Figures and data in Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1

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6 figures, 1 table and 1 additional file

Figures

Figure 1

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Human NPC1: topology, structure, and function.

(A) Predicted topology of human NPC1 based on the data of Davies and Ioannou (2000). Functional domains of the protein are shown in different colors. NTD, N-terminal domain; MLD, middle lumenal domain; CTD, C-terminal domain. (B) Structure of full-length NPC1 as determined by cryo-electron microscopy (Gong et al., 2016) was accessed from the Protein Data Bank (PDB: 3JD8) and color-matched to the topology map in (A). Image was generated using the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC. TM1, transmembrane helix 1; SSD, sterol-sensing domain. (C) Model for cholesterol transfer. (1) NPC2 brings cholesterol to NPC1, which is embedded in the lysosomal membrane. (2) NPC2 binds to the MLD of NPC1 and transfers its cholesterol to the NTD of NPC1 in a hydrophobic handoff (Kwon et al., 2009; Deffieu and Pfeffer, 2011). (3) Cholesterol is transferred from the NTD to the membrane – embedded SSD.

https://doi.org/10.7554/eLife.38564.002

Figure 2

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Mutant versions of human NPC1 used in cholesterol esterification assays.

(A) pNTD-TM1 encodes the signal sequence of human NPC1, followed sequentially by the NTD, TM1, a lysosomal targeting signal, and epitope tags. (B) p∆NTD encodes NPC1 with a deletion of the NTD (amino acids 25–252). The cleaved signal sequence is shown.

https://doi.org/10.7554/eLife.38564.003

Figure 3

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Localization of mutant NPC1 proteins to lysosomes.

SV589 cells were transfected with the indicated plasmids encoding Flag-tagged fragments of NPC1 as described in Materials and methods. Cells were fixed and double stained with 0.8 µg/ml of rabbit monoclonal anti-Flag IgG (*green*) together with 1 µg/ml of mouse monoclonal anti-LAMP-2 IgG (*red*), and images were merged (*yellow*). LAMP-2 is a marker for lysosomes. Immunofluorescence microscopy was performed as described in Materials and methods. Scale bar, 20 µm.

https://doi.org/10.7554/eLife.38564.004

Figure 4

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Trans-complementation between NTD and ∆NTD fragments of NPC1.

On day 0, NPC1^-/- cells (A) and NPC1^-/- cells stably expressing ∆NTD (B) were set up in medium A with 5% FCS at 2.5 × 10⁵ cells/60 mm dish. On day 1, monolayers were switched to fresh medium A (without antibiotics) with 5% LPDS and then transfected with the indicated plasmids encoding either full-length NPC1 or NTD-TM1. All dishes received FuGENE HD and a total of 2 µg DNA/dish adjusted with pcDNA3.1. After incubation for 24 hr, cells were washed once with PBS and switched to medium A with 5% LPDS containing 50 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 5% LPDS or 10% FCS containing lipoproteins. After incubation for 4 hr at 37°C, each cell monolayer was pulse-labeled for 2 hr with 0.1 mM sodium [¹⁴C]oleate (8568 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [¹⁴C]oleate and [¹⁴C]triglycerides as described in Materials and methods. Each bar indicates the mean of duplicate incubations with individual values shown. The mean cellular content of [¹⁴C]triglycerides in the presence of FCS was not significantly different in NPC1^-/- and ∆NTD cells (11.0 and 11.8 nmol/hr per mg protein, respectively). The bottom panel shows immunoblots of whole cell extracts (40 μg) using 0.36 μg/ml of rabbit monoclonal anti-NPC1 and 1.8 μg/ml of mouse monoclonal anti-NPC2. ‡ denotes the endogenous, stably transfected ∆NTD.

https://doi.org/10.7554/eLife.38564.005

Figure 5

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Restoration of cholesterol transport to ∆NTD sequences requires that the NTD of NPC1 localize to lysosomes and bind cholesterol.

On day 0, ∆NTD cells were set up and transfected on day one as described in Figure 4 with the indicated amount of one of the following plasmids: pcDNA3.1 (control) NPC1 (A), pNTD-TM1 (B), pNTD-TM13 (C), or pNTD*-TM1 (D). After incubation for 24 hr, cells were switched to medium A with 5% LPDS containing 50 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 5% LPDS or 10% FCS. After incubation for 4 hr at 37°C, each cell monolayer was pulse-labeled for 2 hr with 0.1 mM sodium [¹⁴C]oleate (9019 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [¹⁴C]oleate and [¹⁴C]triglycerides. Each bar indicates the mean of duplicate incubations with individual values shown. The mean cellular content of [¹⁴C]triglycerides in the presence of FCS was not significantly different in cells transfected with pNPC1, pNTD-TM1, pNTD-T13, and pNTD*-TM1 (13.3, 12.7, 12.3, and 13.3 nmol per hr/mg protein, respectively). The bottom panel shows immunoblots of whole cell extracts (40 µg/lane) using 0.36 µg/ml of rabbit monoclonal anti-NPC1 and 1.8 µg/ml of mouse monoclonal anti-NPC2. ‡ denotes the endogenous, stably transfected ∆NTD.

https://doi.org/10.7554/eLife.38564.006

Figure 6

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Transfer of cholesterol from NPC1(∆Ω) to ∆NTD as determined by cholesterol esterification assay.

(A) Model showing how the NTD of NPC1(∆Ω) might transfer its cholesterol to the SSD of ∆NTD. (B) Cholesterol esterification assay. On day 0, NPC1^-/- cells and ∆NTD cells were set up for experiments as described in Figure 4 and transfected on day one with the indicated plasmids. All dishes contained a total of 3 µg of DNA adjusted with pcDNA3.1. After incubation for 24 hr, cells were washed once with PBS and switched to medium A with 5% LPDS containing 50 µM sodium compactin and 50 µM sodium mevalonate. On day 3, the cells received fresh medium B containing compactin and mevalonate in the presence of either 5% LPDS or 10% FCS. After incubation for 4 hr at 37°C, each cell monolayer was pulse-labeled for 2 hr with 0.1 mM sodium [¹⁴C]oleate (9452 dpm/nmol). The cells were then harvested for measurement of their content of cholesteryl [¹⁴C]oleate and [¹⁴C]triglycerides. Each bar indicates the mean of duplicate incubations with individual values shown. The mean cellular content of [¹⁴C]triglycerides in the presence of FCS was not significantly different in NPC1^-/- and ∆NTD cells (11.8 and 14.8 nmol/hr per mg protein, respectively). The bottom panel shows immunoblots of whole cell extracts (40 μg) using 3.6 μg/ml rabbit polyclonal anti-NPC1(NTD) that detects both NPC1 ((lanes 2–4, 9–11) and NPC1 (∆Ω) (lanes 5–7, 12–14), 0.36 μg/ml rabbit monoclonal anti-NPC1 that detects ∆NTD* (lanes 8–14), and 0.2 μg/ml of mouse monoclonal anti-β-actin. ‡ denotes the endogenous, stably transfected ∆NTD.

https://doi.org/10.7554/eLife.38564.007

Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Chemical compound	Sodium dodecyl sulfate (SDS)	Sigma-Aldrich	71736
Chemical compound	Benzonase nuclease	Sigma-Aldrich	E1014
Chemical compound	Bovine serum albumin	Sigma-Aldrich	A7284
Chemical compound	[1-¹⁴C]Oleic acid (50 mCi/mmol)	PerkinElmer, Waltham, MA	NEC317050UC
Chemical compound	SuperSignal West Pico Chemiluminescent Substrate	Thermo Fisher Scientific	34580
Chemical compound	Zeocin	Life Technologies, Grand Island, NY	R25005
Chemical compound	FuGENE HD	Promega Corporation, Madison, WI	E2311
Chemical compound	Formaldehyde	Sigma-Aldrich	F8775
Chemical compound	Penicillin-Streptomycin Solution	Corning	30–002 Cl
Chemical compound	Methanol	Fisher Scientific, Hampton, NH	A412
Chemical compound	Hexane	Fisher Scientific	H292
Chemical compound	Isopropanol	Fisher Scientific	A416
Chemical compound	Heptane	Fisher Scientific	H350
Chemical compound	Ethyl ether	Fisher Scientific	E138
Chemical compound	Acetic acid	Fisher Scientific	A38C
Chemical compound	Sodium compactin	Brown et al. (1978)	NA
Chemical compound	Sodium mevalonate	Brown et al. (1978)	NA
Other	L-Glutamine-free DMEM	Sigma-Aldrich	D5546	culture medium
Other	DMEM-low glucose (1000 mg/l)	Sigma-Aldrich	D6046	culture medium
Other	Ham’s F-12 medium and Dulbecco’s modified Eagle’s medium containing 2.5 mM L- glutamine (DMEM)	Corning, Manassas, VA	10–090-CV	culture medium
Other	Newborn calf lipoprotein-deficient serum (LPDS, d < 1.215 g/mL)	Goldstein et al. (1983)	NA	culture serum
Commercial assay or kit	Bolt 4–12% Bis-Tris Plus gradient gels	Thermo Fisher Scientific, Waltham, MA	NW04125BOX
Commercial assay or kit	QuikChange II XL Site-Directed Mutagenesis Kit	Agilent Technologies, Santa Clara, CA	200522
Antibody	Rabbit monoclonal IgG against Flag	Sigma-Aldrich, St. Louis, MO	F7425, RRID: AB_439687
Antibody	Mouse monoclonal IgG against LAMP-2	BD Biosciences, Franklin Lakes, NJ	555803, RRID: AB_396137
Antibody	Rabbit monoclonal IgG against amino acids 1261–1278 of human NPC1	Abcam, Cambridge, UK	ab134113
Antibody	Rabbit polyclonal IgG against NPC1(NTD)-His8-FLAG	Infante et al. (2008)	Clone 491B
Antibody	Goat anti-rabbit IgG conjugated to AlexaFluor 488	Invitrogen, Carlsbad, CA	A-11008, RRID: AB_143165
Antibody	Goat anti-mouse IgG conjugated to AlexaFluor 594	Invitrogen	A-11005, RRID: AB_141372
Antibody	Mouse monoclonal HRP-conjugated IgG against β-actin	Cell Signaling Technology, Danvers, MA	12262, RRID: AB_2566811
Antibody	Horse anti-mouse IgG conjugated to HRP	Cell Signaling Technology	7076, RRID: AB_330924
Antibody	Goat anti-rabbit IgG conjugated to HRP	Cell Signaling Technology	7074, RRID: AB_2099233
Antibody	Mouse monoclonal IgG against human NPC2	Wang et al. (2010)	Clone 13G4