NR ASFB
AU Vey,M.; Pilkuhn,S.; Wille,H.; Anderson,R.G.W.; Prusiner,S.B.
TI Detergent-resistant, glycosphingolipid-enriched, membranous microdomains contain both the cellular and scrapie prion proteins
QU Molecular Biology of the Cell 1996; 7(Supplement, Poster Sessions, Pt. III, 90a-129a): 522
IA http://www.pubmedcentral.nih.gov/pagerender.fcgi?artid=526995&pageindex=1
PT Meeting Abstract
AB GPI-anchored proteins cluster in detergent-resistant membranous microdomains called DIGs (detergent-insoluble, glycosphingolipidenriched complexes) which are enriched for both cholesterol and glycosphingolipids. To determine if prion protein (PrP) also associates with these membrane domains, we used two independent methods for isolation of DIGs from scrapie-infected and uninfected mouse neuroblastoma cells. In the detergent-based method, cells were lysed in cold Triton-X100 and the DIGs were separated from the lysates by flotation in sucrose gradients. Alternatively, postnuclear supernatants were prepared from cell homogenates in the absence of detergent and fractionated on Percoll gradients. Isolated plasma membrane fractions were further disrupted by sonication and the DIGs separated from membranous debris by flotation in OptiPrep gradients. Analysis of DIGs showed that the cellular prion protein (PrPc)was concentrated in DIGs. Prion infection did not interfere with formation of DIGs and even after conversion, the pathologic, scrapie isoform PrPsc was still associated with them. The PrP 27-30 core of PrPsc was detected after limited digestion with proteinase K. Furthermore, a 17 kDa degradation product of PrPc could be identified. These results raise the possibility that prion propagation occurs in microdomains of cellular membranes which are enriched for cholesterol, glycosphingolipids and GPI-anchored proteins.
ZR 0
AD Martin Vey, Susanne Pilkuhn, Holger Wille, Stanley B. Prusiner, University of California, San Francisco, CA 94143, Richard G. W. Anderson, University of Texas Southwestern Medical Center, Dallas, TX, 75235, USA
SP englisch