NR ANLA
AU Baron,G.S.; Caughey,B.W.
TI Effect of glycosylphosphatidylinositol anchor-dependent and -independent prion protein association with model raft membranes on conversion to the protease-resistant isoform
QU The Journal of Biological Chemistry 2003 Apr 25; 278(17): 14883-92
IA http://www.jbc.org/cgi/content/full/278/17/14883
PT journal article
AB Prion protein (PrP) is usually bound to membranes by a glycosylphosphatidylinositol (GPI)-anchor that associates with detergent-resistant membranes (DRMs), or rafts. To examine the effect of membrane association on the interaction between the normal protease-sensitive PrP isoform (PrP-sen) and the protease-resistant isoform (PrPres), a model system was employed using PrP-sen reconstituted into sphingolipid-cholesterol-rich raft-like liposomes (SCRLs). Both full length (GPI+) and GPI-anchor-deficient (GPI-) PrP-sen produced in fibroblasts stably associated with SCRLs. The latter, alternative mode of membrane association was not detectably altered by glycosylation and was markedly reduced by deletion of residues 34-94. The SCRL-associated PrP molecules were not removed by treatments with either high salt or carbonate buffer. However, only GPI+ PrP-sen resisted extraction with cold Triton X-100. PrP-sen association with SCRLs was pH-independent. PrP-sen was also one of a small subset of phosphatidylinositol-specific phospholipase C (PI-PLC)-released proteins from fibroblast cells found to bind SCRLs. A cell-free conversion assay was used to measure the interaction of SCRL-bound PrP-sen with exogenous PrPres as contained in microsomes. SCRL-bound GPI+ PrP-sen was not converted to PrPres until PI-PLC was added to the reaction, or the combined membrane fractions were treated with the membrane-fusing agent polyethylene glycol (PEG). In contrast, SCRL-bound GPI- PrP-sen was converted to PrPres without PI-PLC or PEG treatment. Thus, of the two forms of raft membrane association by PrP-sen, only the GPI-anchor-directed form resists conversion induced by exogenous PrPres.
MH Animals; Cricetinae; Endopeptidases/*metabolism; Fibroblasts; Glycosylphosphatidylinositols/*metabolism/pharmacology; Hydrophobicity; Membrane Microdomains/*metabolism; Mice; Octoxynol/pharmacology; Phosphatidylinositol Diacylglycerol-Lyase; Phospholipase C/metabolism; PrPc Proteins/*metabolism; Protein Binding; Protein Isoforms/metabolism; Research Support, Non-U.S. Gov't
AD Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840, USA
SP englisch
PO USA