Binding between the Niemann–Pick C1 protein and a photoactivatable cholesterol analog requires a functional sterol-sensing domain

Proceedings of the National Academy of Sciences of the United States of America - Tập 101 Số 34 - Trang 12473-12478 - 2004
Nobutaka Ohgami1, Dennis C. Ko1, Matthew A. M. Thomas1, Matthew P. Scott1, Catherine C.Y. Chang1, Ta‐Yuan Chang1
1Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755; and Department of Developmental Biology, Clark Center W252, 318 Campus Drive, Stanford University School of Medicine, Stanford, CA 94305-5439

Tóm tắt

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]ACin vitroand 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.

Từ khóa


Tài liệu tham khảo

Patterson, M. C., Vanier, M. T., Suzuki, K., Morris, J. A., Carstea, E., Neufeld, E. B., Blanchette-Mackie, J. E. & Pentchev, P. G. (2001) in The Metabolic and Molecular Bases of Inherited Disease, eds. Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D. (McGraw–Hill, New York), Vol. III, pp. 3611–3633.

10.1074/jbc.M205406200

10.1002/path.1345

10.1194/jlr.D300032-JLR200

10.1083/jcb.110.2.295

10.1074/jbc.275.6.4013

10.1016/S0021-9258(18)42914-6

10.1074/jbc.M003180200

10.1093/oxfordjournals.jbchem.a002932

10.1126/science.277.5323.228

10.1126/science.290.5500.2298

10.1073/pnas.0530027100

10.1073/pnas.0437840100

10.1074/jbc.274.31.21861

10.1091/mbc.12.3.601

10.1016/S1097-2765(02)00822-5

10.1016/S0003-9861(03)00168-1

10.1016/S0092-8674(00)81362-8

10.1101/gad.938601

10.1126/science.1093131

10.1097/00041433-199804000-00010

10.1016/S0168-9525(02)02640-9

10.1073/pnas.081070898

10.1006/mgme.1999.2882

10.1074/jbc.274.14.9627

10.1074/jbc.M300488200

10.1074/jbc.M300542200

10.1074/jbc.273.30.18915

10.1074/jbc.M008272200

10.1194/jlr.M300009-JLR200

10.1016/S0960-9822(01)00396-7

10.1083/jcb.200310046

10.1126/science.290.5500.2295

10.1016/0962-8924(96)40001-0

10.1038/71366

10.1194/jlr.M200015-JLR200

10.1194/jlr.M200230-JLR200

10.1074/jbc.273.8.4635

10.1083/jcb.146.4.765

10.1016/S0039-128X(02)00042-9

10.1016/S0021-9258(18)80136-3

10.1016/S0021-9258(19)47429-2

10.1038/343425a0

10.1172/JCI0215593

Vanier, M. T. & Suzuki, K. (1998) Brain Pathol. 8, 163–174.9458174

10.1023/A:1022575511354

Yano, T., Taniguchi, M., Akaboshi, S., Vanier, M. T., Tai, T., Sakuraba, H. & Ohno, K. (1996) Proc. Jpn. Acad. 72B, 214–219.

10.1074/jbc.M005393200

10.1016/j.molcel.2004.06.019

Thông tin
Thông tin xuất bản

Proceedings of the National Academy of Sciences of the United States of America

Tập 101 Số 34

12473-12478

Thông tin tác giả