Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein.
We have investigated the intracellular traffic of PrP(c), a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrP(c) 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-PrP(c), 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 PrP(c) trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrP(c) accumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrP(c) but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrP(c) 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-PrP(c) is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrP(c) is delivered to classic endosomes after internalization.
Magalhães
Ana C
AC
Laboratório de Neurofarmacologia, Departamento de Farmacologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais 31270-910, Brazil.
Silva
Juliana A
JA
Lee
Kil S
KS
Martins
Vilma R
VR
Prado
Vania F
VF
Ferguson
Stephen S G
SS
Gomez
Marcus V
MV
Brentani
Ricardo R
RR
Prado
Marco A M
MA
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D034284:Dynamin I; D034281:Dynamins; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005799:Genes, Dominant; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D018621:PrPC Proteins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D008164:Luminescent Proteins; D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003300:Copper; D034284:Dynamin I; D020558:GTP Phosphohydrolases; D034281:Dynamins