Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells

The cellular prion protein (PrPc) is a glycosylphosphatidylinositol (GPI)-a nchored plasma membrane protein whose conformational altered forms (PrPsc) are known to cause neurodegenerative diseases in mammals. In order to inves tigate the intracellular traffic of mammalian PrPc in living cells, we have generated a green fluorescent protein (GFP) tagged version of PrPc. The re combinant protein was properly anchored at the cell surface and its distrib ution pattern was similar to that of the endogenous PrPc, with labeling at the plasma membrane and in an intracellular perinuclear compartment. Compar ison of the steady-state distribution of GFP-PrPc and two N-terminal deleti on mutants (Delta 32-121 and Delta 32-134), that cause neurological symptom s when expressed in PrP knockout mice, was carried out. The mutant proteins accumulated in the plasma membrane at the expense of decreased labeling in the perinuclear region when compared with GFP-PrPc. In addition, GFP-PrPc, but not the two mutants, internalized from the plasma membrane in response to Cu2+ treatment and accumulated at a perinuclear region in SN56 cells. O ur data suggest that GFP-PrPc can be used to follow constitutive and induce d PrPc traffic in living cells.