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-PrPcaccumulates 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, PrPcis delivered to classic endosomes after internalization. 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-PrPcaccumulates 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, PrPcis delivered to classic endosomes after internalization. cellular prion protein glycosylphosphatidylinositol green fluorescent protein minimal essential medium differential interference contrast transferrin The cellular prion protein (PrPc)1 is a glycosylphosphatidylinositol (GPI)-plasma membrane-anchored protein whose function is still under debate. Potential roles of PrPc in signaling events (1Shmerling D. Hegyi I. Fischer M. Blattler T. Brandner S. Gotz J. Rulicke T. Flechsig E. Cozzio A. von Mering C. Hangartner C. Aguzzi A. Weissmann C. Cell. 1998; 93: 203-214Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 2Herms J.W. Korte S. Gall S. Schneider I. Dunker S. Kretzschmar H.A. J. Neurochem. 2000; 75: 1487-1492Crossref PubMed Scopus (64) Google Scholar, 3Mouillet-Richard S. Ermonval M. Chebassier C. 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FEBS Lett. 2000; 482: 257-260Crossref PubMed Scopus (100) Google Scholar), protection against oxidative insults (8Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar, 9Brown D.R. Trends Neurosci. 2001; 24: 85-90Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 10Klamt F. Dal-Piolzz F. Conte da Frota M.L.J.R. Walz R. Andrades M.E. da Silva E.G. Brentani R.R. Izquierdo I. Fonseca Moreira J.C. Free Radic. Biol. Med. 2001; 30: 1137-1144Crossref PubMed Scopus (217) Google Scholar), and copper metabolism (8Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar, 11Pauly P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar) have been suggested. Conversion of PrPc from an α-helix- to a β-sheet-rich structure causes relevant biophysical changes to the protein that have been related to brain dysfunction in prion diseases (12Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5131) Google Scholar, 13Weissmann C. J. Biol. Chem. 1999; 274: 3-6Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 14Aguzzi A. Glatzel M. Montrasio F. Prinz M. Heppner F.L. Nat. Rev. Neurosci. 2001; 2: 745-749Crossref PubMed Scopus (80) Google Scholar). The mechanisms involved in this conversion are unknown, but accumulating evidence suggests that the process occurs after PrPc reaches the plasma membrane, and it may involve PrPc entry into intracellular acidic organelles (15Caughey B. Raymond G.J. J. Biol. Chem. 1991; 266: 18217-18223Abstract Full Text PDF PubMed Google Scholar, 16Caughey B. Raymond G.J. Ernst D. Race R.E. J. Virol. 1991; 65: 6597-6603Crossref PubMed Google Scholar, 17Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avrahami D. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (516) Google Scholar). The mechanisms of PrPc trafficking are poorly understood. A chicken PrPc has been shown to cycle through the plasma membrane and endosomes (18Shyng S.L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Abstract Full Text PDF PubMed Google Scholar), and this process has been suggested to involve clathrin-mediated endocytosis (19Shyng S.L. Heuser J.E. Harris D.A. J. Cell Biol. 1994; 125: 1239-1250Crossref PubMed Scopus (223) Google Scholar). However, other evidence suggests that mammalian PrPc may follow a similar endocytic pathway as that of most GPI-anchored proteins. In particular, PrPc can be found in lipid rafts at the plasma membrane that are isolated as detergent-insoluble glycolipid vesicles (17Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avrahami D. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (516) Google Scholar,20Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar, 21Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar, 22Madore N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (333) Google Scholar). Moreover, it has been suggested that internalization of PrPc occurs via a clathrin-independent mechanism, probably through “caveolae” (20Vey M. Pilkuhn S. Wille H. Nixon R. DeArmond S.J. Smart E.J. Anderson R.G. Taraboulos A. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14945-14949Crossref PubMed Scopus (489) Google Scholar, 21Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar). Internalization of GPI-anchored proteins is a complicated cellular event, because proteins intracellular that are relevant with endocytic proteins. A minimum fluorescent GPI-anchored protein, fluorescent through the plasma membrane and the Golgi compartment through classic endocytic organelles in a clathrin-independent suggesting that is a trafficking pathway that is by GPI-anchored proteins R. K. J. J. Cell Biol. 2001; PubMed Google Scholar). However, other GPI-anchored proteins as the through recycling endosomes to the plasma membrane R. K. J. J. Cell Biol. 2001; PubMed Google Scholar, S. G.J. J. Cell Biol. 1996; PubMed Scopus Google Scholar, S. S. EMBO J. 1998; PubMed Scopus Google Scholar). rafts are and not GPI-anchored proteins are in the rafts in N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (333) Google Scholar). it is that endocytic to the internalization of different GPI proteins. we K.S. Zanata S.M. Brentani R.R. Martins V.R. M.A. J. Neurochem. 2001; PubMed Scopus Google Scholar) and L. S. T. Harris D.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. A. Mol. Cell. Neurosci. 2001; PubMed Scopus Google Scholar, O. Kretzschmar H.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) have fluorescent PrPc The fluorescent protein is targeted to the plasma membrane, it is by GPI K.S. Zanata S.M. Brentani R.R. Martins V.R. M.A. J. Neurochem. 2001; PubMed Scopus Google Scholar, A. C. A. Mol. Cell. Neurosci. 2001; PubMed Scopus Google Scholar) and is in rafts A. C. A. Mol. Cell. Neurosci. 2001; PubMed Scopus Google Scholar). copper GFP-PrPc internalization K.S. Zanata S.M. Brentani R.R. Martins V.R. M.A. J. Neurochem. 2001; PubMed Scopus Google Scholar) in a similar to on PrPc P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, W. S. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that the fluorescent protein is functional and can be to PrPc traffic in cells. In the we the intracellular of GFP-PrPc and of the endocytic pathway to intermediates involved in Moreover, we the endocytic intermediates PrPc trafficking in the trafficking of a minimum GPI-anchored protein, We found that the steady-state distribution of GFP-PrPc is and that internalized GFP-PrPc is to Rab5-positive early endocytic vesicles and endosomes. In GFP-GPI is not found in the endocytic organelles as we suggest that PrPc trafficking from that of other GPI-anchored proteins and may depend on internalization in the The SN56 a from of of in medium and in in a at as K.S. Zanata S.M. Brentani R.R. Martins V.R. M.A. J. Neurochem. 2001; PubMed Scopus Google M.A. J. Neurochem. 1999; Google Scholar). The SN56 from Lee Res. PubMed Scopus Google Scholar) and a of of proteins M.A. J. Neurochem. 1999; Google Scholar) and C. A. M.A. Res. 2001; PubMed Scopus Google Scholar). are by differentiation C. A. M.A. Res. 2001; PubMed Scopus Google Scholar, A. D.A. Lee J. Neurosci. PubMed Google Scholar). The GFP-PrPc has been K.S. Zanata S.M. Brentani R.R. Martins V.R. M.A. J. Neurochem. 2001; PubMed Scopus Google Scholar). The Rab5 mutant dynamin and the dominant-negative dynamin mutant a from of Cell and GFP-GPI a from J. and J. of and Cell and of The SN56 on Cell by the to the of and of cells. of in medium and In we of a of a of dynamin GFP-PrPc and the other cell at on in essential and to a in the the of a with a the to a with a as K.S. Zanata S.M. Brentani R.R. Martins V.R. M.A. J. Neurochem. 2001; PubMed Scopus Google Scholar). and with Confocal and has been and shown to be on the of the of PrPc K.S. Zanata S.M. Brentani R.R. Martins V.R. M.A. J. Neurochem. 2001; PubMed Scopus Google Scholar). with after the with and endocytic we the with at in and by microscopy as F. C. S.S. M.A. J. Neurochem. 2001; PubMed Scopus Google Scholar). of endosomes by with transferrin at in with and with in Golgi with as in with and at and at with with with and at of and the in SN56 and by A. Moreover, in with a fluorescent of PrPc different have suggested that the fluorescent protein the Golgi as by the of the protein with a of Golgi markers L. S. T. Harris D.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. A. Mol. Cell. Neurosci. 2001; PubMed Scopus Google Scholar, O. Kretzschmar H.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of with GFP-PrPc and observations an of of GFP-PrPc in the Golgi in SN56 However, a of GFP-PrPc to that by the endosomal compartment A of the endosomes the in to the plasma membrane with the that the structure can be with this endocytic These observations suggest that intracellular GFP-PrPc is accumulating not in the Golgi but in endosomal the involved with PrPc we the of a dynamin I-K44A mutant GFP-PrPc and GFP-GPI of endocytosis R. K. J. J. Cell Biol. 2001; PubMed Google distribution in SN56 cells. of the dynamin I-K44A dominant-negative mutant has been as a to of endocytic intermediates H. T. S.L. J. Cell Biol. 1994; PubMed Scopus Google Scholar). dynamin I-K44A with GFP-PrPc in SN56 GFP-PrPc in to the plasma membrane surface In in dynamin of not The internalization of endocytic in dynamin with suggesting that most of the accumulation in the on dynamin of dynamin I-K44A by the internalization of fluorescent transferrin by suggesting that this mutant clathrin-mediated endocytosis L. M. M. M. S. S. M. A. and F. J. in Scholar). in the that in dynamin I-K44A cells. vesicles with GFP-PrPc to the plasma membrane be with The from cell to but of that with of to the of the cell that most to be to the and to the because are to the the of the cell the of fluorescent in with the plasma membrane and The with GFP-PrPc in dynamin similar to plasma membrane that traffic between the and the J.L. L.B. S.S. Mol. 2000; Google Scholar). In the of the GFP-PrPc to with of the plasma membrane, suggesting that the GFP-PrPc is In under of the dominant-negative dynamin I-K44A mutant of into SN56 is in the of GFP-GPI A and GFP-GPI a similar of distribution in and mutant These observations indicate that the with GFP-PrPc not in the internalization of The that GFP-PrPc, but not GFP-GPI, in vesicles to the plasma membrane in the of dynamin I-K44A to the cellular organelles the trafficking of We events of between GFP-PrPc and in to the plasma membrane in with a in the Therefore, we GFP-PrPc the Rab5-positive early endosomal compartment to the compartment of cells. Rab5 is involved in endosomal traffic and A mutant that the of Rab5 and of endocytic vesicles into H. R.G. O. A. J. M. EMBO J. 1994; PubMed Scopus Google Scholar, E. F. J.M. M. Nat. Cell Biol. 1999; PubMed Scopus Google Scholar). this be in the distribution of the protein in the of of that GFP-PrPc in vesicles to the plasma membrane of the vesicles with the with and in we of GFP-PrPc with the endocytic and These suggest the early endocytic of the In with a R. K. J. J. Cell Biol. 2001; PubMed Google Scholar), GFP-GPI did not in vesicles with in the of and with in Rab5 cells. SN56 with GFP-PrPc and the Rab5 with GFP-GPI and after with with and by of GFP-PrPc and are shown in A and of a cell GFP-GPI and are shown and D. GFP-PrPc and GFP-GPI are in is in and is in in the to vesicles of GFP-PrPc and The between is shown at the The are of and GFP-PrPc and GFP-GPI in suggest that traffic of GFP-PrPc is from that of GFP-GPI to and in Rab5-positive endosomes. However, did not GFP-PrPc early endosomes internalization from the plasma membrane the fluorescent protein is to endosomes. is that copper to an of (8Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. 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Neurochem. 2001; PubMed Scopus Google Scholar). observations suggest that under through the early endosomal compartment and that the of endosomal trafficking is GFP-PrPc in early endosomes. may be the of the GFP-PrPc, and may accumulation of protein in early endosomes GFP-GPI, is not found in endosomes R. K. J. J. Cell Biol. 2001; PubMed Google Scholar), did not internalized through of to and The GFP-PrPc on the mechanisms the internalization and trafficking of We that GFP-PrPc internalization is from the endocytosis GFP-GPI R. K. J. J. Cell Biol. 2001; PubMed Google Scholar, R. K. J. J. Cell Biol. 2001; PubMed Scopus Google Scholar), suggesting that internalization and the pathway by the GPI anchor. We that PrPc internalization is dynamin a involved with the of but not J. L. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, R.G. von M. J. Cell Biol. 1999; PubMed Scopus Google Scholar), endocytic vesicles from the plasma Moreover, we Rab5-positive early endosomes as the of internalized GFP-PrPc in cells. of PrPc in intracellular compartments has been in S.J. Prusiner S.B. PubMed Google Scholar, J. M. PubMed Google Scholar, J. H. J. Neurosci. 2001; PubMed Google Scholar), with in the Golgi J. H. J. Neurosci. 2001; PubMed Google Scholar). of GFP-PrPc by has the of the protein in the Golgi compartment L. S. T. Harris D.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, A. C. A. Mol. Cell. Neurosci. 2001; PubMed Scopus Google Scholar, O. Kretzschmar H.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We have a Golgi by endocytic markers and a Rab5 mutant we found that of the intracellular GFP-PrPc can be found in the endosomal compartment. have been the mechanisms involved in the internalization of chicken and mammalian chicken PrPc to via endocytic vesicles (18Shyng S.L. Huber M.T. Harris D.A. J. Biol. Chem. 1993; 268: 15922-15928Abstract Full Text PDF PubMed Google Scholar), mammalian PrPc to depend on a of endocytosis (17Taraboulos A. Scott M. Semenov A. Avrahami D. Laszlo L. Prusiner S.B. Avrahami D. J. Cell Biol. 1995; 129: 121-132Crossref PubMed Scopus (516) Google Scholar, S.L. Heuser J.E. Harris D.A. J. Cell Biol. 1994; 125: 1239-1250Crossref PubMed Scopus (223) Google Scholar, 21Kaneko K. Vey M. Scott M. Pilkuhn S. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2333-2338Crossref PubMed Scopus (236) Google Scholar). The of mammalian PrPc to classic endosomes is under Linden R. Walz R. Izquierdo I. Brentani R.R. 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Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (678) Google Scholar). The suggest that, a of internalization of PrPc, as is involved in the dynamin-dependent internalization of PrPc, it of that PrPc to the early endocytic is that vesicles are involved with mammalian similar to has been chicken PrPc (19Shyng S.L. Heuser J.E. Harris D.A. J. Cell Biol. 1994; 125: 1239-1250Crossref PubMed Scopus (223) Google Scholar). In with the mammalian PrPc in is found in N. Smith K.L. Graham C.H. Jen A. Brady K. Hall S. Morris R. EMBO J. 1999; 18: 6917-6926Crossref PubMed Scopus (333) Google Scholar, J. H. J. Neurosci. 2001; PubMed Google Scholar). These with the observations that both intracellular of of the occurs by PrPc with S. Ermonval M. Chebassier C. Laplanche J.L. Lehmann S. Launay J.M. Kellermann O. Science. 2000; 289: 1925-1928Crossref PubMed Scopus (678) Google Scholar). indicate that the of PrPc to pre-endocytic is a and may through PrPc is a protein (8Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar), but the of with the protein function are poorly understood. has been suggested that PrPc is to to intracellular compartments P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar) that the protein in a of protection from oxidative insults (8Brown D.R. Qin K. Herms J.W. Madlung A. Manson J. Strome R. Fraser P.E. Kruck T. von Bohlen A. Schulz-Schaeffer W. Giese A. Westaway D. Kretzschmar H. Nature. 1997; 390: 684-687Crossref PubMed Scopus (37) Google Scholar, 9Brown D.R. 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