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Endocytic Intermediates Involved with the Intracellular Trafficking of a Fluorescent Cellular Prion Protein
2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês
10.1074/jbc.m203661200
ISSN1083-351X
AutoresAna C. Magalhães, Juliana Silva, Kil Sun Lee, Vilma R. Martins, Vânia F. Prado, Stephen Ferguson, Marcus V. Gomez, Helena Brentani, Marco A. M. Prado,
Tópico(s)Biotin and Related Studies
ResumoWe 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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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. 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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 these proteins lack intracellular oriented sequences that are relevant for interaction with endocytic adaptor proteins. A minimum fluorescent GPI-anchored protein, GFP (green fluorescent protein)-GPI continuously cycles through the plasma membrane and the Golgi compartment without passing through classic endocytic organelles in a clathrin-independent fashion, suggesting that there is a default trafficking pathway that is followed by some GPI-anchored proteins (23Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-542Crossref PubMed Google Scholar). However, other GPI-anchored proteins such as the folate receptor pass through recycling endosomes before returning to the plasma membrane (23Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-542Crossref PubMed Google Scholar, 24Rijnboutt S. Jansen G. Posthuma G. Hynes J.B. Schornagel J.H. Strous G.J. J. Cell Biol. 1996; 132: 35-47Crossref PubMed Scopus (176) Google Scholar, 25Mayor S. Sabharanjak S. Maxfield F.R. EMBO J. 1998; 17: 4626-4638Crossref PubMed Scopus (270) Google Scholar). Lipid rafts are heterologous structures and not all GPI-anchored proteins are clustered in the same rafts in cells (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). Thus, it is possible that multiple endocytic pathways contribute to the internalization of different GPI proteins. Recently, we (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar) and others (27Ivanova L. Barmada S. Kummer T. Harris D.A. J. Biol. Chem. 2001; 276: 42409-42421Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 28Negro A. Ballarin C. Bertoli A. Massimino M.L. Sorgato M.C. Mol. Cell. Neurosci. 2001; 17: 521-538Crossref PubMed Scopus (61) Google Scholar, 29Lorensz Windl O. Kretzschmar H.A. J. Biol. Chem. 2002; 277: 8508-8516Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) have generated distinct fluorescent PrPc molecules (GFP-PrPc). The fluorescent protein is correctly targeted to the plasma membrane, where it is anchored by GPI (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar, 28Negro A. Ballarin C. Bertoli A. Massimino M.L. Sorgato M.C. Mol. Cell. Neurosci. 2001; 17: 521-538Crossref PubMed Scopus (61) Google Scholar) and is present in rafts (28Negro A. Ballarin C. Bertoli A. Massimino M.L. Sorgato M.C. Mol. Cell. Neurosci. 2001; 17: 521-538Crossref PubMed Scopus (61) Google Scholar). Importantly, copper induces GFP-PrPc internalization (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar) in a similar way to its effect on PrPc (11Pauly P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar, 30Sumudhu W. Perera S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), suggesting that the fluorescent protein is functional and can be used to infer PrPc traffic in living cells. In the present work we examined the intracellular localization of GFP-PrPc and disrupted distinct steps of the endocytic pathway to uncover intermediates involved in PrPctrafficking. Moreover, we tested whether the same endocytic intermediates required for PrPc trafficking also participate in the trafficking of a minimum GPI-anchored protein, GFP-GPI. We found that the steady-state distribution of GFP-PrPc is dynamin-regulated and that internalized GFP-PrPc is localized to Rab5-positive early endocytic vesicles and endosomes. In contrast, GFP-GPI is not found in the same endocytic organelles as GFP-PrPc. Consequently, we suggest that PrPc trafficking differs from that of other standard GPI-anchored proteins and may depend on additional internalization signals present in the protein. The SN56 cells were a generous gift from Prof. Bruce Wainer (Department of Pathology, Emory University School of Medicine, Atlanta, GA). Cells were maintained in Dulbecco's modified Eagle's medium (Sigma Chemical Co., St. Louis, MO), 10% fetal bovine serum (Invitrogen), 2 mml-glutamine, and 1% penicillin/streptomycin in 25-cm2 culture bottles in a 5% CO2 atmosphere at 37 °C as described previously (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar,32Barbosa J., Jr. Massensini A.R. Santos M.S. Meireles S.I. Gomez R.S. Gomez M.V. Romano-Silva M.A. Prado V.F. Prado M.A.M. J. Neurochem. 1999; 73: 1881-1893PubMed Google Scholar). The SN56 cells were derived from septum neurons (31Hammond D.N. Lee H.J. Tonsgard J.H. Wainer B.H. Brain Res. 1990; 512: 190-200Crossref PubMed Scopus (89) Google Scholar) and present a number of neuronal features, including expression of synaptic vesicle proteins (32Barbosa J., Jr. Massensini A.R. Santos M.S. Meireles S.I. Gomez R.S. Gomez M.V. Romano-Silva M.A. Prado V.F. Prado M.A.M. J. Neurochem. 1999; 73: 1881-1893PubMed Google Scholar) and neuronal type calcium channels (33Kushmerick C. Romano-Silva A. Gomez M.V. Prado M.A. Brain Res. 2001; 916: 199-210Crossref PubMed Scopus (26) Google Scholar). Such features are increased by differentiation (33Kushmerick C. Romano-Silva A. Gomez M.V. Prado M.A. Brain Res. 2001; 916: 199-210Crossref PubMed Scopus (26) Google Scholar, 34Blusztajn J.K. Venturini A. Jackson D.A. Lee H.J. Wainer B.H. J. Neurosci. 1992; 12: 793-799Crossref PubMed Google Scholar). The GFP-PrPc vector has been described previously (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar). The constitutively activated Rab5 mutant Q79L (Q79L), dynamin I (dynamin), and the dominant-negative dynamin I mutant K44A (K44A) plasmids were a gift from Prof. Marc G. Caron (Department of Cell Biology, Duke University and Howard Hughes Medical Institute). GFP-GPI was a gift from Benjamin J. Nichols and J. Lippincott-Schwartz (Medical Research Council Laboratory of Molecular Biology, United Kingdom, and Cell Biology and Metabolism Branch, NICHD, National Institutes of Health). The SN56 cells were plated on coverslips 1 day before transfection. Cell transfection was performed by the liposome-mediated method (LipofectAMINE 2000, Invitrogen, Gaithersburg, MD) according to the manufacturer's instructions. One microgram of plasmid and 2.5 μl of LipofectAMINE 2000 were used for 5.5 × 104 cells. After 4 h of transfection, cells were maintained in serum-free medium and differentiated for 2 days. In co-transfection experiments we used 3–4 μg of DNA (with a proportional change of LipofectAMINE 2000), following a plasmid ratio of 1:3 (Rab5-Q79L) or 1:2 (dynamin I or dynamin I-K44A) for GFP-PrPc and the other plasmids. Live cell experiments were performed at room temperature (20–25 °C). Cells on coverslips were washed in MEM (minimal essential medium, without phenol red) and transferred to a custom holder in which the coverslip formed the bottom of a 400-μl bath. Imaging was performed with a Bio-Rad MRC 1024 laser scanning confocal system running the software Lasersharp 3.0 coupled to a Zeiss microscope (Axiovert 100) with a water immersion objective (40×, 1.2 numerical aperture) as described previously (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar). Image analysis and processing were performed with Lasersharp (Bio-Rad), Confocal Assistant, Adobe Photoshop, and Metamorph (Universal Imaging) software. This assay has been previously described and shown to be dependent on the presence of the intact octarepeat Cu2+ binding region of PrPc (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar). Cells were perfused with MEM and, after obtaining the first Z series (0 min), MEM with or without 250–500 μm Cu2+ was perfused and another Z series was acquired (15 min). To label endocytic organelles, we used the styryl dye FM4-64 (Molecular Probes, Eugene, OR). Cells were incubated with 16 μm FM4-64 for 15–40 min at 37 °C in 5% CO2 and then visualized by confocal microscopy as previously described (35Santos M.S. Barbosa J., Jr. Veloso G.S. Ribeiro F. Kushmerick C. Goámez M.V. Ferguson S.S. Prado V.F. Prado M.A. J. Neurochem. 2001; 78: 1104-1113Crossref PubMed Scopus (34) Google Scholar). Labeling of endosomes was performed by incubating cells with 40 μg/ml Alexa Fluor 568-labeled transferrin (Tfn-568, Molecular Probes) at 37 °C in 5% CO2 for 20 min. After incubation, cells were washed three times with ice-cold phosphate-buffered saline and then either imaged or fixed with 3% paraformaldehyde in phosphate-buffered saline for 20 min for posterior imaging. Golgi complex was identified with Ceramide-Bodipy TR (Molecular Probes) as follows: cells were washed in HEPES-buffered salt solution (in millimolar: 137 NaCl, 4 KCl, 2 CaCl2, 1.2 MgSO4, 10 glucose, 10 HEPES, pH 7.4, adjusted with NaOH) and preincubated for 15 min at 37 °C and then 15 min at 4 °C with 5 μm Bodipy TR complexed with bovine serum albumin. Cells were then washed with HEPES-buffered salt solution and incubated for further 30 min at 37 °C before imaging. Two types of Ceramide-Bodipy (FL and TR) labeled the same structures in SN56 cells, and labeling was completely abolished by Brefeldin A. Moreover, Ceramide-Bodipy FL, which fluoresces in green, co-localized with a red fluorescent variant of PrPc (not shown). Previous experiments using different GFP-PrPcconstructions have suggested that the fluorescent protein labels the Golgi compartment, as assessed by the co-localization of the GFP-tagged protein with a number of Golgi markers (27Ivanova L. Barmada S. Kummer T. Harris D.A. J. Biol. Chem. 2001; 276: 42409-42421Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 28Negro A. Ballarin C. Bertoli A. Massimino M.L. Sorgato M.C. Mol. Cell. Neurosci. 2001; 17: 521-538Crossref PubMed Scopus (61) Google Scholar, 29Lorensz Windl O. Kretzschmar H.A. J. Biol. Chem. 2002; 277: 8508-8516Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Optical sections of living cells double-labeled with GFP-PrPc and Ceramide-Bodipy TR confirmed these previous observations showing an excellent degree of localization of GFP-PrPc in the Golgi apparatus in SN56 cells (Fig. 1,A–C). However, a significant proportion of GFP-PrPc was localized to vesicular structures that were labeled by the endosomal compartment marker Tfn-568 (Fig. 1,D–F). A large proportion of the double-labeled endosomes were packed within the perinuclear region, although rare puncta in close proximity to the plasma membrane were also observed (Fig. 1D, arrow). Experiments with the vital dye FM4-64 showed that the GFP-PrPc-labeled perinuclear structure can be partially labeled with this endocytic tracer (Fig. 1,G–J). These observations suggest that intracellular GFP-PrPc is accumulating not only in the Golgi but also in endosomal compartments. To determine the initial steps involved with PrPc trafficking, we examined whether the expression of a dynamin I-K44A mutant might perturb GFP-PrPc and GFP-GPI (a marker of non-clathrin-mediated endocytosis (23Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-542Crossref PubMed Google Scholar)) distribution in SN56 cells. Expression of the dynamin I-K44A dominant-negative mutant has been used extensively as a tool to block fission of endocytic intermediates (36Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1037) Google Scholar). When dynamin I-K44A was co-expressed with GFP-PrPc in SN56 cells, GFP-PrPc was localized in structures close to the plasma membrane surface (Fig. 2,E–G). In contrast, in cells co-expressing wild-type dynamin I, labeling of these structures was not observed (Fig. 2,A–D). The internalization of FM4-64 (an endocytic tracer dye) was also decreased in dynamin I-K44A-expressing cells (Fig. 2, compare B with F), suggesting that most of the FM4-64 accumulation in the perinuclear region depends on dynamin activity. As expected, expression of dynamin I-K44A also inhibited by 75% the internalization of fluorescent transferrin by these cells, suggesting that this mutant potently inhibits clathrin-mediated endocytosis (37Barbosa, J., Jr., Ferreira, L. T., Martins-Silva, C., Santos, M. S., Torres, G. E., Caron, M. G., Gomez, M. V., Ferguson, S. S. G., Prado, M. A. M., and Prado, V. F. (2002) J. Neurochem., in press.Google Scholar). Fig. 2 (I–L) shows in more detail the GFP-PrPcstructures that appear in dynamin I-K44A co-transfected cells. Few vesicles labeled with GFP-PrPc close to the plasma membrane could also be labeled with FM4-64 (Fig. 2, I–L). The labeling varied from cell to cell, but some cells showed large numbers of GFP-positive structures that were also stained with FM4-64 (see Fig.2J). Optical sectioning of regions close to the top of the cell shows that these structures most often appeared to be connected to the plasmalemma and perhaps to the exterior milieu, because they are accessible to the impermeant dye FM4-64 (Fig. 2, I andJ). Sections toward the middle of the cell show the presence of fluorescent GFP puncta in close association with the plasma membrane (Fig. 2, K and L). The structures labeled with GFP-PrPc in dynamin I-K44A-expressing cells were similar to those described for some plasma membrane receptors that continuously traffic between the plasmalemma and the cytoplasm (38Anborgh P.H. Seachrist J.L. Dale L.B. Ferguson S.S. Mol. Endocrinol. 2000; 14: 2040-2053PubMed Google Scholar). In some cells (Fig. 2E), the expression of the GFP-PrPc was limited to intense vesicular patches with very little diffuse labeling of the plasma membrane, suggesting that the GFP-PrPc is constitutively endocytosed. In contrast, under conditions where expression of the dominant-negative dynamin I-K44A mutant blocked uptake of FM4-64 into SN56 cells, there is little change in the subcellular localization of GFP-GPI (compare Fig.3, A and E). Thus, GFP-GPI exhibits a similar pattern of distribution in wild-type and mutant dynamin-expressing cells (Fig. 3). These observations indicate that the structures labeled with GFP-PrPc do not participate in the internalization of GFP-GPI. The observation that GFP-PrPc, but not GFP-GPI, accumulated in vesicles connected to the plasma membrane in the presence of dynamin I-K44A prompted us to investigate the cellular organelles underlying the constitutive trafficking of PrPc. We detected some events of co-localization between GFP-PrPc and Tfn-568 in puncta close to the plasma membrane in cells (Fig. 1), with a more extensive co-localization observed in the perinuclear region. Therefore, we examined whether GFP-PrPc either transits or bypasses the Rab5-positive early endosomal compartment to reach the perinuclear compartment of cells. Rab5 is involved in endosomal traffic and fusion. A Rab5-Q79L mutant that mimics the GTP-bound form of Rab5 and exhibits constitutive activity promotes homotypic fusion of endocytic vesicles into enlarged vesicular structures (39Stenmark H. Parton R.G. Steele-Mortimer O. Lutcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (771) Google Scholar, 40Nielsen E. Severin F. Backer J.M. Hyman A.A. Zerial M. Nat. Cell Biol. 1999; 1: 376-382Crossref PubMed Scopus (395) Google Scholar). If GFP-PrPcbypasses this compartment, there should be no change in the subcellular distribution of the GFP-tagged protein in the presence of Rab5-Q79L. Images of cells overexpressing Rab5-Q79L show that GFP-PrPc was readily identified in vesicles close to the plasma membrane (Fig. 4, A–C, green). Some of the GFP-PrPc-positive vesicles were also labeled with the vital dye FM4-64 (Fig. 4, A–C, superimposed images labeled with arrows), and in many cases we detected co-localization of GFP-PrPc with the endocytic marker Tfn-568 (Fig. 5,A and B). These results suggest the early endocytic origin of the GFP-PrPc-labeled vesicles. In agreement with a previous report (23Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-542Crossref PubMed Google Scholar), GFP-GPI did not localize in vesicles filled with FM4-64 or Tfn-568 in the presence of Rab5-Q79L (Figs. 4, D–F, and 5, C andD).Figure 5GFP-PrPc co-localizes with Tfn-568 in Rab5 Q79L-expressing cells. SN56 cells were transfected with GFP-PrPc and the Rab5 Q79L mutant, or with GFP-GPI and Rab5-Q79L. Forty-eight hours after transfection, cells were labeled with Tfn-568 for 20 min, fixed with paraformaldehyde 3%, and examined by laser scanning confocal microscopy. Two optical sections of representative cells expressing GFP-PrPc and Rab5-Q79L are shown in A and B, whereas two optical sections of a representative cell expressing GFP-GPI and Rab5-Q79L are shown inC and D. GFP-PrPc and GFP-GPI are presented in green, Tfn-568 is in red, and co-localization is seen in yellow in the superimposed images (lower panel). Arrows point to vesicles presenting co-localization of GFP-PrPc and Tfn-568. The Z axis distance between each image is shown at the bottom. The results are representative of 35 and 28 cells, respectively, for GFP-PrPc and GFP-GPI imaged in three independent experiments. Scale bar, 20 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Our experiments suggest that constitutive traffic of GFP-PrPc is distinct from that of GFP-GPI for its sensitivity to K44A and presence in Rab5-positive endosomes. However, these studies did not determine whether GFP-PrPc enters early endosomes directly following internalization from the plasma membrane or whether the fluorescent protein is indirectly redistributed to earlier endosomes. It is known that copper binds to PrPcthrough an octarepeat region of amino acids (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, 41Viles J.H. Cohen F.E. Prusiner S.B. Goodin D.B. Wright P.E. Dyson H.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2042-2047Crossref PubMed Scopus (510) Google Scholar), and among other changes Cu2+ induces PrPc internalization (11Pauly P.C. Harris D.A. J. Biol. Chem. 1998; 273: 33107-33110Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar,26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar, 30Sumudhu W. Perera S. Hooper N.M. Curr. Biol. 2001; 11: 519-523Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). We thus used Cu2+ to evoke synchronized GFP-PrPc internalization in cells overexpressing Rab5-Q79L to determine whether these vesicles receive internalized GFP-PrPc. Fig. 6(A and B) shows that GFP-PrPcresponded to copper and accumulated in endocytic organelles in cells overexpressing Rab5-Q79L (arrows). In the absence of Rab5 Q79L there was only limited accumulation of GFP-PrPc in early endocytic vesicles (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar). However, short term exposure to Cu2+ in the absence of Rab5-Q79L induced the rapid accumulation of GFP-PrPc in the perinuclear compartment (Fig. 6, G and H) (26Lee K.S. Magalhães A.C. Zanata S.M. Brentani R.R. Martins V.R. Prado M.A. J. Neurochem. 2001; 79: 79-87Crossref PubMed Scopus (109) Google Scholar). Taken together, these observations suggest that under normal conditions GFP-PrPctransits very rapidly through the early endosomal compartment and that only following the perturbation of endosomal trafficking is GFP-PrPc detected in early endosomes. This may be the consequence of the detection limits for GFP-PrPc, and its localization may require increased accumulation of protein in early endosomes following Rab5-Q79L expression. As expected, GFP-GPI, which is not found in Tfn-positive endosomes (23Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-542Crossref PubMed Google Scholar), did not become internalized through exposu
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