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Binding between the Niemann–Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain
- Source :
- Proceedings of the National Academy of Sciences. 101:12473-12478
- Publication Year :
- 2004
- Publisher :
- Proceedings of the National Academy of Sciences, 2004.
-
Abstract
- 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 [ 3 H]7,7-azocholestanol ([ 3 H]AC). After immunoprecipitation, 3 H-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 [ 3 H]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 [ 3 H]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.
- Subjects :
- congenital, hereditary, and neonatal diseases and abnormalities
Light
Immunoprecipitation
Recombinant Fusion Proteins
Vesicular Transport Proteins
Photoaffinity Labels
Analogs & derivatives
Plasma protein binding
Biology
Cholesterol analog
Cell Line
Niemann-Pick C1 Protein
hemic and lymphatic diseases
Animals
Humans
Glycoproteins
Niemann-Pick Diseases
Membrane Glycoproteins
Multidisciplinary
Molecular Structure
Photoaffinity labeling
Cell Membrane
Intracellular Signaling Peptides and Proteins
nutritional and metabolic diseases
Biological Sciences
Sterol
Protein Structure, Tertiary
Cholestanol
Luminescent Proteins
Transmembrane domain
Cholesterol
Biochemistry
lipids (amino acids, peptides, and proteins)
NPC1
Carrier Proteins
Azo Compounds
Protein Binding
Subjects
Details
- ISSN :
- 10916490 and 00278424
- Volume :
- 101
- Database :
- OpenAIRE
- Journal :
- Proceedings of the National Academy of Sciences
- Accession number :
- edsair.doi.dedup.....82ede40321c682409a956581969be428