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First published online 25 September 2007
doi: 10.1242/jcs.03487

Journal of Cell Science 120, 3640-3652 (2007)
Published by The Company of Biologists 2007
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The subcellular localization of the Niemann-Pick Type C proteins depends on the adaptor complex AP-3

Adam 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,{ddagger}

1 Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
2 Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA
3 Graduate Program in Biochemistry, Cell, and Developmental Biology, Emory University Graduate Division of Biological and Biomedical Sciences, Atlanta, GA 30322, USA
4 Departments of Physiology and Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA


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

 

Figure 2
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  		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{Delta}gga2{Delta} deletion yeast cells. The GFP-tagged proteins were visualized by direct fluorescence microscopy. Arrows indicate areas of mislocalization. A magnification of the boxed area is depicted. The corresponding DIC images are shown.

 

Figure 3
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  		Fig. 3. The AP-2 complex is not required for the localization of scNcr1p or scNpc2p. GFP-ALP (top), scNCR1-GFP (middle) and scNPC2-GFP (bottom) plasmids were transformed into a yeast deletion of the AP-2 complex (apl1{Delta}). The GFP-tagged proteins were visualized by direct fluorescence microscopy. The corresponding DIC images are shown.

 

Figure 4
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  		Fig. 4. The AP-3 complex directs the localization of scNcr1p and scNpc2p. GFP-ALP (top), scNCR1-GFP (middle) and scNPC2-GFP (bottom) plasmids were transformed into yeast deletions of the AP-3 complex (apl6{Delta}, apl5{Delta}, apm3{Delta} and aps3{Delta}). The GFP-tagged proteins were visualized by direct fluorescence microscopy. Arrows indicate areas of mislocalization. The corresponding DIC images are shown.

 

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

 

Figure 6
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  		Fig. 6. Quantification of the co-distribution of mNPC1 and hNPC2 with various organellar markers. All images were quantified using Metamorph software. Values from AP-3+/+ cells are denoted with white bars and those from AP-3–/– cells with gray bars. All results are the average of 10-20 quantified images, except for adaptor co-localizations, for which 30 images were analyzed. The results from one of three independent experiments are depicted. Standard deviations are indicated. (A) Percentages of co-localization of mNPC1 with AP-3, AP-1, LAMPI, syntaxin 8 (Syn8), Vti1b or transferrin receptor (TfR) in AP-3+/+ and AP-3–/– cells. P values are *1 and *2 P<0.0001. (B) Percentages of co-localization of mNPC1 with Vti1b and Syn8 in freshly isolated primary skin fibroblasts from 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 Syn8 in 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.

 

Figure 7
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  		Fig. 7. Mammalian NPC1 transport is dependent on AP-3. (A) Non-permeabilized AP-3+/+ and AP-3–/– mouse fibroblasts were biotinylated (Biot.) in order to label surface proteins. Immunoblot analysis of streptavidin-precipitated proteins was performed for endogenous mNPC1, LAMPI, TfR, caveolin 1 (Cav1) and beta actin. As a control, 2% of the total input is shown. (B) Quantification of mNPC1 surface content was determined by normalizing the (surface content genotype/input content genotype)/(surface content wild type/input content wild type) to Cav1. The white bars are AP-3+/+ and the gray are AP-3–/–. beta actin is shown as a loading control. Numbers in parentheses are the number of independent determinations. Standard deviations are indicated.

 

Figure 8
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  		Fig. 8. Cholesterol is decreased in AP-3–/– fibroblasts. (A) The distribution of unesterified cholesterol is unaffected by the loss of AP-3. AP-3+/+ and AP-3–/– mouse fibroblasts were stained with filipin to detect unesterified cholesterol and were imaged by epifluorescence and deconvolution microscopy. Representative images are shown. (B) Total unesterified cholesterol levels in AP-3+/+ and AP-3–/– mouse fibroblasts were measured by flow cytometry of 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 filipin fluorescence 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 cholesterol levels in AP-3+/+ and AP-3–/– fibroblasts was assessed using an Amplex Red Cholesterol Assay Kit. Fibroblasts were either untreated or treated with MbetaCD. Cholesterol content (µg cholesterol/µg 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 three independent experiments (n=11). Standard deviations and statistically significant differences (P<0.002) are indicated.

 

Figure 9
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  		Fig. 9. Localization of hNPC2. AP-3+/+ and AP-3–/– mouse fibroblasts were infected with an adenovirus encoding hNPC2-GFP. The GFP-tagged protein (green) was assessed for co-distribution with various organellar markers (AP-3, AP-1, LAMPI, Syn8) (red) by indirect confocal microscopy using an anti-GFP antibody. Co-distribution is visualized in yellow in the merged images. Boxed areas are shown magnified by 400% (Mag). Bar, 10 µm.

 

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

 

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© The Company of Biologists Ltd 2007