1. Biochemistry and Chemical Biology
  2. Cell Biology
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Lysosomal cholesterol export reconstituted from fragments of Niemann-Pick C1

  1. Michael Nguyen Trinh
  2. Michael S Brown  Is a corresponding author
  3. Joachim Seemann
  4. Joseph L Goldstein  Is a corresponding author
  5. Feiran Lu
  1. University of Texas Southwestern Medical Center, United States
Research Article
Cite this article as: eLife 2018;7:e38564 doi: 10.7554/eLife.38564
6 figures, 1 table, 1 data set and 1 additional file

Figures

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
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
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
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
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
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 resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compoundSodium dodecyl
sulfate (SDS)
Sigma-Aldrich71736
Chemical compoundBenzonase nucleaseSigma-AldrichE1014
Chemical compoundBovine serum
albumin
Sigma-AldrichA7284
Chemical compound[1-14C]Oleic acid
(50 mCi/mmol)
PerkinElmer,
Waltham, MA
NEC317050UC
Chemical compoundSuperSignal West Pico
Chemiluminescent
Substrate
Thermo Fisher
Scientific
34580
Chemical compoundZeocinLife Technologies,
Grand Island, NY
R25005
Chemical compoundFuGENE HDPromega Corporation,
Madison, WI
E2311
Chemical compoundFormaldehydeSigma-AldrichF8775
Chemical compoundPenicillin-Streptomycin
Solution
Corning30–002 Cl
Chemical compoundMethanolFisher Scientific,
Hampton, NH
A412
Chemical compoundHexaneFisher ScientificH292
Chemical compoundIsopropanolFisher ScientificA416
Chemical compoundHeptaneFisher ScientificH350
Chemical compoundEthyl etherFisher ScientificE138
Chemical compoundAcetic acidFisher ScientificA38C
Chemical compoundSodium compactinBrown et al. (1978)NA
Chemical compoundSodium mevalonateBrown et al. (1978)NA
OtherL-Glutamine-free
DMEM
Sigma-AldrichD5546culture
medium
OtherDMEM-low glucose
 (1000 mg/l)
Sigma-AldrichD6046culture
medium
OtherHam’s F-12 medium
and Dulbecco’s
modified Eagle’s medium
containing 2.5 mM L-
glutamine (DMEM)
Corning,
Manassas, VA
10–090-CVculture
medium
OtherNewborn calf
lipoprotein-deficient serum
(LPDS, d < 1.215 g/mL)
Goldstein et al. (1983)NAculture
serum
Commercial assay or kitBolt 4–12% Bis-Tris
Plus gradient gels
Thermo Fisher Scientific,
Waltham, MA
NW04125BOX
Commercial assay or kitQuikChange
II XL Site-Directed
Mutagenesis Kit
Agilent Technologies,
Santa Clara, CA
200522
AntibodyRabbit monoclonal
IgG against Flag
Sigma-Aldrich,
St. Louis, MO
F7425, RRID:
AB_439687
AntibodyMouse monoclonal
IgG against LAMP-2
BD Biosciences,
Franklin Lakes, NJ
555803, RRID:
AB_396137
AntibodyRabbit monoclonal
IgG against
amino acids 1261–1278
of human NPC1
Abcam,
Cambridge, UK
ab134113
AntibodyRabbit polyclonal
IgG against
NPC1(NTD)-His8-FLAG
Infante et al. (2008)Clone 491B
AntibodyGoat
anti-rabbit
IgG conjugated
to AlexaFluor 488
Invitrogen,
Carlsbad, CA
A-11008, RRID:
AB_143165
AntibodyGoat anti-mouse
IgG conjugated to
AlexaFluor 594
InvitrogenA-11005, RRID:
AB_141372
AntibodyMouse monoclonal
HRP-conjugated
IgG against β-actin
Cell Signaling
Technology,
Danvers, MA
12262, RRID:
AB_2566811
AntibodyHorse anti-mouse
IgG conjugated
to HRP
Cell Signaling
Technology
7076, RRID:
AB_330924
AntibodyGoat anti-rabbit
IgG conjugated
to HRP
Cell Signaling
Technology
7074, RRID:
AB_2099233
AntibodyMouse monoclonal
IgG against
human NPC2
Wang et al. (2010)Clone 13G4

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1 and 6.

The following previously published data sets were used
  1. 1
    Cryo-EM Structure of the Full-Length human NPC1 at 4.4 Angstrom
    1. Gong X
    2. Qian HW
    3. Zhou XH
    4. Wu JP
    5. Zhou Q
    6. Yan N
    (2016)
    Publicly available at RCSB Protein Data Bank (accession no. 3JD8).

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