consistent withthe NP-C proteins playing a role in cholesterol metabolism,AP-3-deficient cells have reduced levels of cholesterol.These results provide information about how the NP-Cproteins are targeted to their sites of action and illustratethe possibility that defective sorting of the NP-C proteinsalong the endocytic route can alter cellular cholesterol.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/20/3640/DC1

Key words: Niemann-Pick disease Type C, NPC1, NPC2, AP-3,Endosomal trafficking

Summary

The subcellular localization of the Niemann-Pick TypeC proteins depends on the adaptor complex AP-3Adam C. Berger1,2,3,*, Gloria Salazar1, Melanie L. Styers1,3, Karen A. Newell-Litwa1,3, Erica Werner1,Robert A. Maue4, Anita H. Corbett2 and Victor Faundez1,‡

1Department of Cell Biology and 2Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA3Graduate Program in Biochemistry, Cell, and Developmental Biology, Emory University Graduate Division of Biological and Biomedical Sciences,Atlanta, GA 30322, USA4Departments of Physiology and Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA*Present address: NIH, NCI Experimental Immunology Branch, Bethesda, MD 20892, USA‡Author for correspondence (e-mail: faundez@cellbio.emory.edu)

Accepted 13 August 2007Journal of Cell Science 120, 3640-3652 Published by The Company of Biologists 2007doi:10.1242/jcs.03487

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3641NP-C protein trafficking is adaptor dependent

mentioned above, hNPC2 is modified by the addition of amannose-6-phosphate group (Naureckiene et al., 2000), whichis a signal for targeting soluble molecules to the lysosomewhen recognized by the mannose-6-phosphate receptor(Kornfeld and Mellman, 1989; Pohlmann et al., 1995).Consistent with this modification, the targeting of NPC2 isregulated by mannose-6-phosphate receptors (Willenborg etal., 2005).

For intracellular trafficking, different adaptor proteins (APs)recognize intrinsic targeting signals in proteins and sort thesecargo molecules into vesicles for transport between organelles(Bresnahan et al., 1998; Dietrich et al., 1997; Fujita et al., 1999;Heilker et al., 1996; Hofmann et al., 1999; Honing et al., 1998;Peden et al., 2001). There are several types of adaptors,including the heterotetrameric AP complexes AP-1, AP-2, AP-3 and AP-4 as well as the monomeric Golgi-localized, � ear-containing, ARF-binding (GGA) proteins (reviewed in Boehmand Bonifacino, 2002). The AP-1, AP-2, AP-3 and GGAproteins are, for the most part, functionally and structurallyconserved in all eukaryotes, including in Saccharomycescerevisiae (reviewed in Boehm and Bonifacino, 2002). Incontrast to what has been observed in multicellular organisms,none of the components of these complexes are essential forcell viability in yeast (reviewed in Boehm and Bonifacino,2002). The finding that both of the yeast homologs of the NP-C proteins are able to functionally replace the loss of theirrespective mammalian homologs (Berger et al., 2005b; Malathiet al., 2004), in conjunction with the conservation of adaptorfunction, makes yeast an ideal model system in which toexamine the effects of the AP complexes on NP-C proteinsorting. We can then use the studies from the simple yeastmodel system to guide our studies of mammalian NPC1 andNPC2 trafficking.

In this study we examine the mechanisms that control theproper sorting of mouse NPC1 (mNPC1) and hNPC2 to theirtarget organelles. We first exploit the yeast model system toanalyze the requirements for trafficking of the yeast NP-Chomologs S. cerevisiae Ncr1p (scNcr1p) (Berger et al., 2005a;Malathi et al., 2004; Zhang et al., 2004) and scNpc2p (Bergeret al., 2005b). Results of the survey of APs in yeast implicatethe AP-3 complex in trafficking both scNcr1p and scNpc2p tothe yeast vacuole. These findings in yeast were then used as aguide to examine the role that the AP-3 complex plays in thetransport of the mNPC1 and hNPC2 proteins in mammaliancells. We describe the role that the AP-3 complex plays in thelocalization and function of the NP-C proteins and identify anendocytic compartment in which the NP-C proteins andregulatory sorting machinery reside together. This studyindicates that a single sorting mechanism, mediated by AP-3,can regulate the targeting of proteins that contain topologicallydistinct sorting information by both direct and indirectmechanisms.

ResultsAdaptor protein complexes direct the proper targeting ofyeast Ncr1p and Npc2p to the vacuoleAs a first step towards understanding the mechanisms thatgovern mammalian NP-C protein localization, we tookadvantage of the genetic tractability of S. cerevisiae to analyzethe machinery required to traffic the yeast NP-C proteins,scNcr1p and scNpc2p, to the vacuole (Berger et al., 2005b;

Malathi et al., 2004). To investigate the transport pathways forthe yeast NP-C homologs, a genetic analysis was performed byexploiting deletion mutants of the conserved, but non-essential,AP complexes GGA1 and GGA2, AP-1, AP-2 and AP-3(Boehm and Bonifacino, 2002). We analyzed the effect thatdeletion of the individual members of each of the APcomplexes had on the localization of both scNcr1p, which isnormally localized to the vacuolar membrane (Berger et al.,2005a; Malathi et al., 2004; Zhang et al., 2004), and scNpc2p,which is normally localized to the vacuolar lumen (Berger etal., 2005b) (Fig. 1). As a control, we also examined thelocalization of alkaline phosphatase (ALP), a known AP-3cargo molecule (Cowles et al., 1997; Stepp et al., 1997) thatlocalizes to the yeast vacuolar membrane in wild-type cells(Fig. 1).

Individual deletion mutants of the yeast AP-1 componentsAPL4, APL2, APM1, APM2 and APS1, homologous to �, �1,�1A, �1B and �1A of mammalian AP-1, respectively (Boehmand Bonifacino, 2002), were transformed with individualmulticopy plasmids encoding the GFP fusion proteinsscNcr1p-GFP or scNpc2p-GFP, or, as a control, GFP-ALP.The subcellular localization of each protein was assessed bydirect fluorescence microscopy. Consistent with previousobservations that individual deletions of the AP-1 complexmembers yield no overt phenotypes (Nakai et al., 1993; Phanet al., 1994; Rad et al., 1995; Stepp et al., 1995; Yeung et al.,1999), we found that, in the majority of mutants tested, thelocalization of scNcr1p, scNpc2p or ALP was not affected(supplementary material Fig. S1). AP-1-dependent proteinmis-sorting has been identified when individual AP-1 mutantsare combined with a clathrin heavy chain mutant; however,there is variability in the penetrance of these phenotypesdepending on which subunit is studied (Valdivia et al., 2002;Yeung et al., 1999). We therefore examined the role of the

Fig. 1. Localization of ALP, scNcr1p and scNpc2p. GFP-ALP (top),scNCR1-GFP (middle) and scNPC2-GFP (bottom) plasmids weretransformed into wild-type yeast cells. The GFP-tagged proteinswere visualized by direct fluorescence microscopy. Thecorresponding DIC images are shown.

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GGA proteins GGA1 and GGA2 in the trafficking of the yeastNP-C proteins. GGA proteins mediate protein sorting from theGolgi in a way similar to AP-1 complex sorting from the Golgi(Boehm and Bonifacino, 2002). We analyzed the localizationof scNcr1p-GFP, scNpc2p-GFP or control GFP-ALP by directfluorescence microscopy in cells deficient for both GGA1 andGGA2 (gga1�gga2�). Much like AP-1/clathrin heavy chaindouble-mutant cells, gga1�gga2� cells exhibit defects incarboxypeptidase Y sorting (Hirst et al., 2000; Zhdankina etal., 2001). Our results indicate that neither scNcr1p nor ourcontrol ALP was mislocalized (Fig. 2). However, scNpc2p waslocalized to the lumen of the vacuole and also accumulated insmall punctate structures that were absent in wild-type cells.These structures were observed in close proximity to theplasma membrane as well as surrounding the vacuole (Fig. 2).These results suggest that scNpc2p is trafficked throughprevacuolar compartments prior to reaching the vacuole,similar to what is observed for other proteins dependent uponAP-1 or the GGA proteins in yeast (Boehm and Bonifacino,2002). Furthermore, these results highlight the conservationbetween the NPC2 orthologs, because both are targeted by AP-1/GGA-controlled pathways in both yeast (this work) andmammals (Willenborg et al., 2005).

The role of the AP-2 complex was assessed by examiningthe localization of scNcr1p-GFP, scNpc2p-GFP or GFP-ALPin each of the following strains: apl3�, apl1�, apm4� oraps2�. These yeast mutants contain a deletion of the geneencoding the homolog of �, �2, �2 or �2 of mammalian AP-2, respectively (Boehm and Bonifacino, 2002). These cellswere examined by direct fluorescence microscopy forlocalization of each GFP fusion protein. Results from this

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analysis indicate that single deletions of the AP-2 complexmembers have no effect on the localization of scNcr1p or ALPto the vacuolar membrane, nor does it affect the targeting ofscNpc2p to the vacuolar lumen (Fig. 3 and supplementarymaterial Fig. S2). These results suggest that the AP-2 complexin yeast is not required for localization of the NP-C proteins.

AP-3 complex involvement in NP-C protein trafficking wasassessed by examining the localization of scNcr1p-GFP,scNpc2p-GFP or GFP-ALP in apl5�, apl6�, apm3� or aps3�cells, deletion mutants of genes that encode homologs of �,�3A, �3A and �3A of mammalian AP-3, respectively (Boehmand Bonifacino, 2002). The subcellular localization of the GFPfusion proteins was assessed by direct fluorescencemicroscopy. The AP-3 complex traffics ALP in yeast (Cowleset al., 1997; Piper et al., 1997; Stepp et al., 1997), as evidencedby GFP-ALP accumulation in punctate structures throughoutthe cell in addition to the vacuolar membrane in all AP-3pathway mutants (Fig. 4). When the localization of scNcr1pand scNpc2p was assessed, these proteins were alsomislocalized in the majority of the AP-3 mutant cells (Fig. 4).Notably, scNpc2p, a soluble protein, accumulated in bothpunctate and tubular structures surrounding the vacuole in allAP-3 mutants tested (Fig. 4). scNcr1p accumulated in punctatestructures, similar to what was observed for ALP, in all AP-3mutants except for apm3�. The finding that scNcr1p localizescorrectly in the apm3� mutant background is consistent withpreviously published work in which apm3� was the only AP-3 mutant examined (Zhang et al., 2004).

Based on the vacuolar localization of scNcr1p in an apm3�mutant background, Zhang et al. concluded that scNcr1ptrafficked through the vacuolar protein sorting (VPS) pathway

Fig. 2. The GGA proteins direct the localization of scNpc2p. GFP-ALP (top), scNCR1-GFP (middle) and scNPC2-GFP (bottom)plasmids were transformed into gga1�gga2� deletion yeast cells.The GFP-tagged proteins were visualized by direct fluorescencemicroscopy. Arrows indicate areas of mislocalization. Amagnification of the boxed area is depicted. The corresponding DICimages are shown.

Fig. 3. The AP-2 complex is not required for the localization ofscNcr1p or scNpc2p. GFP-ALP (top), scNCR1-GFP (middle) andscNPC2-GFP (bottom) plasmids were transformed into a yeastdeletion of the AP-2 complex (apl1�). The GFP-tagged proteinswere visualized by direct fluorescence microscopy. Thecorresponding DIC images are shown.

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3643NP-C protein trafficking is adaptor dependent

and not the ALP pathway (Zhang et al., 2004). However,because scNcr1p and scNpc2p mislocalize in all other AP-3mutants (Fig. 4), we investigated whether scNcr1p andscNpc2p can traffic through either pathway to the vacuole. TheVPS pathway was assessed by examining the localization ofboth scNcr1p and scNpc2p in a vps4� mutant background(Zahn et al., 2001). Results from this analysis showed nodiscernible mislocalization of scNcr1p from the vacuolarmembrane or accumulation in enlarged multilamellar pre-vacuolar compartments typical of this class (E) of VPS mutants(Raymond et al., 1992) (supplementary material Fig. S3). Bycontrast, scNpc2p accumulated in these compartments whilestill remaining luminal, suggesting that scNpc2p can trafficthrough the VPS pathway as well as the AP-3 pathway(supplementary material Fig. S3).

Taken together, our analysis of NP-C protein trafficking inyeast supports a model in which scNcr1p traffics to the vacuolein an AP-3-dependent manner, whereas proper scNpc2plocalization depends on both the AP-1/GGA/VPS and AP-3pathways.

Mammalian NPC1 is targeted by the AP-3 complexBased upon the finding that yeast Ncr1p, which canfunctionally replace mammalian NPC1 (Malathi et al., 2004),is trafficked to the vacuole via an AP-3-mediated mechanism,we hypothesized that the mammalian NPC1 protein is alsotrafficked in an AP-3-dependent manner. To test thishypothesis, we first examined whether mNPC1 and the AP-3 complex co-localize. Because we lack an antibody capableof detecting endogenous mNPC1 by indirectimmunofluorescence, we assessed the localization ofmNPC1-GFP expressed from an adenovirus constructcapable of rescuing Npc1–/– phenotypes (Paul et al., 2005).Furthermore, and as shown below (see Fig. 5 and Fig. 6A),mNPC1-GFP expressed from an adenoviral vectorrecapitulated the known localization of NPC1 to LAMPI-positive late endosomal compartments (Paul et al., 2005).Adenovirally delivered mNPC1-GFP was co-localized withAP-3 by infecting immortalized AP-3 �–/– mocha mousefibroblasts transduced with a retrovirus encoding the �subunit of AP-3 (AP-3+/+ cells) or an empty retrovirus (AP-3–/– cells) as a control. The localization of both mNPC1 andthe � subunit of AP-3 was assessed in the AP-3+/+ cells byindirect immunofluorescence confocal microscopy. Resultsfrom this analysis show that ~20% of all mNPC1-GFP-positive puncta co-localize with AP-3 and indicate that thisco-localization is specific because mNPC1-GFP co-localization with the adaptor complex AP-1 is 3.8-fold lowerthan with AP-3 (Fig. 5 and Fig. 6A). Furthermore, theabsence of AP-3 did not increase the extent of co-localizationbetween AP-1 and mNPC1 (Fig. 5 and Fig. 6A). As a control,we detected no AP-3 staining in the AP-3–/– cells (Fig. 5).Collectively, these findings indicate that mNPC1 and AP-3reside in a subset of the same organelles.

To analyze the effect that loss of the AP-3 complex has onprotein localization, mNPC1 was co-localized with markers ofvarious organelles in the endocytic pathway. Early/recyclingendosomes were revealed with antibodies directed against thetransferrin receptor (TfR) (supplementary material Fig. S4)(Dunn et al., 1989); the transition between early and lateendosomes was identified using antibodies against theSNAREs Vti1b and syntaxin 8 (Fig. 5 and supplementarymaterial Fig. S4, Syn8) (Atlashkin et al., 2003; Hirst et al.,2004; Kreykenbohm et al., 2002; Prekeris et al., 1999; Pryoret al., 2004); and late endosomes/lysosomes were labeled usingantibodies against LAMPI (Fig. 5) (Lewis et al., 1985), whichtraffics in an AP-3-dependent manner (Dell’Angelica et al.,1999; Yang et al., 2000). Immunolocalization of mNPC1-GFPwith the AP-3 cargo molecule LAMPI in AP-3+/+ and AP-3–/–

cells indicates that, consistent with previous studies (Garver etal., 2000; Higgins et al., 1999; Neufeld et al., 1999), there isalmost total (~80%) co-localization between these proteins,irrespective of the presence or absence of AP-3 (Fig. 5 and Fig.6A). mNPC1 localization to LAMPI-positive late endosomal/lysosomal compartments is selective, because only a tenth ofmNPC1-GFP-positive puncta co-localized with TfR-positive

Fig. 4. The AP-3 complex directs the localization of scNcr1p andscNpc2p. GFP-ALP (top), scNCR1-GFP (middle) and scNPC2-GFP(bottom) plasmids were transformed into yeast deletions of the AP-3complex (apl6�, apl5�, apm3� and aps3�). The GFP-taggedproteins were visualized by direct fluorescence microscopy. Arrowsindicate areas of mislocalization. The corresponding DIC images areshown.

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early/recycling endosomes (Fig. 6A andsupplementary material Fig. S4).

AP-3 budding profiles containing LAMPI arepresent in domains of early endosomes (Peden etal., 2004). Therefore, we hypothesized that in theabsence of AP-3, mNPC1 could accumulate inendocytic compartments upstream of LAMPI-positive late endosomes. To explore thishypothesis, we analyzed the co-localization ofmNPC1-GFP with TfR and the endosomalSNAREs Vti1b and syntaxin 8 in AP-3–/– cells,and, as a control, AP-3+/+ cells (Fig. 5 and Fig.6A,B and supplementary material Fig. S4).Results demonstrated that the co-localizationbetween mNPC1 and TfR was not affected by theabsence of AP-3. However, the co-localization ofmNPC1 with the early/late endocytic SNAREsVti1b and syntaxin 8 increased two- and three-fold, respectively, in AP-3–/– cells (Fig. 5 and Fig.6A,B and supplementary material Fig. S4).Similar findings were obtained in freshly isolatedprimary skin fibroblasts from two different AP-3-deficient mice, grizzled and mocha (Peden etal., 2002), respectively (Fig. 6B). Importantly,the same phenotype was also observed when theAP-3 cargo molecule LAMPI was co-localizedwith Vti1b and syntaxin 8 (Fig. 6C andsupplementary material Fig. S5). Theseobservations indicate that, at steady state,mNPC1 preferentially localized with the adaptorcomplex AP-3 and its cargoes, such as LAMPI.Furthermore, our findings suggest that, much likeLAMPI, the subcellular distribution of mNPC1in Vti1b–syntaxin-8-positive endosomes isaffected by the lack of AP-3.

Previous studies have shown that AP-3deficiency leads to increased targeting of AP-3cargoes to the plasma membrane and thisenrichment has been widely used as a reliableassay for AP-3 function (Dell’Angelica et al.,1999; Di Pietro et al., 2006; Janvier andBonifacino, 2005; Peden et al., 2004; Salazar etal., 2006; Styers et al., 2004). To determinewhether mNPC1 also has a similar AP-3-dependent increase in plasma membranedistribution, surface levels of endogenousmNPC1 were examined by biotinylation of AP-3–/– and AP-3+/+ cells followed by streptavidin-precipitation of biotin-conjugated proteins.Precipitates were resolved by SDS-PAGE andanalyzed by immunoblotting using an antibodydirected against NPC1 to detect endogenousmNPC1. As controls, we also examined thesurface levels of endogenous LAMPI, an AP-3-dependent cargo molecule, as well as that of theTfR and caveolin 1 (Cav1), neither of which istransported in an AP-3-dependent manner(Dell’Angelica et al., 1999). Furthermore, Cav1and � actin were used to examine the behaviorof proteins that, although not containing extracellular epitopes,could co-precipitate with biotinylated transmembrane proteins.

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Results indicate that, as expected, there is an increase inLAMPI distribution to the plasma membrane in AP-3–/– cells

Fig. 5. mNPC1 co-localizes with AP-3. AP-3+/+ and AP-3–/– mouse fibroblasts wereinfected with an adenovirus encoding mNPC1-GFP. The GFP-tagged protein(green) was assessed for co-distribution with AP-3 and with the various organellarmarkers [AP-1, LAMPI and syntaxin 8 (Syn8)] (red) by indirect confocalmicroscopy using an anti-GFP antibody. Co-distribution is visualized in yellow inthe merged images. Boxed areas are shown magnified by 400% (Mag). Bar, 10 �m.

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3645NP-C protein trafficking is adaptor dependent

as compared with AP-3+/+ cells, with no difference detectablefor TfR or Cav1 (Fig. 7A). When the distribution of mNPC1was examined, as was discovered with LAMPI, there was anincrease in the surface level of mNPC1 (Fig. 7A).Quantification of Cav1 normalized surface content indicates aneven greater increase for mNPC1 (sevenfold) than thatobserved for LAMPI (fivefold; Fig. 7B). This finding indicatesthat mNPC1 possesses a similar targeting behavior to thatdescribed for known AP-3 cargoes, such as LAMPI, whichsuggests that mNPC1 is trafficked to the late endosome by theAP-3 complex, as was suggested by results with yeast Ncr1p.

AP-3 deficiency leads to decreased detection ofunesterified cholesterolTo determine whether cholesterol levels can be regulated in an

AP-3-dependent manner, we examined the cellular distributionand content of cholesterol in AP-3+/+ and AP-3–/– cells usingfilipin, a fluorescent marker for unesterified cholesterol (Demeland Van, 1965), and Amplex Red, a cholesterol oxidase-basedassay to quantitatively measure total cholesterol levels(Amundson and Zhou, 1999). Cholesterol distribution wasqualitatively analyzed by epifluorescence and deconvolutionmicroscopy. A comparison of the localization pattern ofcholesterol between AP-3–/– and AP-3+/+ cells indicates thatthere are no appreciable qualitative differences in thesubcellular compartmentalization of unesterified cholesterol(Fig. 8A). By contrast, quantitative assessment of totalunesterified cholesterol by filipin flow cytometry demonstratedthat filipin staining of AP-3–/– cells was diminished by ~50%as compared with AP-3+/+ cells (Fig. 8B,C). Similarly, totalcellular cholesterol was reduced by 30% (n=11, P<0.002; Fig.8D) as determined using Amplex Red. Taken together with theincrease in plasma membrane mNPC1 in AP-3–/– cells, thisdecrease in cellular cholesterol suggests that the AP-3 complexregulates the function of cholesterol transport proteins, such asNPC1, by affecting their spatial distribution within the cell.

AP-3 deficiency affects the fate of mammalian NPC2Our analysis of the yeast Npc2p protein reveals that itslocalization to the vacuolar lumen is dependent on AP-3adaptor complex and AP-1/GGA sorting machinery (see Figs2 and 4). To test the hypothesis that the AP-3 complexfacilitates the trafficking of the non-membrane-associatedhNPC2 protein in mammalian cells, we examined whetherthere was co-localization between the AP-3 complex andhNPC2. We assessed the co-localization of the functionalfusion protein hNPC2-GFP, expressed from an adenovirusconstruct (Berger et al., 2005b), with AP-3 in AP-3+/+ cells or,as a control, AP-3–/– cells. As an additional control, we co-localized hNPC2 with the adaptor complex AP-1, an adaptorthat binds to the cytosolic domains of the mannose-6-phosphate receptors that preferentially deliver NPC2 from theGolgi complex to lysosomal organelles (Willenborg et al.,2005). Protein distribution was analyzed by indirectimmunofluorescence confocal microscopy. Results from thisanalysis show that half of all hNPC2 puncta were positive forAP-1 (Fig. 6D) and that these AP-1-positive puncta wereconcentrated around the perinuclear area, consistent with a

Fig. 6. Quantification of the co-distribution ofmNPC1 and hNPC2 with various organellarmarkers. All images were quantified usingMetamorph software. Values from AP-3+/+ cellsare denoted with white bars and those from AP-3–/– cells with gray bars. All results are theaverage of 10-20 quantified images, except foradaptor co-localizations, for which 30 imageswere analyzed. The results from one of threeindependent experiments are depicted. Standarddeviations are indicated. (A) Percentages of co-localization of mNPC1 with AP-3, AP-1,LAMPI, syntaxin 8 (Syn8), Vti1b or transferrinreceptor (TfR) in AP-3+/+ and AP-3–/– cells. Pvalues are *1 and *2 P<0.0001. (B) Percentages of co-localization of mNPC1 with Vti1b and Syn8 in freshly isolated primary skin fibroblastsfrom grizzled and mocha mice. P values are *1 P<0.001 and *2 P<0.0001. (C) Percentages of co-localization of LAMPI with Vti1b and Syn8in AP-3+/+ and AP-3–/– cells. P values are *1 P<0.02 and *2 P<0.03. (D) Percentages of co-localization of hNPC2 with AP-3, AP-1, LAMPI,Syn8, Vti1b and TfR in AP-3+/+ and AP-3–/– cells.

Fig. 7. Mammalian NPC1 transport is dependent on AP-3. (A) Non-permeabilized AP-3+/+ and AP-3–/– mouse fibroblasts werebiotinylated (Biot.) in order to label surface proteins. Immunoblotanalysis of streptavidin-precipitated proteins was performed forendogenous mNPC1, LAMPI, TfR, caveolin 1 (Cav1) and � actin.As a control, 2% of the total input is shown. (B) Quantification ofmNPC1 surface content was determined by normalizing the (surfacecontent genotype/input content genotype)/(surface content wildtype/input content wild type) to Cav1. The white bars are AP-3+/+

and the gray are AP-3–/–. � actin is shown as a loading control.Numbers in parentheses are the number of independentdeterminations. Standard deviations are indicated.

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Golgi-endosome distribution (Fig. 9).The distribution and extent of co-localization between AP-1 and hNPC2was not affected by the absence of AP-3 (Fig. 6D and Fig. 9). As predictedfrom our yeast data, hNPC2 also co-localized with AP-3-positive organelles(Fig. 6D and Fig. 9), although to alesser extent than that observed formNPC1 (Fig. 6A,D and see Fig. 5). NoAP-3 fluorescence was detected in thecontrol AP-3–/– cells (Fig. 9).

We, and others, have previously co-localized hNPC2 with markers forlysosomal and Golgi structures (Bergeret al., 2005b; Blom et al., 2003). To testwhether loss of the AP-3 complex altersthe steady-state localization of hNPC2,we co-localized hNPC2-GFP with AP-1 �, an adaptor found in Golgi andendosomes, and the endosomal markersTfR, Vti1b, syntaxin 8 and LAMPIin either AP-3+/+ or AP-3–/– cells.Consistent with our previous reports(Berger et al., 2005b), co-localizationof hNPC2 with LAMPI and TfR wasrestricted to a few discrete puncta thatrepresent ~10% and ~20% of allhNPC2-positive structures, respectively(Fig. 6D and Fig. 9; supplementarymaterial Fig. S6). In addition,immunolocalization of hNPC2indicated that there was no detectableeffect of the loss of AP-3 on thesteady-state distribution of hNPC2 inAP-3–/– cells compared with AP-3+/+

cells (Fig. 6D). hNPC2 co-localizedpredominantly (~50%) with theendosome markers Vti1b and syntaxin8 (Fig. 6D and Fig. 9; supplementarymaterial Fig. S6). However, and incontrast to mNPC1, the subcellulardistribution of hNPC2 in these Vti1b-and syntaxin 8-positive compartmentswas minimally affected by the absenceof AP-3 (Fig. 6D and Fig. 9; supplementary material Fig. S6),consistent with the preferential localization of hNPC2 with AP-1.

Because hNPC2 is a secreted protein, we hypothesized that,if the AP-3 complex was involved in transporting this protein,there might be an alteration in the amount of hNPC2 detectedin the media similar to the changes in mNPC1 surface levelsobserved in AP-3–/– cells. To test this hypothesis, we infectedequal numbers of AP-3+/+ and AP-3–/– cells with adenovirusencoding hNPC2-GFP or GFP alone. The infected cells weregrown for 72 hours and the media was collected in order toassess the amount of hNPC2-GFP present. Cell lysates wereprepared as controls for cellular hNPC2 protein levels fromeach sample. Immunoblot analysis revealed the expected 47kDa hNPC2-GFP detectable in the culture media from AP-3+/+

cells with no detectable changes in hNPC2-GFP levels in cell

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lysates from wild-type and AP-3-deficient cells (Fig. 10).However, the amount of hNPC2 detected in the media fromAP-3–/– cells is greatly diminished compared with media fromAP-3+/+ cells (Fig. 10). As a control, an equal amount of theresidual GFP was detected in the media from either cell type.These results support the hypothesis that, in addition to the AP-1/GGA sorting machinery (see Fig. 2) (Willenborg et al.,2005), the subcellular fate of mammalian NPC2 is alsoregulated by the AP-3 complex, probably by an indirectmechanism.

DiscussionMammalian NPC1 and NPC2 are hypothesized to function inretrograde lipid transport from late endosomes and lysosomes(Liscum, 2000), suggesting that the localization of NPC1 andNPC2 to these organelles is of primary importance to their

Fig. 8. Cholesterol is decreased in AP-3–/– fibroblasts. (A) The distribution of unesterifiedcholesterol is unaffected by the loss of AP-3. AP-3+/+ and AP-3–/– mouse fibroblasts werestained with filipin to detect unesterified cholesterol and were imaged by epifluorescence anddeconvolution microscopy. Representative images are shown. (B) Total unesterifiedcholesterol levels in AP-3+/+ and AP-3–/– mouse fibroblasts were measured by flow cytometryof filipin-labeled cells. A representative histogram is shown with unlabeled and labeled AP-3–/– and AP-3+/+ cells in black and gray, respectively. (C) Quantification of filipinfluorescence intensities for AP-3+/+ and AP-3–/– fibroblasts normalized to unlabeled cells.Results are the average of three independent experiments each performed in triplicate.Standard deviations are indicated. (D) Biochemical quantification of total cellular cholesterollevels in AP-3+/+ and AP-3–/– fibroblasts was assessed using an Amplex Red CholesterolAssay Kit. Fibroblasts were either untreated or treated with M�CD. Cholesterol content (�gcholesterol/�g protein for each genotype calculated as a percentage of AP-3+/+) for AP-3+/+

and AP-3–/– cells are shown. All determinations were performed at least in triplicate in threeindependent experiments (n=11). Standard deviations and statistically significant differences(P<0.002) are indicated.

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function. Unraveling the mechanisms that regulate NP-Cprotein transport is therefore crucially important tounderstanding the pathology of the disease. Furthermore, thesetrafficking pathways might offer new therapeutic targets.

We have exploited yeast cell biology to assess the routes bywhich the two NP-C homologs, scNcr1p and scNpc2p, traffic

to the vacuole. Our analysis indicates that there is arequirement for the AP-1/GGA pathway in trafficking scNpc2pto the vacuolar lumen. Interestingly, there is mis-targeting ofscNpc2p to punctate vesicles throughout the cell as well as totubular structures that surround the vacuole in gga1�gga2�mutants. This localization to structures outside of the vacuole

suggests that scNpc2p might normally trafficthrough intermediary compartments, such as theprevacuolar compartment, which become easilydetectable when the GGA proteins are deficient.The probability that scNpc2p traffics throughintermediary compartments is strengthened byour finding that scNpc2p accumulates in thelumen of class E compartments, whichaccumulate in a vps4� background (Zahn et al.,2001). These results are consistent with thefinding that hNPC2 binds to and is dependentupon the mannose-6-phosphate receptor forlysosomal targeting (Naureckiene et al., 2000;Willenborg et al., 2005) and with ourobservation that hNPC2 extensively co-localizes with AP-1.

In contrast to the GGA proteins, which seemto only affect scNpc2p localization, our findingsindicate that the AP-3 pathway is required forthe proper transport of both NP-C proteins. Wefound that, as seen for ALP (a protein traffickedspecifically by the AP-3 complex in yeast)(Cowles et al., 1997; Piper et al., 1997; Stepp etal., 1997), scNcr1p and scNpc2p are alsodependent on the AP-3 complex for propertrafficking. When each individual deletion ofthe AP-3 complex was evaluated for its effectsupon scNcr1p localization, except for apm3�,there was a clear mislocalization of the proteinto cytoplasmic punctate structures in addition tothe normal vacuolar membrane localization inthe majority of mutants, similar to what wasseen for ALP (see Fig. 4).

scNcr1p is found throughout the cytoplasm inpunctate structures in all AP-3 mutants tested,except for apm3� (see Fig. 4), consistent withprevious work in which scNcr1p localizationwas examined only in this single AP-3 mutantbackground (Zhang et al., 2004). The findingthat this single deletion mutant does not alterthe scNcr1p trafficking pattern suggests that the� subunit of the yeast AP-3 complex might bedispensable for the transport of specific cargomolecules, because the localization of bothALP and scNpc2p is altered in the apm3�

Fig. 9. Localization of hNPC2. AP-3+/+ and AP-3–/–

mouse fibroblasts were infected with an adenovirusencoding hNPC2-GFP. The GFP-tagged protein(green) was assessed for co-distribution with variousorganellar markers (AP-3, AP-1, LAMPI, Syn8)(red) by indirect confocal microscopy using an anti-GFP antibody. Co-distribution is visualized inyellow in the merged images. Boxed areas areshown magnified by 400% (Mag). Bar, 10 �m.

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mutant (see Fig. 4). The observation that a deficiency in theyeast Apm3p subunit does not manifest evident scNcr1pphenotypes is not unprecedented. Deficiencies in theCaenorhabditis elegans AP-2 �2 (Boehm and Bonifacino,2002; Grant and Hirsh, 1999) and AP-3 �3 (Shim and Lee,2005) subunits do not fully reproduce the extent and quality ofthe phenotypes observed with deficiencies in any of the otherthree AP-2 and AP-3 adaptor subunits. Although themechanism for the phenotypic differences among subunitdeficiencies remains unknown, a possible explanation is thepresence of partial adaptor complexes constituted by just twosubunits, which has been described for mouse models deficientin the AP-3 �3 chain (Peden et al., 2002; Yang et al., 2000).In this regard, recent evidence indicates that, in some cases,yeast AP-3 complexes assembled with mutant Apm3p subunitsare unable to support the targeting of membrane proteinscontaining tyrosine-based motifs to the same extent asobserved for apm3� cells, in which the Apm3p subunit isabsent altogether (Wen et al., 2006). However, these samemutant complexes show no defect in the targeting of an AP-3-trafficking-dependent protein containing a di-leucine-basedmotif to the vacuolar membrane (Wen et al., 2006), suggestingthat deficiencies in the Apm3p subunit might lead to targetingdefects in only a subset of AP-3 cargoes. Alternatively, thedifferential effect on scNcr1p localization might be due todifferences in the recognition of the protein, because theendosomal sorting signal for scNcr1p has not been defined;although, the mammalian NPC1 protein contains a di-leucinemotif at its C-terminus (Watari et al., 1999) and separatesorting information in the sterol sensing domain (Scott et al.,2004). Further analysis will be necessary to determine why theloss of APM3 does not alter the trafficking pattern of scNcr1p.

Zhang et al. reported that scNcr1p did not traffic through theAP-3 pathway, based on their analysis of apm3� cells, butrather through intermediary compartments that are a part of the

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VPS pathway (Zhang et al., 2004). When we examinedwhether scNcr1p could transit through the VPS pathway, wefound that, unlike scNpc2p localization, there was nodetectable defect in the localization of scNcr1p. In contrast toZhang et al., our analysis of the VPS route was restricted tostudies just in a vps4� background (supplementary materialFig. S3). Therefore, a distinctive possibility is that mutationsin the VPS pathway as well as in the AP-3 route could bothaffect the localization of scNcr1p but to different degreesdepending on the specific nature of the mutations, the geneticloci affected or the assays used in our studies and those byZhang et al. Thus, much like scNpc2p, the targeting of scNcr1pcould be modulated by other targeting mechanisms in additionto the AP-3 route.

The finding that the AP-3 complex facilitates thelocalization of the NP-C proteins in yeast directed us toexamine whether the trafficking of the mammalian NP-Cproteins was also AP-3-dependent in mammalian cells.Previous work has demonstrated that a deficiency in the AP-3complex leads to an accumulation of AP-3-dependent cargomolecules at the cell surface without affecting theirendosomal/lysosomal steady-state localization (Dell’Angelicaet al., 1999). Indeed, our results indicate that, as seen for theAP-3-transported LAMPI protein (see Fig. 7) (Dell’Angelicaet al., 1999), mNPC1 is mis-targeted to the plasma membranein AP-3-deficient mouse fibroblasts to an even greater extentthan LAMPI. Concomitant with mNPC1 redistribution to theplasma membrane, mNPC1 co-localization with the early/lateendosome markers Vti1b and syntaxin 8 increases, suggestingthat mNPC1 might be delayed in these compartments. Similarresults are seen for LAMPI. Importantly, both Vti1b andsyntaxin 8 levels are not affected by a deficiency in AP-3(Salazar et al., 2006), with Vti1b transport from endosomesbeing dependent on EpsinR-AP-1 mechanisms (Hirst et al.,2004). These results are consistent with the notion thatmNPC1, like scNcr1p, is an AP-3 cargo molecule. This findingis very interesting in light of our evidence showing that AP-3–/– cells have less detectable unesterified and total cholesterolthan AP-3+/+ fibroblasts, with no obvious effect on the steady-state cholesterol distribution (see Fig. 8). The precisemechanism leading to decreased cholesterol content in AP-3-deficient cells remains unclear, but it is reasonable tohypothesize that it is related to the redistribution of NPC1,NPC2 and/or other late endosomal proteins that are mis-targeted in the absence of AP-3. Of particular interest is theobservation that genetic ablation of mouse LAMPI andLAMPII, which are both AP-3 cargoes (Bonifacino and Traub,2003), alters the cellular content of cholesterol detected byfilipin staining (Eskelinen et al., 2004). Alternatively, otherproteins, such as MLN64, a membrane protein targeted to lateendosomes that is predicted to bind cholesterol, based uponstructural studies (Alpy et al., 2001; Murcia et al., 2006), couldcontribute to the phenotypes observed in AP-3–/– cells.Irrespective of the precise mechanism that leads to reducedcellular cholesterol levels, targeted perturbation of AP-3complex function might help ameliorate cholesterolaccumulation defects seen in a number of disorders, includingin NP-C.

We also examined AP-3-dependent trafficking of hNPC2.Based upon our results showing increased trafficking ofmNPC1 to the plasma membrane, we hypothesized that, if the

Fig. 10. Localization of hNPC2 is dependent on the AP-3 complex.Equal numbers of AP-3+/+ and AP-3–/– mouse fibroblasts wereinfected with adenovirus containing hNPC2-GFP (top set) or, as acontrol, GFP alone (top set). Media and cell lysates were collectedfor immunoblot analysis. As a control for cell type and loading, blotswere analyzed for the presence of the AP-3 � subunit and tubulin,respectively. Samples are shown in duplicate.

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transport of hNPC2 was AP-3-dependent, we would detect anincrease in hNPC2 in media. Surprisingly, although ouranalysis indicated that hNPC2 trafficking is affected by AP-3,we found that our ability to detect hNPC2 in the media of AP-3-deficient cells was greatly diminished as compared to mediafrom AP-3+/+ cells. How can the AP-3 pathway affect amannose-6-phosphate-modified luminal protein? It isspeculative at this point, but it is attractive to consider thepossibility that AP-3 plays an indirect role in the transport ofhNPC2, with a membrane-bound protein, such as NPC1, oranother AP-3-interacting membrane protein, binding the smallsoluble protein. Alternatively, AP-3 might regulate thetargeting of a factor that does not bind hNPC2, yet is requiredfor hNPC2 targeting via an AP-3-independent route; such afactor could be, for example, a vesicular SNARE protein thatdirectly binds AP-3 (Martinez-Arca et al., 2003), such asVAMP7 (also known as SYBL1). The trafficking andexpression of this vesicular SNARE has recently been shownto be affected by AP-3 deficiencies (Salazar et al., 2006). Thistype of mechanism has been postulated to explain thephenotypes observed with carboxypeptidase Y, a mannose-6-phosphate-modified luminal protein, in AP-3-deficient yeastcells (Bonangelino et al., 2002).

Recent studies show that the lysosomal targeting of NPC2is dependent upon the presence of the mannose-6-phosphatereceptors in fibroblasts (Willenborg et al., 2005). Thisobservation is consistent with our findings in yeast andmammalian cells. Yeast Npc2p is trafficked in a GGA-dependent as well as an AP-3-dependent manner (see Figs 2and 4). Similarly, hNPC2 predominantly co-localizes with theadaptor complex AP-1 and to a lesser extent with AP-3 inmouse fibroblasts. These observations support a model inwhich AP-3 might play an indirect role in the targeting of thesoluble hNPC2 protein, possibly by increasing the surfacedisplay of a hNPC2-binding partner that allows for greateruptake of the free protein from the media. However, we cannotrule out the possibility that the loss of hNPC2 detection couldoccur by a change in the secretion of the protein. Whether the

effect of AP-3 deficiency on hNPC2 detection in the media isdue to enhanced uptake into endosomes or the inability ofhNPC2 to be transported into an appropriate compartment forefflux from the cell remains to be explored. Irrespective of themechanism, the factors that can most readily influence thefunction and targeting of NPC2 are those that reside with it ina common compartment. It is of interest to point out thathNPC2 is present in Vti1b–syntaxin-8-positive endosomes, alocalization that, in the case of mNPC1, becomes evident incells that lack functional AP-3 complexes. Further analyseswill be necessary to delineate the exact role that AP-3 plays inthe transport of hNPC2.

Our studies indicate that the AP-3 complex facilitates thetrafficking of the NP-C proteins. These findings indicate thatthe targeting of the NP-C proteins is controlled by multiplesorting machineries (AP-1/GGA/VPS and AP-3). In addition,the finding that hNPC2 transport is AP-3-dependentdemonstrates that the sorting of a non-membrane-boundprotein might be controlled indirectly by the AP-3 complex.This complex and the proteins that are transported by it(Dell’Angelica et al., 1998; Dell’Angelica et al., 1999;Faundez and Kelly, 2000; Honing et al., 1998; Robinson, 2004;Salazar et al., 2005; Salazar et al., 2004a) could provide newinsight into the function of NPC1 and NPC2, and could providenovel therapeutic targets for the treatment of NP-C disease.

Materials and MethodsStrains, plasmids and cell cultureAll DNA manipulations were performed according to standard protocols (Sambrookand Russell, 2001) and all yeast media was prepared by standard methods (Adamset al., 1997). All yeast strains and plasmids are described in Table 1. Chemicalswere obtained from Fisher Scientific (Pittsburgh, PA), Sigma Chemical (St Louis,MO), Stratagene (La Jolla, CA) or USBiological (Swampscott, MA) unlessotherwise noted.

HEK293 cells were grown in DMEM (4.5 g/l glucose) (Cellgro, Herndon, VA)supplemented with 10% FBS (Hyclone, Logan, UT), 100 units/ml penicillin and100 �g/ml streptomycin (Cellgro). Immortalized AP-3 �–/– mocha mouse fibroblaststransduced with a retrovirus encoding the � subunit of AP-3 (AP-3+/+ cells) or withan empty retrovirus (AP-3–/– cells) were grown in DMEM supplemented with 10%FBS, 100 units/ml penicillin, 100 �g/ml streptomycin and 200 �g/ml hygromycinB (Peden et al., 2004).

Table 1. Yeast strains, cell lines, plasmids and viruses used in this studyStrains/cell lines/plasmids/viruses Genotype/description Source

ACY402 (BY4741) (wild type) MATa his3 leu2 met15 ura3 Research Genetics, Invitrogen Corporation, Carlsbad, CA

ACY969 MATa ade2::hisG his3 leu2 met15 trp1 ura3 GGA1::TRP1 GGA2::HIS3 (Zhdankina et al., 2001)ACY1084 MATa his3 leu2 met15 ura3 APL3::KanMX4 Research GeneticsACY1085 MATa his3 leu2 met15 ura3 APL1::KanMX4 Research GeneticsACY1086 MATa his3 leu2 met15 ura3 APM4::KanMX4 Research GeneticsACY911 MATa his3 leu2 met15 ura3 APS2::KanMX4 Research GeneticsACY1087 MATa his3 leu2 met15 ura3 APL6::KanMX4 Research GeneticsACY909 MATa his3 leu2 met15 ura3 APL5::KanMX4 Research GeneticsACY1088 MATa his3 leu2 met15 ura3 APM3::KanMX4 Research GeneticsACY1089 MATa his3 leu2 met15 ura3 APS3::KanMX4 Research GeneticsHEK293 E1+/+ V.F.AP-3+/+ mocha mouse fibroblasts transduced with a retrovirus encoding the AP-3 � subunit (Peden et al., 2004)AP-3–/– mocha mouse fibroblasts transduced with an empty retrovirus (Peden et al., 2004)grizzled Primary skin fibroblasts V.F.mocha Primary skin fibroblasts V.F.pAC1184 pNCR1-NCR1-GFP URA 2 � (Berger et al., 2005a)pAC1185 pNPC2-NPC2-GFP URA 2 � (Berger et al., 2005b)pAC1439 GFP-ALP (Cowles et al., 1997)mNPC1-GFP adenovirus pCMV-mNPC1-GFP (Paul et al., 2005)hNPC2-GFP adenovirus pCMV-hNPC2-GFP (Berger et al., 2005b)GFP adenovirus pCMV-GFP V.F.

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AntibodiesPrimary antibodies used in this study were: anti-mouse LAMPI (1D4B) used at1:1000 and monoclonal anti-AP-3 � (SA4) ascites used at 1:500 (DevelopmentalStudies Hybridoma Bank, University of Iowa); mouse monoclonal anti-TfR used at1:1000 and anti-NPC1 used at 1:500 (Zymed Laboratories, San Francisco, CA);anti-�-tubulin used at 1:1000 (DM1A) and anti-� actin (actin) used at 1:5000(Sigma); rabbit anti-GFP used at 1:5000 (Synaptic Systems, Goettingen, Germany);anti-mouse Caveolin1 (Cav1) used at 1:2000 and mouse monoclonal anti-AP1 �used at 1:500 (BD Biosciences Transduction Labs); mouse monoclonal anti-syntaxin 8 used at 1:500 and mouse monoclonal anti-Vti1b used at 1:500 (BDBioscience Pharmigen). Secondary antibodies used were: Alexa-Fluor-568-conjugated goat anti-mouse used at a 1:1000 dilution (Molecular Probes, InvitrogenDetection Technologies, Carlsbad, CA), and goat anti-rabbit HRP and goat anti-mouse HRP used at 1:7000 (Zymed Laboratories, Invitrogen Immunodetection,Carlsbad, CA).

Adenovirus productionAn adenovirus encoding hNPC2-GFP was created as previously described (Bergeret al., 2005b). Briefly, hNPC2 was amplified by PCR from human peripheral bloodmononuclear cell cDNA (kind gift of Silvija I. Staprans, Emory University, Atlanta,GA). hNPC2 was first cloned into the pEGFP vector (Clontech, Palo Alto, CA) tocreate an in-frame C-terminal GFP fusion. PCR was used to amplify the entirecDNA fused to GFP followed by cloning into the AdEasy Adenoviral Vector system(Stratagene). In short, hNPC2-GFP was cloned NotI-HindIII into the pShuttle-CMVvector followed by homologous recombination into the pAdEasy-1 adenoviralbackbone. The resulting recombinant DNA was transfected into HEK293 cells usingLipofectamine 2000 (Invitrogen, Carlsbad, CA) for plaque formation. Virus wasamplified in HEK293 cells and purified using the BD Adeno-X Virus PurificationKit (Clontech) as directed.

Flow cytometryUnesterified cholesterol levels for mocha (AP-3–/–) and rescued (AP-3+/+) cells (Pedenet al., 2004) were quantified by flow cytometry. Briefly, cells were placed insuspension using PBS with 5 mM EDTA followed by fixation in 4%paraformaldehyde in PBS at 4°C for 20 minutes. After fixation, the paraformaldehydewas quenched by washing the cells with PBS supplemented with 25 mM glycine.Cells were incubated with 50 �g/ml of filipin for 2 hours at 37°C to label unesterifiedcholesterol. Excess filipin was removed by washing the cells with PBS three timesfor 5 minutes each. Unesterified cholesterol levels were quantified using a MoFloHigh Performance Cell Sorter (DakoCytomation, Fort Collins, CO). Results wereanalyzed using FlowJo software version 4.4.4 (Tree Star, Ashland, OR).

Cholesterol quantitationTotal cellular cholesterol was determined using an Amplex Red Cholesterol AssayKit (Molecular Probes, A12216) and following manufacturer’s instructions. Briefly,cells that had been seeded in six-well plates were incubated with or without methyl-�-cyclodextrin (M�CD) at 10 mg/ml for 1 hour at 37°C in DMEM media to partiallydeplete cholesterol. Incubating cells for longer periods of time with M�CDcompromised cell viability. Cells were washed twice in ice-cold PBS and lysed in100 �l of buffer A (150 mM NaCl, 10 mM HEPES, 1 mM EGTA and 0.1 mMMgCl2, pH 7.4) containing 0.5% Triton X-100 plus Complete anti-protease mixture.Both free cholesterol and cholesterol esters were analyzed in 5 �l of whole-cellextract. Fluorescence was measured with a Synergy HT (Biotek, Winooski, VT)microplate reader using an excitation of 540 nm and a fluorescence detection of 575nm. Results were expressed as �g cholesterol/�g protein and then calculated as thepercentage of control. All determinations were performed at least in triplicate inthree independent experiments (n=11).

Direct fluorescence microscopyscNcr1p and scNpc2p were localized in living yeast cells as the C-terminal GFPfusion proteins, scNcr1p-GFP (pAC1184) and scNpc2p-GFP (pAC1185), aspreviously described (Berger et al., 2005a; Berger et al., 2005b). Alkalinephosphatase (ALP) was localized in living yeast cells as the N-terminal GFP fusionprotein, GFP-ALP (Cowles et al., 1997). Wild-type yeast cells (ACY402),gga1�gga2� yeast cells (ACY969), deletions of AP-1 complex members (apl4�,apl2�, apm1�, apm2� and aps1�), deletions of AP-2 complex members (apl3�,apl1�, apm4� and aps2�) or deletions of AP-3 complex members (apl5�, apl6�,apm3� and aps3�) were transformed with scNCR1-GFP, scNPC2-GFP or GFP-ALP plasmids and viewed by direct fluorescence microscopy using an OlympusBX60 epifluorescence microscope equipped with an Olympus UPlan Apo100/1.35 oil iris DIC objective, a GFP optimized barrier filter and a PhotometricsQuantix digital camera. All images were collected using IPlab Spectrum software.

Indirect immunofluorescenceImmunofluorescence of cultured cells was performed as previously described(Faundez et al., 1997; Wei et al., 1998). Briefly, cells were placed on ice and fixedin 4% paraformaldehyde in PBS for 20 minutes. Following fixation, cells werequenched by washing twice with PBS containing 25 mM glycine and then once in

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PBS. Cells were permeabilized by incubating with block containing 15% horseserum, 0.02% saponin in PBS, 2% BSA and 1% fish skin gelatin for 1 hour at roomtemperature. Primary antibody incubation in block was performed for 1 hour at37°C. Cells were washed three times with block and incubated with secondaryantibodies diluted in block for 1 hour at room temperature. Cells were washed threetimes with block, once with PBS and were then mounted in gelvatol. Cells werevisualized by confocal microscopy using a Zeiss Axiovert 100 M microscopecoupled to HeNe1 and argon ion lasers. All images were viewed and acquired usinga Plan Apochromat 63/1.4 oil DIC objective and Zeiss LSM 510 sp1 software.Co-localization analyses were performed using Metamorph software (UniversalImaging, Downingtown, PA) and obtained from at least ten images collected fromtwo coverslips per condition and per independent experiment. All determinationswere performed in three independent experiments.

Deconvolution microscopyImmunoflorescence was performed as described (Salazar et al., 2004b). Imageswere acquired with a scientific-grade cooled charge-coupled device (Cool-Snap HQwith ORCA-ER chip) on a multi-wavelength, wide-field, three-dimensionalmicroscopy system (Intelligent Imaging Innovations, Denver, CO), based on a 200Minverted microscope using a 63 numerical aperture 1.4 lens (Carl Zeiss,Thornwood, NY). Immunofluorescent samples were imaged at room temperatureusing a Sedat filter set (Chroma Technology, Rockingham, UT), in successive 0.25�m focal planes. Out-of-focus light was removed with a constrained iterativedeconvolution algorithm (Swedlow et al., 1997).

BiotinylationAP-3–/– and AP-3+/+ cells were grown to confluence and biotinylated as a monolayer(Salazar and Gonzalez, 2002). Cells were lifted off plates at 4°C with PBS-10 mMEDTA and sedimented at 800 g for 5 minutes. Cell pellets were resuspended in 5%SDS, 0.15 M Tris-HCl pH 6.7, 30% glycerol diluted 1:3 (vol:vol) with RIPA buffer(25 mM Tris-HCl pH 8.2, 50 mM NaCl, 0.5% NP-40, 0.5% DOC, 0.1% SDS, 0.1%azide) plus Complete anti-protease mixture. After a brief sonication (2 times 5seconds each) samples were diluted 1:10 with PBS containing 0.5% NP-40. Celldebris was removed by sedimentation at 15,000 g for 20 minutes. A sample of 500�g of each supernatant was precipitated with streptavidin-conjugated Sepharosebeads over 5 hours at 4°C. After extensive washes with PBS containing 0.5% NP-40, bead-bound material was eluted with SDS-sample buffer, and proteins wereresolved by SDS-PAGE and analyzed by immunoblot. Immunoblots were probedwith an anti-NPC1 antibody, an anti-LAMPI antibody, an anti-TfR antibody and ananti-Cav1 antibody, or, as a control, an anti-� actin antibody. Immunoreactive bandswere visualized by chemiluminescence.

Media detection assaysDetection of hNPC2 in media was assessed by infecting 2105 fibroblasts withadenovirus encoding either human NPC2-GFP or GFP alone. After 7 hours, the cellswere washed with DMEM and then grown in 800 �l of fresh media. After 72 hours,the media was collected and cell lysates were prepared by washing cells twice withPBS followed by lysis. 65 �l of media and 30 �g of lysate (5% of total) were usedfor immunoblot analysis. Proteins were resolved by SDS-PAGE and transferred toa PVDF membrane. Immunoblotting was performed by standard methods (Towbinet al., 1979). Blots were probed with an anti-GFP rabbit polyclonal antibody, ananti-AP-3 � antibody or an anti-tubulin antibody. Immunoreactive bands werevisualized by chemiluminescence.

Statistical analysisExperimental conditions were compared with the non-parametric Wilcoxon-Mann-Whitney rank sum test using Synergy KaleidaGraph v3.6.2 (Reading, PA).

We thank Annette L. Boman, Todd R. Graham, Andrew A. Pedenand Silvija I. Staprans for supplying reagents, and members of theFaundez and Corbett labs for helpful discussions. This work wassupported by a pre-doctoral NRSA fellowship from the NationalInstitute of Neurological Disorders and Stroke, National Institutes ofHealth, to A.C.B. (NS044743), a research training grant inDermatology from the National Institute of Arthritis andMusculoskeletal and Skin Diseases, National Institutes of Health, toM.L.S. (2T32AR007587-11), grants from the National Niemann-PickDisease Foundation to A.H.C. and to R.A.M., and grants from theNational Institutes of Health to R.A.M. (NS40564) and to V.F.(NS42599 and GM 077569). An award from the American HeartAssociation supported E.W.

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