Essential Role of the Prion Protein N Terminus in Subcellular Trafficking and Half-life of Cellular Prion Protein*

Aberrant metabolism and conformational alterations of the cellular prion protein (PrPc) are the underlying causes of transmissible spongiform encephalopathies in humans and animals. In cells, PrPc is modified post-translationally and transported along the secretory pathway to the plasma membrane, where it is attached to the cell surface by a glycosylphosphatidylinositol anchor. In surface biotinylation assays we observed that deletions within the unstructured N terminus of murine PrPc led to a significant reduction of internalization of PrP after transfection of murine neuroblastoma cells. Truncation of the entire N terminus most significantly inhibited internalization of PrPc. The same deletions caused a significant prolongation of cellular half-life of PrPc and a delay in the transport through the secretory pathway to the cell surface. There was no difference in the glycosylation kinetics, indicating that all PrP constructs equally passed endoplasmic reticulum-based cellular quality control. Addition of the N terminus of the Xenopus laevis PrP, which does not encode a copper-binding repeat element, to N-terminally truncated mouse PrP restored the wild type phenotype. These results provide deeper insight into the life cycle of the PrPc, raising the novel possibility of a targeting function of its N-proximal part by interacting with the secretory and the endocytic machinery. They also indicate the conservation of this targeting property in evolution.

Transmissible spongiform encephalopathies in humans and animals can be manifested as sporadic, familial and acquired disorders and include Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle. These neurodegenerative diseases are caused by the accumulation of a conformationally altered isoform of the cellular prion protein (PrP c ), 1 denoted PrP Sc (1)(2)(3). During biogenesis, PrP c is directed cotranslationally into the lumen of the endoplasmic reticulum by a 22-amino acid N-terminal signal peptide. This is then removed together with a 23-amino acid C-terminal signal sequence promoting attachment of a GPI anchor. The protein undergoes further post-translational modifications with the addition of two N-linked carbohydrate chains. Properly folded PrP c transits through the Golgi compartment and the secretory pathway and is attached to the outer leaflet of the plasma membrane by its GPI anchor (4 -6).
Conversion of PrP c into PrP Sc has been reported to occur close to the plasma membrane along the endocytic pathway, probably in caveolae-like domains or in rafts, membranous domains or invaginations of the plasma membrane rich in cholesterol and glycosphingolipids (7). Here also the first steps in PrP c degradation occur (8,9) before reaching acidic compartments for final degradation. Cell surface localization of PrP c is thought to be essential for subsequent conversion into PrP Sc (4 -6), and studies in transgenic mice suggest a direct interaction between the two PrP isoforms, possibly in a complex with auxiliary factors (1). The cellular function of the prion protein is still unknown, although binding of copper to the octapeptide repeat sequence located at its N terminus suggests a role of PrP c related to this phenomenon (10,11). A superoxide dismutase activity or a role as a carrier protein for uptake and delivery of metal ions from the extracellular space into the cell have also been discussed (12,13).
Studies performed on the subcellular trafficking of GPIanchored proteins have revealed that their sorting is not a simple default process because specific signals are required for transport from the Golgi to the cell surface and for endocytosis. These targeting signals are still poorly characterized. Although sorting and endocytosis of most transmembrane proteins require recognition of specific motifs in their cytoplasmic domains by multimeric adaptor protein complexes, other targeting elements are obviously necessary for the trafficking of GPI proteins which lack such cytoplasmic extensions. Several elements have been shown to influence PrP c trafficking. Studies done with PrP constructs lacking the GPI anchor revealed slower transport through the secretory pathway compared with wild type PrP c (wtPrP c ) (14), although conversion of these constructs into PrP Sc was previously not found to be compromised significantly (15). Prevention of glycosylation affected the transport to the cell surface and changed the biochemical properties of PrP c (16,17). Addition of a transmembrane moiety to the C terminus of PrP c has been shown to affect subcellular trafficking and to inhibit conversion into the scrapie isoform (7).
In this report we were interested in devising a physiological function for the N-terminal part of PrP c in subcellular trafficking. We therefore performed various metabolic labeling and surface biotinylation assays to follow the intracellular trafficking and turnover of the PrP. We expressed specific N-terminal PrP deletion constructs and one chimeric Xenopus laevis-mouse construct in murine neuroblastoma cells and compared their internalization from the exofacial plasma membrane, their transport along the secretory pathway, and half-life with those of transfected wtPrP c . Our results showed significant differences in the behavior of these proteins, although all constructs effectively passed the cellular quality control in the endoplasmic reticulum/Golgi. On the other hand, the chimeric protein consisting of the short N-terminal segment of Xenopus (amino acids 23-69) fused to the truncated mouse PrP nearly restored wild type secretory and endocytic kinetics. These data indicate that the N-proximal domain of the PrP functions as a putative targeting element and is essential for both transport to the plasma membrane and modulation of endocytosis. These targeting functions of the N terminus are also highly conserved in evolution.

EXPERIMENTAL PROCEDURES
Reagents-Monoclonal PrP-specific antibody 3F4 (Signet Pathology) recognizes the sequence encompassing amino acids 109 and 112 in hamster and human PrP and has been described before. Polyclonal anti-PrP-specific antibody A7 was obtained in our laboratories after immunization of rabbits with recombinant dimeric mouse PrP. Cell culture media and trypsin-EDTA were obtained from Invitrogen. [ 35 S]Met/Cys (Promix; 1,000 Ci/mmol) for labeling of proteins, protein A-Sepharose, and an enhanced chemoluminescence blotting kit were from Amersham Biosciences. Endoglycosidase H (Endo-H), PNGase F, and Pefabloc proteinase inhibitor were all obtained from Roche Molecular Biochemicals. Biotin sulfo-NHS and horseradish peroxidase-conjugated streptavidin were obtained from Pierce, and trypsin inhibitor was from Sigma. Transient and stable transfections were carried out using FuGENE (Roche Molecular Biochemicals) or Effectene (Qiagen) transfection reagents.
Cell Culture and Transient Expression of wtPrP c and N-terminally Truncated PrP Constructs-The mouse neuroblastoma cell line N2a (ATCC CCL 131) has been described (18). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, penicillin/streptomycin, and glutamine in a 5% CO 2 atmosphere. The medium was changed every 48 h. wtPrP c and PrP chimeric constructs were all cloned into the pcDNA3.1/Zeo expression vector (Invitrogen). Substitution of amino acids 109 and 112 (numbering referring to human PrP according to Ref. 19) in murine wtPrP with methionine using site-directed mutagenesis allows recognition of transfected mouse PrP by the monoclonal antibody 3F4 and discrimination from endogenous PrP because the antibody does not recognize murine wtPrP c . Construction of PrP chimeric constructs was done by PCR-based standard techniques using 3F4-tagged mouse PrP as a template. Insertion of appropriate restriction sites allowed cloning of PCR fragments into the multiple cloning site of the vector pcDNA3.1 using standard cloning techniques. Briefly, PrP⌬(23-90) was obtained by deleting amino acids 23-90 of full-length PrP (numbering referring to human PrP); in PrP⌬(48 -93) residues 48 -93 were deleted; in PrP⌬(23-51) and PrP⌬(68 -91) amino acids 23-51 and 68 -91 were absent, respectively. For cloning of PrPXen(23-69) a PCR fragment encompassing residues 23-69 of X. laevis was generated from an Image cDNA clone using primers allowing insertion of the fragment between amino acids 22 and 92 of murine PrP. Constructs were always confirmed by nucleic acid sequencing. Plasmid pEGFP (Clontech) was used as a control for specificity of results. All cloned constructs were transiently transfected into N2a cells by lipofection according to manufacturer's directions, and cells were lysed for experiments 72 h post-transfection. For selected constructs, stable transfections were performed in addition, using Zeocin as a selection marker.
Detergent Solubility Assay and Western Blot Analysis-Confluent transfected cells were lysed in cold lysis buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholate). Postnuclear cell lysates were supplemented with 0.5 mM Pefabloc protease inhibitor and N-lauryl sarcosine to 1% and centrifuged for 1 h at 100,000 ϫ g at 4°C in a Beckman TL-100 centrifuge. Soluble fractions (supernatants) were precipitated with ethanol. Insoluble fractions (pellets) were resuspended in 50 l of TNE (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA). Ethanol-precipitated samples were centrifuged for 30 min at 2,500 ϫ g, and the pellets were redissolved in TNE buffer with the addition of gel loading buffer. Samples were then boiled for 10 min, and an aliquot was analyzed on 12.5% SDS-PAGE. Proteins were electrotranferred to a polyvinylidene difluoride membrane. This was blocked with non-fat dry milk (5%) in TBST (0.05% Tween 20, 100 mM NaCl, 10 mM Tris-Cl, pH 7.8), incubated overnight with antibody 3F4 at 4°C, and stained using an enhanced chemoluminescence blotting kit from Amersham Biosciences.
Proteinase K (PK) Digestion-Aliquots of postnuclear lysates were incubated for 30 min at 37°C with 20 g/ml PK; the digestion was stopped by the addition of Pefabloc. Samples were precipitated with ethanol and analyzed in an immunoblot assay.
Metabolic Labeling and Immunoprecipitation Assay-Confluent transfected N2a cells were washed twice with phosphate-buffered saline (PBS) and starved for 1 h in RPMI medium without methionine/ cysteine containing 1% fetal calf serum. Labeling was carried out by adding 400 Ci/ml [ 35 S]Met/Cys to the medium for 5 min or, for half-life studies, for 1 h. After incubation, cells were washed twice in cold PBS and harvested with lysis buffer on ice for 10 min or incubated at 37°C for different lengths of time in complete culture medium to allow transport to the cell surface. After appropriate chase times, cells were washed with PBS and either harvested directly or 1 ml of trypsin-EDTA was added on the dishes for 10 min on ice. Trypsinized cells were transferred to polypropylene tubes, and the reaction was stopped by centrifugation of the cells twice at 900 ϫ g in PBS containing 20% fetal calf serum and 100 l of trypsin inhibitor. Cells were then lysed in cold lysis buffer and cell debris removed by centrifugation for 40 s at 18,000 ϫ g. After addition of 1% N-lauryl sarcosine postnuclear lysates were boiled at 95°C for 10 min. Samples were placed on ice, and Pefabloc protease inhibitor was added; lysates were incubated with antibody A7 or 3F4, as indicated, overnight at 4°C (dilution 1:300). Protein A-Sepharose beads were added to the protein-antibody complexes for 90 min at 4°C. The beads were centrifuged at 18,000 ϫ g for 1 min and washed in radioimmune precipitation assay buffer (0.5% Triton X-100, 0.5% deoxycholate in PBS) supplemented with 1% SDS at 4°C. All samples were treated with 0.1 unit/l PNGase F at 37°C overnight to remove N-linked oligosaccharides and analyzed by 12.5% SDS-PAGE. Gels were exposed to an x-ray film (Kodak), or the autoradiographic signals were quantified by PhosphorImager analysis of the gel (Molecular Dynamics). The amount of total or intracellular (after surface trypsin digestion) PrP present at each time point after the chase was expressed as a percentage of nascent PrP rescued from the cell lysate directly at the end of the labeling period.
Internalization Assay Using Surface Biotinylation-Endocytosis of wtPrP c and PrP mutants located on the cell surface was assessed by surface biotinylation. Briefly, confluent transfected N2a cells were washed twice with cold PBS and incubated on ice for 15 min with 1 ml of PBS containing 250 g of biotin sulfo-NHS. Cells were washed again three times with cold PBS to remove unbound biotin and were either harvested directly with cold lysis buffer on ice for 10 min, or culture medium was added for appropriate chase times as indicated in the respective experiments at 37°C to allow internalization. Cells were washed with PBS and either lysed directly or treated with 1 ml of trypsin-EDTA on ice for 10 min before lysis and immunoprecipitated as described above, using monoclonal antibody 3F4 for detection of transfected constructs. Immunoadsorbed proteins were subjected to 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Biosciences). Blots were developed with horseradish peroxidase-conjugated streptavidin and visualized with enhanced chemoluminescence. Films were digitized using an APB Image Scanner and images quantified using Image Master 1D software (both from Amersham Biosciences).

Treatment with Endo-H, PNGase F, and Immunoblot Analysis-For
Endo-H digestion, aliquots of protein lysates were incubated after immunoprecipitation with 0.1 M ␤-mercaptoethanol and 0.1% SDS and heated at 95°C for 10 min. After centrifugation at 18,000 ϫ g, supernatants were supplemented with Endo-H buffer (0.1 M sodium citrate, pH 5.5, 0.5% phenylmethylsulfonyl fluoride, 6 milliunits of Endo-H) and incubated overnight at 37°C. All samples were then subjected to SDS-PAGE. PNGase F treatment was carried out by mixing 100 l of postnuclear lysate or immunoprecipitated proteins with 20 l of mercaptoethanol and 0.5% SDS and heating at 95°C for 10 min. Supernatants from immunoprecipitation were incubated with lysis buffer, and all samples were supplemented with protease inhibitor and 0.1 unit/l PNGase F overnight at 37°C before analysis on SDS-PAGE.

Internalization of Mouse PrP c Is Impaired by Progressive
N-terminal Deletions-To assess the relevance of the N-terminal part of PrP c in its intracellular trafficking, we transiently transfected murine neuroblastoma cells with a series of PrP constructs in which amino acids within the N terminus had been progressively deleted (Fig. 1A). All experiments were performed in parallel with and compared with transfected wtPrP c , to rule out possible effects on the kinetics caused by overexpression or metabolic stress upon transfection. The constructs were first characterized biochemically in a solubility assay. Postnuclear lysates were ultracentrifuged in a buffer containing 1% sarcosyl for separation of soluble from insoluble proteins. Aliquots of the lysates were subjected to a mild proteolytic treatment with PK or to PNGase F digestion. All samples were then run on a polyacrylamide gel and examined by Western blot analysis. The PrP deletion constructs were detected in the supernatant after ultracentrifugation and were therefore soluble ( Fig. 1B, lanes 2 , 5, 8, 11, and 14). They were also completely degraded by PK (lanes 3, 6, 9, 12, and 15) and, upon PNGase F digestion, migrated as single unglycosylated bands (Fig. 1C). Taken together these results indicate that deletions within the N-terminal parts do not alter the biochemical features of PrP c . We next focused on the endocytic arm of the PrP c life cycle and evaluated the kinetics of PrP c internalization. We labeled proteins expressed at the outer leaflet of the plasma membrane by adding membrane-impermeable biotin sulfo-NHS to the culture medium for 15 min on ice and either lysed the cells immediately or incubated them at 37°C for different time periods to allow internalization. Subsequent digestion with trypsin on ice allowed separation of internalized proteins from those still present on the plasma membrane. Cells were then lysed and the wild type and mutant PrPs immunoprecipitated with the antibody 3F4, which allows discrimination of transfected constructs (all containing a 3F4 epitope) from endogenous PrP and to visualize endocytosis. We first compared the internalization of wtPrP c with that of mutant PrPs presenting deletions of different length in their N-terminal part: PrP⌬(23-51) lacked the 29 amino acids between the signal peptide and the octapeptide repeats, in PrP⌬(48 -93) a segment of 44 residues encompassing the octapeptides was deleted, and in PrP⌬(23-90) the complete N terminus (67 residues after the signal peptide, therefore comprising octapeptide repeats and the region preceding them) were missing. Immediately after the label, all constructs were expressed on the cell surface because they could all be biotin-labeled ( Fig. 2A, lanes 1, 8, 14, and 18) and could not be detected after treatment with trypsin, which only digests surface-located proteins (lanes 2, 9, 15, and 19). Lane 7 shows lysate from cells transfected with the plasmid pEGFP, as a control, and precipitated with antibody 3F4, to show specificity of our results. Trypsinization of the wtPrP c and of PrP⌬(23-51) revealed that after 45 min of chase, only wtPrP c had been internalized efficiently ( Fig. 2A, lanes 4 and  11). On the other hand, after 60 min both constructs had entered the cells although in different amounts: quantification of the blots revealed that ϳ72% of wtPrP c had been endocytosed whereas PrP⌬(23-51) only measured ϳ32% (Fig. 2B). In contrast, neither PrP⌬(48 -93) nor PrP⌬(23-90) could be detected intracellularly (lanes 17 and 21). To evaluate the kinetics of internalization for PrP⌬ (48 -93) and PrP⌬(23-90) in more detail, we prolonged the chase times using an analogous biotinylation assay (Fig. 3). This experiment revealed an extremely impaired endocytosis for both constructs: only small amounts of PrP⌬ (48 -93) were detectable inside the cell after 6 h of chase, rising to 65% after 10 h (lane 16). On the contrary, the level of intracellular PrP⌬(23-90) remained extremely low throughout the chase (lanes 4, 6, and 8) and only reached ϳ7% of total amount of PrP after 10 h. Fig. 3B shows a quantitative evaluation of two experiments. Because an altered endocytosis was observed in all PrP mutants analyzed, the stronger impairment clearly caused by the longer deletions, our results argue for a correlation between the length of the N-terminal truncation and the efficacy of PrP internalization.
Deletion of N-proximal Sequences Significantly Prolongs the Half-life of PrP c -Because the partial or complete removal of the N-terminal part of PrP significantly prolonged the presence of PrP c on the plasma membrane we wondered whether altered internalization also interfered with the degradation and cellular stability of the proteins. In pulse-chase experiments we compared the half-lives of wtPrP c , PrP⌬(23-90), and PrP⌬(48 -93). N2a cells expressing these constructs were metabolically labeled with [ 35 S]methionine/cysteine for 1 h and either har-FIG. 1. Biochemical characterization of PrP deletion constructs. A, wt-PrP c and PrP deletion constructs used in this study were derived from murine PrP c , and all contain the 3F4 epitope (amino acids 109 -112, vertical bars), which allows detection by the monoclonal antibody 3F4. Numbers refer to the human PrP sequence. Black and open bars represent the signal peptide and GPI anchor, respectively; hatched bars, the octapeptide repeats (48 -93); and dotted bars, the amino acids preceding the octapeptide region . B, postnuclear lysates of N2a cells transiently transfected with wtPrP c or with PrP deletion constructs were subjected to a solubility assay or treatment with 20 g/ml PK. The detergent-insoluble pellets (lanes 1, 4, 7, 10, and 13), the supernatants (lanes 2, 5, 8, 11, and 14), and the PK-treated fractions (lanes 3, 6, 9, 12, and 15) were all analyzed by immunoblotting using the monoclonal antibody 3F4. Molecular mass markers are indicated in kDa on the left. C, 200-l fractions of postnuclear lysates described in B were treated overnight with PNGase F for deglycosylation of the proteins and then subjected to immunoblotting with the antibody 3F4. The molecular masses are shown in kDa on the left. vested directly or chased for different intervals of time in 35 Sfree culture medium before lysis. PrP present in the lysates was immunopurified with the anti-PrP antibody A7, deglycosylated with PNGase F, and analyzed by SDS-PAGE (Fig. 4A). The use of the polyclonal antibody A7 in this and in the following experiments did not alter the results due to cross-reaction with endogenous PrP c because differences in molecular mass allowed discrimination between deletion mutants and endogenous wtPrP c . The autoradiogram was evaluated by densitometric analysis and the specific bands quantified as fractions of the signal observed in the absence of chase. Time points of 2, 3, 6, 8, and 22 h show that for all constructs the signal declined as a function of the chase. Curves in Fig. 4B show the mean values from three independent experiments: wtPrP c revealed a halflife of ϳ2.6 h. On the other hand, the turnover of our PrP mutants proved to be significantly prolonged. Our measurements indicated a half-life of ϳ4.8 h for PrP⌬(23-90) and ϳ4.2 for PrP⌬ (48 -93), both values almost twice as long as for wt-PrP c . These data corroborate the idea that prolonged accumulation of N-terminal deleted mutants on the plasma membrane indeed affects the turnover of these proteins.
N-proximal Region of PrP Modulates Transport to the Plasma Membrane-Having found that the N-terminal part of PrP c plays an essential role in modulation of endocytosis and stability, we next focused on the secretory pathway and on the transport of PrP to the cell surface to identify a possible function of the N-proximal segment in this arm of the PrP life cycle. Using pulse-chase experiments we evaluated the time required by wtPrP c and PrP deletion constructs to reach the plasma membrane. After transfection, N2a cells were metabolically labeled with a short pulse of 5 min, to have a more homogeneous population, and then harvested immediately or incubated in [ 35 S]Met-free medium at 37°C for different time periods to allow transport to the cell surface. Molecules that had reached the plasma membrane were separated from those still undergoing synthesis or transport to the cell surface by trypsin treatment on ice for 10 min and were then immunoprecipitated with an anti-PrP antibody. All samples were deglycosylated with PNGase F for simplifying quantitative comparison of the constructs. Immediately after the pulse, wtPrP c and the PrP constructs were still protected from extracellular trypsin digestion (Fig. 5, lanes 2, 8, and 14), consistent with their presence in the endoplasmic reticulum and/or Golgi apparatus. Our assay revealed that most of the wtPrP c molecules reach the cell surface within 1 h after synthesis (Fig. 5A). In Fig. 5B the mean data from three independent experiments show that although after 45 min 50% of total wtPrP can still be detected after trypsin treatment, the amount rapidly decreases to ϳ25% after 60 min. The transport of PrP⌬(23-90) and PrP⌬ (48 -93) to the cell surface showed important differences compared with wt-PrP. Even after 60 min of chase, both constructs were still detectable intracellularly in considerable amounts (Fig. 5A); PrP⌬(48 -93) was still clearly detectable after 75 min of chase. Quantification of the signals verified that 50% of labeled proteins were not susceptible to trypsin treatment after 65 and 75 min, respectively, for PrP⌬(23-90) and PrP⌬ (48 -93). This analysis corroborated the idea that deletion of N-terminal sequences negatively affects transport of PrP c even along the secretory pathway. For further analysis of the domains involved in trafficking of PrP c , we compared the transport of PrP⌬(23-51) with that of another construct where three of the five repeats (residues 68 -91) had been removed, in a pulsechase assay combined with trypsin treatment of transfected cells using the same time points for easier comparison (Fig. 6,  A and B). Because in these constructs segments of similar lengths were deleted in distinct sections of the N terminus, different kinetics of transport should reveal the presence of a domain with a dominant role in the secretory pathway. The time for detection of 50% of intracellular PrP was 65 and 70 min for PrP⌬(68 -91) and PrP⌬(23-51), respectively, which was longer than that measured for wtPrP c (45 min). These data confirmed that truncation of even small segments within the N terminus encompassing or preceding the octapeptide repeat region negatively interferes with the efficiency of transport of PrP c to the plasma membrane.
X. laevis N Terminus Restores Wild Type Phenotype in Truncated Mouse PrP-In light of the results described above, we studied the influence of the N-terminal part of a PrP belonging to a different species on the trafficking of the analyzed mouse PrP. We therefore expressed a chimeric PrP comprising the first 47 amino acids after the signal peptide (residues 23-69) of the X. laevis PrP inserted between residues 22 and 92 of mouse PrP (Fig. 7A). This chimeric protein, (Xen(23-69)), was first characterized biochemically in a solubility assay and upon PK digestion. The construct appeared to be soluble and entirely PK-sensitive (Fig. 7B, lanes 2 and 3). After deglycosylation with PNGase F, the construct migrated as a single band of about 23 kDa, consistent with its shorter amino acid sequence (lane 4). Further examination showed that Xen(23-69) was expressed on the cell surface, as seen in surface fluorescenceactivated cell sorter analysis (data not shown). To characterize the subcellular trafficking of this construct we first addressed its internalization in a surface biotinylation assay (Fig. 7C). Trypsinization and immunoprecipitation with antibody 3F4 revealed that this construct behaved neither like PrP⌬(23-90) nor like PrP⌬(48 -93) because we could detect considerable amounts of this protein after trypsinization following a 1-h chase (lane 6), showing more similarity to mouse wtPrP c . Analysis of the transport of Xen(23-69) along the secretory pathway in a pulse-chase experiment followed by trypsin digestion and immunoprecipitation led to similar results. After a short label of 5 min, 50% of Xen(23-69) moiety reached the cell surface after ϳ43 min (Fig. 7D), a value very similar to that of wild type mouse PrP. These data suggest that the N terminus of X. laevis, like the corresponding segment of the mouse PrP, has the same intrinsic sorting function and is able to restore the wild type trafficking kinetics.

N-terminal PrP Deletions Affect the Secretory Pathway after PrP Has Reached the Mid-Golgi
Compartment-To identify a specific compartment along the secretory pathway where N-terminal truncation of PrP c might result in a delayed transport to the cell surface, we subjected immunoprecipitated samples to Endo-H treatment, which removes high mannose glycans from glycoproteins before they reach the mid-Golgi. This assay therefore enables us to associate the localization of a protein with its glycosylation state. In pulse-chase experiments we measured at which time points wtPrP c , PrP⌬(23-90), and PrP⌬(48 -93) acquired Endo-H resistance (Fig. 8). After metabolic labeling of proteins, we either immediately harvested or chased them for different time intervals before lysis. After immunoprecipitation of proteins with antibody 3F4, all samples were subjected to Endo-H treatment. As shown in Fig. 8A, treatment of wtPrP c with Endo-H immediately after labeling resulted in a shift of the 33 and 28 kDa bands to a band of ϳ25 kDa (lane 2). We could detect initial signs of resistance after 30 min, when the monoglycosylated form appeared (data not shown). Complete Endo-H resistance was achieved between 40 and 50 min (lanes 4 and 6). Observing the other two constructs, we could detect the conversion to unglycosylated molecules of 19 and 22 kDa for PrP⌬(23-90) and PrP⌬(48 -93), respectively, when treated with Endo-H immediately after the pulse (Fig.  8A, lane 12, and Fig. 8B, lane 2). Partial resistance was detected after 40 min for PrP⌬(23-90) (lane 14) and for PrP⌬ (48 -93) (Fig. 8B, lane 4), and both mutants became fully Endo-H-resistant after 50 min. We therefore conclude from these results that deletions within the N-terminal part of PrP c do not significantly affect kinetics of glycosylation and transport of PrP c to the Golgi. DISCUSSION The structure of the PrP c displays enigmatic features because it seems to be composed of two highly different parts: the C-terminal part has a defined secondary structure consisting of three ␣-helices and two short ␤-strands. In contrast, the N-terminal region (i.e. amino acids 23-89) is devoid of any defined secondary structure and has the properties of a flexible random coiled polypeptide (20 -22). After conversion of PrP c into PrP Sc , residues 23-89 of PrP c remain entirely susceptible to PK digestion, whereas the rest of the protein becomes partly protease-resistant (1). Regions within the N-terminal part of PrP c have been suggested to influence interactions required for PrP Sc formation and to stabilize the conformation of PrP c (23). The hypothesis for a biological function of the N-terminal part of PrP, besides the copper binding mentioned earlier, is also supported by the fact that this domain, despite its lack of ordered structure, is highly conserved in evolution. Interspecies here mainly concern insertions and deletions (19). The current study aimed to assess a possible cellular function of the N-terminal part of PrP c in the life cycle of PrP and therefore to characterize the subcellular trafficking of N-terminally deleted PrP constructs.
Progressive Deletions within the N Terminus of PrP c Result in Highly Reduced Endocytosis and Prolonged Turnover-In preliminary studies, the biochemical properties of PrP constructs lacking segments of different length in their N-terminal end were compared with those of wtPrP c to exclude that conformational changes or overexpression might influence the outcome of the subsequent analysis. All of the mutants analyzed were properly glycosylated and did not aggregate. Therefore, significant alterations in their conformations were not likely. Our following analysis of internalization kinetics revealed delayed endocytosis for all PrP deletion constructs compared with For both constructs one plate was lysed immediately after the pulse or treated with trypsin, and the others were incubated for 45, 60, 75, and 90 min at 37°C and then digested with trypsin before harvesting. Proteins were immunoprecipitated with antibody A7, deglycosylated with PNGase F, and then subjected to SDS-PAGE. Bars on the right indicate PrP-specific signals. Both constructs migrated as doublets, probably because of incomplete deglycosylation. On the left a molecular mass marker is shown in kDa. B, diagrams show the percentage of protein detected after treatment with trypsin. Signals from autoradiograms were quantified by PhosphorImager analysis and depicted as a percentage of protein precipitated immediately after the pulse without trypsin treatment.
wtPrP c with a degree of impairment dependent on the length of the deletion. The effect of the N-terminal truncation on the endocytosis of PrP was particularly evident when we analyzed the internalization of PrP⌬(23-90) and PrP⌬ (48 -93). Our biotinylation studies support other work on endocytosis of chicken PrP c (24), although the murine PrP seems to have slower internalization kinetics. These differences are probably because of relatively poor identity between mammalian and avian PrP (ϳ30%) and to the longer N terminus of chicken PrP, which contains eight hexapeptide repeats (25). The endocytosis of murine PrP has also been analyzed in other studies, and the binding of copper to the histidine residues within the octapeptide repeats of PrP c has been seen to affect the cell surface localization of the protein by stimulating endocytosis (11,26,27). Our studies show that the octapeptide repeats are not the only region in PrP c with an internalization-promoting function because we could detect considerable differences between the internalization kinetics of PrP⌬(23-90) and PrP⌬ (48 -93). This hypothesis was confirmed when we examined PrP⌬(23-51) because this construct also showed impairment in its endocytosis, although not as strong as PrP⌬ . Because the PrP constructs analyzed were expressed on the cell surface and their biochemical behavior did not significantly differ from that of the wild type protein, we conclude that the effects described here on the trafficking of PrP mutants are not a consequence of folding abnormalities. An endoplasmic reticulum-based cellular quality control resulting in degradation by proteasomes in the cytosol as described for mutated secretory proteins does not seem to be the case (28). Interestingly, in our biotinylation assays, the glycosylated forms of wtPrP c and of the deletion constructs were particularly evident, compared with the unglycosylated one. This might be because the relative amount of glycosylation seems to be dependent on the cellular localization of PrP c . The labeled proteins localized at the plasma membrane could therefore represent a subpopulation with specific glycan ratios compared with the total cellular PrP (29). Endocytosis of most transmembrane proteins is mediated by direct recognition of tyrosine-based signal motifs in their cytoplasmic domain by specific sorter proteins. Different mechanisms are responsible for internalization of GPI-anchored proteins such as PrP because they are devoid of such domains. Lipid rafts where PrP c is localized on the cell surface appear to form a marginal boundary of semiordered lipids flanking the glycerolipid domain in which transmembrane proteins are embedded (30). Numerous transmembrane receptors have been shown to bind to GPI-anchored proteins and to translocate between different  35 S-free culture medium for 0, 45, 60, and 75 min, respectively. One plate was harvested immediately after the pulse or treated with trypsin and then lysed. All other plates were subjected to digestion with trypsin before lysis. Proteins were immunoprecipitated with antibody 3F4, deglycosylated with PNGase F, and subjected to SDS-PAGE. The blot shows PrP signals before (Ϫ) and after (ϩ) treatment with trypsin. Molecular mass markers are shown in kDa on the left. Lower panel, PhosphorImager evaluation of autoradiograms representing the amounts of protein detected after digestion with trypsin. Each point represents a mean value of two independent experiments. Quantities are calculated as a percentage of protein precipitated immediately after the pulse without trypsin treatment. plasma membrane domains (31,32). Clustering of PrP c in an intermediate semiordered lipid domain could promote binding to transmembrane proteins leading to endocytic trafficking. Our results support a similar model in which the N-terminal half of PrP c binds, as a single motif or as part of a larger epitope, with other elements of the protein, to the extracellular part of a transmembrane receptor containing internalization motifs. Impairment in endocytosis seen with N-terminal deletion constructs could therefore be explained by reduced affinity of these PrPs for a putative transmembrane receptor caused by deletions within the binding epitope.
The extremely prolonged presence of PrP molecules with deletions in their N-terminal part on the outer leaflet of the plasma membrane led to the assumption that this phenomenon might influence the turnover of these proteins. Our results revealed a half-life of ϳ2.6 h for wtPrP c , confirming published data on PrP c degradation in primary cell cultures derived from lymphoid and nervous tissues (33) and neuroblastoma cells (34). On the other hand, the half-life monitored for N-terminal deletion mutants was almost twice as long as that of wtPrP c . To our knowledge, these findings are new because we show that inefficient internalization of PrP c correlates with a significantly prolonged turnover. Studies done with PrP constructs encoding pathogenic mutations showed that these mutations can affect some biochemical properties and intracellular trafficking of PrP c (35), but prolonged half-life was not reported.
The N Terminus of PrP c and the Secretory Pathway-Given the importance of the N-terminal part of PrP c for internalization and rate of degradation, we next focused on the transport of PrP c along the secretory pathway. Because the targeting of proteins to the cell surface requires specific sorting determinants similar to those involved in endocytosis, we addressed whether the same PrP segment, having a significant role in endocytosis, could also display a targeting function in the transport to the cell surface. Our assays revealed a delay in the transport of the PrP mutants to the cell surface, and even relative small truncations within the N-terminal segment could impair trafficking through the secretory pathway. The N-terminal deletions affected PrP trafficking with different impacts, depending on the pathway analyzed: the strongest impairment was monitored in internalization, whereas the secretory pathway was less compromised. These findings argue for the specificity of our results.
In a first attempt to characterize this impairment in the secretory pathway in more detail we subjected immunoprecipitated proteins to an Endo-H digestion. This analysis revealed that complete resistance was achieved for all constructs after ϳ50 min. Our experiments also evidenced that wtPrP c as well as deletion mutants were fully glycosylated after the 5-min pulse. Endo-H resistance implies that glycoproteins have left the mid-Golgi, therefore our results indicate that the delay in the transport to the cell surface takes place in the trans-Golgi or in a post-trans-Golgi compartment. Numerous work has shown the trans-Golgi network represents the principal sorting station for transport of proteins to the cell surface (36). Here, sphingolipid-and cholesterol-rich rafts in the luminal leaflet of the Golgi membrane act as microdomains for inclusion of proteins destined for the plasma membrane (37). For proteins lacking cytoplasmic domains, sorting to specific cellular destinations is not based exclusively on protein sequence: N-and O-glycosylations (38), lipid attachment (e.g. palmitoylation) (39), and the GPI anchor (40,41) can act as sorting determinants for recruitment into vesicles targeted to the cell surface (42). Of note, the N-terminal segment of the phospholipidbinding protein annexin XIIIb was reported to contain a plasma membrane localization determinant (43). Despite the cell surface localization of the deletion mutants used in our assay, the impaired trafficking described indicates reduced capacity of the cell to deal with these proteins properly. The question therefore arises of whether prolonged permanence of these mutated PrPs on the cell surface and in the endocytic pathway has an impact on certain PrP Sc biogenesis scenarios. Indeed, studies done with transgenic mice expressing PrP lacking residues 32-93 showed that these were still susceptible to scrapie infection but showed altered pathology and longer incubation time. Prion titers and protease-resistant PrP were about 30-fold lower than in wild type mice, with no histopathology typical for scrapie (44). These results are in line with in vitro cell-free conversion studies performed with hamster PrP c , where a truncated form of PrP c lacking amino acids 32-94 influenced the quantity and the conformation of generated PrP Sc (45). In light of the results reported here, these findings could also be explained by altered intracellular trafficking of the mutated PrP because of the alterations in the N-terminal part. Clinical studies have shown that two to nine octapeptides in addition to the normal five segregate with familial forms of Creutzfeldt-Jakob disease (46,47) and nontransmissible prion disease in transgenic mice (48). Studies addressing the effect of these mutations on subcellular trafficking are in progress. In general, the question remains as to whether alterations in the trafficking of PrP and of other neurodegenerative proteins is important in the pathogenesis of sporadic forms of neurodegenerative disorders.
Targeting Function of PrP N Terminus Is Conserved in Evolution-The involvement of the N terminus of murine PrP in targeting and internalization led us to the question whether the corresponding sequence in the PrP c of more remote species might exert the same functions. The recent analysis of X. laevis PrP (49) allowed the construction of a chimeric Xenopus/mouse prion protein and characterization of its trafficking. Analysis of the biochemical properties of this construct did not reveal any abnormalities compared with wtPrP c . Surprisingly, addition of the Xenopus N-terminal stretch to the truncated mouse PrP almost restored the wild type trafficking phenotype, its internalization and secretory kinetics strongly differing from those of PrP⌬(23-90) as well as of PrP⌬ (48 -93). Xenopus PrP is significantly shorter than the mammalian PrP, mainly because of the complete lack of copper-binding repeat elements and only shows about 28% identity to murine PrP c . This segment was nevertheless able to direct subcellular trafficking properly when fused to PrP of another species. These data argue for a conserved, intrinsic sorting function of this segment.
Taken together, our results confirm the role of the N-terminal part of PrP c in mediation of endocytosis. Moreover, They extend its importance, as we show that this conserved segment exerts a more general function as a targeting signal through the secretory pathway, therefore representing a basic sorting motif for intracellular trafficking. Additionally, our finding that the trafficking of the chimeric Xenopus/mouse protein was not altered despite the absence of octarepeats argues in favor of a model in which copper binding and subcellular trafficking represent separate aspects in the life-cycle of PrP.