Abstract
The association of the prion protein (PrP) with sphingolipid- and cholesterol-rich lipid rafts is instrumental in the pathogenesis of the neurodegenerative prion diseases. Although the glycosylphosphatidylinositol (GPI) anchor is an exoplasmic determinant of raft association, PrP remained raft-associated in human neuronal cells even when the GPI anchor was deleted or substituted for a transmembrane anchor indicating that the ectodomain contains a raft localization signal. The raft association of transmembrane-anchored PrP occurred independently of Cu(II) binding as it failed to be abolished by either deletion of the octapeptide repeat region (residues 51-90) or treatment of cells with a Cu(II) chelator. Raft association of transmembrane-anchored PrP was only abolished by the deletion of the N-terminal region (residues 23-90) of the ectodomain. This region was sufficient to confer raft localization when fused to the N terminus of a non-raft transmembrane-anchored protein and suppressed the clathrin-coated pit localization signal in the cytoplasmic domain of the amyloid precursor protein. These data indicate that the N-terminal region of PrP acts as a cellular raft targeting determinant and that residues 23-90 of PrP represent the first proteinaceous raft targeting signal within the ectodomain of a GPI-anchored protein.
Highlights
Lipid rafts represent compositionally and functionally distinct membrane microdomains that serve as the platform for a number of membrane-mediated biological processes including signal transduction and the trafficking and sorting of proteins and lipids [1, 2]
A GPI Anchor Is Not Essential for the Raft Association of prion protein (PrP)—To investigate determinants required for the raft association of PrP, alternatively anchored forms of the protein were stably expressed in the human neuronal cell line SH-SY5Y, which lacks detectable levels of endogenous PrP [33] (Fig. 1A)
The detergent-resistant membranes (DRMs) association of PrP-CTM cannot be attributed to the TM and cytoplasmic domains of ACE, as ACE itself failed to associate with DRMs and remained in the clathrin-containing fractions when stably expressed in SH-SY5Y cells (Fig. 1B), consistent with earlier observations that this TM protein is excluded from rafts [35, 36]
Summary
Lipid rafts represent compositionally and functionally distinct membrane microdomains that serve as the platform for a number of membrane-mediated biological processes including signal transduction and the trafficking and sorting of proteins and lipids [1, 2]. These data indicate that the N-terminal region of PrP acts as a cellular raft targeting determinant and that residues 23–90 of PrP represent the first proteinaceous raft targeting signal within the ectodomain of a GPI-anchored protein.
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