The membrane environment of endogenous cellular prion protein in primary rat cerebellar neurons.
We studied the membrane environment of cellular prion protein in primary cultured rat cerebellar neurons differentiated in vitro. In these cells, about 45% of total cellular prion protein (corresponding to a 35-fold enrichment) is associated with a low-density, sphingolipid- and cholesterol-enriched membrane fraction, that can be separated by flotation on sucrose gradient. Biotinylation experiments indicated that almost all prion protein recovered in this fraction was exposed at the cell surface. Prion protein was efficiently separated from this fraction by a monoclonal antibody immuno-separation procedure. Under conditions designed to preserve lipid-mediated membrane organization, several proteins were found in the prion protein-enriched membrane domains (i.e. the non-receptor tyrosine kinases Lyn and Fyn and the neuronal glycosylphosphatidylinositol-anchored protein Thy-1). The prion protein-rich membrane domains contained, as well, about 50% of the sphingolipids, cholesterol and phosphatidylcholine present in the sphingolipid-enriched membrane fraction. All main sphingolipids, including sphingomyelin, neutral glycosphingolipids and gangliosides, were similarly enriched in the prion protein-rich membrane domains. Thus, prion protein plasma membrane environment in differentiated neurons resulted to be a complex entity, whose integrity requires a network of lipid-mediated non-covalent interactions.
Loberto
Nicoletta
N
Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Segrate, Italy.
Prioni
Simona
S
Bettiga
Arianna
A
Chigorno
Vanna
V
Prinetti
Alessandro
A
Sonnino
Sandro
S
D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D003902:Detergents; D005732:Gangliosides / Q000378:metabolism; D047468:Immunoprecipitation; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D020383:Neutral Glycosphingolipids / Q000378:metabolism; D017830:Octoxynol; D010938:Plant Oils; D011092:Polyethylene Glycols; D011328:Prions / Q000378:metabolism*; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013109:Sphingomyelins / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D003902:Detergents; D005732:Gangliosides; D008563:Membrane Lipids; D008565:Membrane Proteins; D020383:Neutral Glycosphingolipids; D010938:Plant Oils; D011328:Prions; D013109:Sphingomyelins; D011092:Polyethylene Glycols; D017830:Octoxynol; C033084:polyethylene glycol oleyl ether; C494961:Fyn protein, rat; D051076:Proto-Oncogene Proteins c-fyn; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases