Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein

We have investigated the intracellular traffic of PrPc, a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrPc is found at the cell surface and in intracellular compartments in SN56 cells. Confocal microscopy and organelle-specific markers suggest that the protein is found in both the Golgi and the recycling endosomal compartment. Perturbation of endocytosis with a dynamin I-K44A dominant-negative mutant altered the steady-state distribution of the GFP-PrPc, leading to the accumulation of fluorescence in unfissioned endocytic intermediates. These pre-endocytic intermediates did not seem to accumulate GFP-GPI, a minimum GPI-anchored protein, suggesting that PrPc trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrPc accumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrPc but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrPc internalizes via a dynamin-dependent endocytic pathway and that the protein is targeted to the recycling endosomal compartment via Rab5-positive early endosomes. These observations indicate that traffic of GFP-PrPc is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrPc is delivered to classic endosomes after internalization.