title: Contamination of nuclear fractions with plasma membrane lipid rafts.
abstract: Subcellular fractionation is central to a range of cell biological, biochemical and proteomic studies. Purification of nuclear-enriched fractions is critical for studies on nuclear structure and function. Here we show that detergent-based nuclear isolation methods cause the redistribution of proteins associated with plasma membrane lipid rafts into nuclear fractions. The glycosyl-phosphatidylinositol (GPI)-anchored prion protein (PrP(C)) and a GPI-anchored construct of angiotensin converting enzyme (GPI-ACE), as well as the lipid raft markers flotillin-1 and -2, were present in the nuclear fractions derived using three different subcellular fractionation protocols. Incubation of intact cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), which cleaves GPI-anchored proteins from the cell surface, significantly reduced the amount of PrP(C) and GPI-ACE in the nuclear fraction. Buoyant sucrose density gradient centrifugation in the presence of Triton X-100 of the nuclear fraction resulted in a significant proportion of the GPI-anchored proteins being recovered in the low density lipid raft fractions. These data indicate that the nuclear fraction isolated using such subcellular fractionation protocols is contaminated with components of plasma membrane lipid rafts and raises questions as to the integrity of the nuclear fraction isolated by such protocols for use in detailed cell biological studies and proteomics analysis.
authors.affiliation: Proteolysis Research Group, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK.
authors.forename: Yee-How
authors.initials: YH
authors.lastname: Say
authors.forename: Nigel M
authors.initials: NM
authors.lastname: Hooper
mesh_terms: D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D003902:Detergents / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D021962:Membrane Microdomains / Q000737:chemistry*; D008565:Membrane Proteins / Q000378:metabolism; D017830:Octoxynol / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000378:metabolism; D018621:PrPC Proteins / Q000378:metabolism
chemical_list: D003902:Detergents; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D018621:PrPC Proteins; C106354:flotillins; D017830:Octoxynol; D007703:Peptidyl-Dipeptidase A