Binding between the Niemann-Pick C1 Protein and a Photoactivatable Cholesterol Analog Requires a Functional Sterol-Sensing Domain

Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent p...

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2004-08, Vol.101 (34), p.12473-12478
Main Authors: Ohgami, Nobutaka, Ko, Dennis C., Thomas, Matthew, Scott, Matthew P., Catherine C. Y. Chang, Chang, Ta-Yuan, Vagelos, P. Roy
Format: Article
Language:English
Subjects:
Animals
Antibodies
Azo Compounds - chemistry
Azo Compounds - metabolism
Biochemistry
Biological Sciences
Carrier Proteins - chemistry
Carrier Proteins - genetics
Carrier Proteins - metabolism
Cell Line
Cell Membrane - metabolism
Chemical bonds
CHO cells
Cholestanol - analogs & derivatives
Cholestanol - chemistry
Cholestanol - metabolism
Cholesterol
Cholesterol - chemistry
Cholesterol - metabolism
Cholesterols
Gels
Genetic mutation
Glycoproteins - metabolism
Humans
Intracellular Signaling Peptides and Proteins
Light
Lipids
Luminescent Proteins - genetics
Luminescent Proteins - metabolism
Membrane Glycoproteins - chemistry
Membrane Glycoproteins - genetics
Membrane Glycoproteins - metabolism
Membrane proteins
Molecular Structure
Niemann-Pick C1 Protein
Niemann-Pick Diseases
Photoaffinity Labels - chemistry
Photoaffinity Labels - metabolism
Protein Binding
Protein Structure, Tertiary
Proteins
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Sterols
Vesicular Transport Proteins
Western blotting
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Niemann-Pick type C (NPC) 1 protein plays important roles in moving cholesterol and other lipids out of late endosomes by means of vesicular trafficking, but it is not known whether NPC1 directly interacts with cholesterol. We performed photoaffinity labeling of intact cells expressing fluorescent protein (FP)-tagged NPC1 by using [3H]7,7-azocholestanol ([3H] AC). After immunoprecipitation,3H-labled NPC1-GFP appeared as a single band. Including excess unlabeled sterol to the labeling reaction significantly diminished the labeling. Altering the NPC1 sterol-sensing domain (SSD) with loss-of-function mutations (P692S and Y635C) severely reduced the extent of labeling. To further demonstrate the specificity of labeling, we show that NPC2, a late endosomal/lysosomal protein that binds to cholesterol with high affinity, is labeled, whereas mutant NPC2 proteins inactive in binding cholesterol are not. Vamp7, an abundant late endosomal membrane protein without an SSD but with one transmembrane domain, cannot be labeled. Binding between [3H] AC and NPC1 does not require NPC2. Treating cells with either U-18666A, a compound that creates an NPC-like phenotype, or with bafilomycin A1, a compound that raises late endosomal pH, has no effect on labeling of NPC1-YFP, suggesting that both drugs affect processes other than NPC1 binding to cholesterol. We also developed a procedure to label the NPC1-YFP by [3H] AC in vitro and showed that cholesterol is more effective in protection against labeling than its analogs epicholesterol or 5-α-cholestan. Overall, the results demonstrate that there is direct binding between NPC1 and azocholestanol; the binding does not require NPC2 but requires a functional SSD within NPC1.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0405255101