Essential Role of the Prion Protein N Terminus in Subcellular
Trafficking and Half-life of Cellular Prion Protein*
Max
Nunziante,
Sabine
Gilch, and
Hermann M.
Schätzl
From the Gene Center Munich, Max von Pettenkofer Institute for
Virology, Faculty of Medicine, Ludwig-Maximilians-University,
Feodor-Lynen-Strasse 25, D-81377 Munich, Germany
Received for publication, June 25, 2002, and in revised form, October 21, 2002
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ABSTRACT |
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.
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INTRODUCTION |
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
(PrPc),1 denoted
PrPSc (1-3). During biogenesis, PrPc 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
PrPc 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 PrPc into PrPSc 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 PrPc
degradation occur (8, 9) before reaching acidic compartments for final
degradation. Cell surface localization of PrPc is thought
to be essential for subsequent conversion into PrPSc
(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 PrPc 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 GPI- anchored
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 PrPc trafficking. Studies done
with PrP constructs lacking the GPI anchor revealed slower transport
through the secretory pathway compared with wild type PrPc
(wtPrPc) (14), although conversion of these constructs into
PrPSc 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
PrPc (16, 17). Addition of a transmembrane moiety to
the C terminus of PrPc 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 PrPc 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
wtPrPc. 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.
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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.
[35S]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 wtPrPc 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%
CO2 atmosphere. The medium was changed every 48 h.
wtPrPc 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 wtPrPc. 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 × g at 4 °C
in a Beckman TL-100 centrifuge. Soluble fractions (supernatants) were
precipitated with ethanol. Insoluble fractions (pellets) were
resuspended in 50 µl 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 × 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 µg/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 µCi/ml [35S]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 × g in PBS containing 20%
fetal calf serum and 100 µl of trypsin inhibitor. Cells were then
lysed in cold lysis buffer and cell debris removed by centrifugation
for 40 s at 18,000 × 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 × 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/µl 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 wtPrPc 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 µg 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 × 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 µl of
postnuclear lysate or immunoprecipitated proteins with 20 µl 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/µl PNGase F overnight at 37 °C before analysis on
SDS-PAGE.
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RESULTS |
Internalization of Mouse PrPc Is Impaired by
Progressive N-terminal Deletions--
To assess the relevance of the
N-terminal part of PrPc 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
wtPrPc, 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
PrPc. We next focused on the endocytic arm of the
PrPc life cycle and evaluated the kinetics of
PrPc 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 wtPrPc 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 wtPrPc and of PrP
(23-51) revealed
that after 45 min of chase, only wtPrPc 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 ~72% of wtPrPc had been endocytosed
whereas PrP
(23-51) only measured ~32% (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 ~7% 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.

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Fig. 1.
Biochemical characterization of PrP deletion
constructs. A, wtPrPc and PrP deletion
constructs used in this study were derived from murine
PrPc, 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 (23-51). B,
postnuclear lysates of N2a cells transiently transfected with
wtPrPc or with PrP deletion constructs were subjected to a
solubility assay or treatment with 20 µg/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-µl 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.
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Fig. 2.
Comparison of internalization of
wtPrPc and deletion mutants after 45 and 60 min of chase.
A, N2a cells transfected transiently with wtPrPc
or PrP deletion mutants were surface biotinylated on ice for 15 min and
then incubated for 0, 45, or 60 min at 37 °C. Cells where lysed
immediately ( ) or treated with trypsin (+) for 10 min on ice before
harvesting and were immunoprecipitated with antibody 3F4 (to detect
only transfected proteins). Lane 7 describes N2a cells
transfected with the unrelated plasmid pEGFP and precipitated with
antibody 3F4. Samples were subjected to SDS-PAGE, and signals were
detected by streptavidin. Molecular mass markers are depicted in kDa on
the left. B, quantification of the signals shows
the amount of internalized protein after 45 and 60 min calculated as a
percentage of protein without treatment with trypsin at the same time
point (each bar represents the mean values from two
independent experiments). The blot was digitized using an APB Image
Scanner, and specific bands were quantified with Master 1D analysis
software.
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Fig. 3.
Internalization of
PrP (23-90) and
PrP (48-93). A, transiently
transfected N2a cells expressing PrP deletion mutants were surface
biotinylated on ice and then incubated at 37 °C for 0, 3, 6, and
10 h, respectively. Cells were harvested directly or treated with
trypsin for 10 min on ice and lysed. PrPs were immunoprecipitated with
antibody 3F4. The blot shows samples treated (+) and untreated ( )
with trypsin. Numbers on the top indicate the
chase times (in hours) after the pulse. Molecular mass markers are
indicated in kDa on the left. B, mean values from
two independent experiments represent the amount of internalized
protein expressed as a percentage of total labeled protein without
trypsin digestion (at same time points) and plotted as a function of
different time points.
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Deletion of N-proximal Sequences Significantly Prolongs the
Half-life of PrPc--
Because the partial or complete
removal of the N-terminal part of PrP significantly prolonged the
presence of PrPc 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 wtPrPc, PrP
(23-90), and
PrP
(48-93). N2a cells expressing these constructs were
metabolically labeled with [35S]methionine/cysteine for
1 h and either harvested directly or chased for different
intervals of time in 35S-free 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 PrPc because differences in molecular mass allowed
discrimination between deletion mutants and endogenous
wtPrPc. 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: wtPrPc revealed
a half-life of ~2.6 h. On the other hand, the turnover of our PrP
mutants proved to be significantly prolonged. Our measurements indicated a half-life of ~4.8 h for PrP
(23-90) and ~4.2 for
PrP
(48-93), both values almost twice as long as for
wtPrPc. These data corroborate the idea that prolonged
accumulation of N-terminal deleted mutants on the plasma membrane
indeed affects the turnover of these proteins.

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Fig. 4.
Half-life times of wtPrPc and
deletion mutants PrP (23-90) and
PrP (48-93). A, confluent N2a cells
expressing wtPrP or deletion constructs were metabolically labeled with
[35S]Met/Cys for 1 h at 37 °C and were either
lysed after the pulse or incubated in culture medium without
35S at 37 °C for 2, 3, 6, 8, and 22 h,
respectively, before harvesting. Proteins were precipitated with
polyclonal antibody A7 and deglycosylated with PNGase F to facilitate
molecular mass comparison and quantification. Samples were subjected to
SDS-PAGE and autoradiography. Molecular mass markers are designated in
kDa on the left. On the right unglycosylated
PrP-specific bands are indicated. B, evaluation of
autoradiograms from three independent experiments. The amounts of
protein are expressed as percentage of total protein rescued directly
after the labeling period and plotted as a function of the chase time
points. The data points were fitted to an exponential curve using
nonlinear regression analysis.
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N-proximal Region of PrP Modulates Transport to the Plasma
Membrane--
Having found that the N-terminal part of
PrPc 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
wtPrPc 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 [35S]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, wtPrPc 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 wtPrPc 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 ~25% after 60 min. The
transport of PrP
(23-90) and PrP
(48-93) to the cell surface
showed important differences compared with wtPrP. 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 PrPc
even along the secretory pathway. For further analysis of the domains
involved in trafficking of PrPc, 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 pulse-chase 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 wtPrPc (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
PrPc to the plasma membrane.

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Fig. 5.
Kinetics of transport to the cell surface of
wtPrPc, PrP (23-90), and
PrP (48-93). A, wtPrP and
N-terminal truncated constructs were transiently transfected into N2a
cells, metabolically labeled with [35S]Met/Cys for 5 min
on ice, and then chased in 35S-free culture medium for 0, 45, 60, 75, 90 min, respectively. For each construct, 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 A7,
deglycosylated with PNGase F, and subjected to SDS-PAGE. The blot shows
PrP signals before ( ) and after (+) treatment with trypsin. Molecular
mass markers in kDa are shown on the left. B,
PhosphorImager evaluation of autoradiograms representing the amounts of
protein detected after digestion with trypsin. Each point represents a
mean value of three independent experiments. Quantities are calculated
as a percentage of protein precipitated immediately after the pulse
without trypsin treatment.
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Fig. 6.
Kinetics of
PrP (68-91) and
PrP (23-51) transport through the secretory
pathway monitored by pulse-chase experiments. A, PrP
deletion constructs were transfected into N2a cells and metabolically
labeled with [35S]Met/Cys for 5 min. 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.
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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 fluorescence-activated 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 wtPrPc. 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 ~43
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.

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Fig. 7.
Internalization and transport to the cell
surface of Xen(23-69). A, schematic representation of the
chimeric construct Xen(23-69). For construction of Xen(23-69),
residues 23-69 of the Xenopus PrP (vertical
bars) were introduced into the murine PrP (23-90), after
residue 22. Black and open bars represent the
signal peptide and GPI anchor, respectively; vertical bars
are residues 23-69 of Xenopus PrP. B,
biochemical characterization of Xen(23-69). Postnuclear lysates of N2a
cells expressing the chimeric constructs Xen(23-69) were analyzed for
solubility and PK resistance. One aliquot of the lysate was used for
PNGase F digestion. The resulting fractions were examined by Western
blot analysis with antibody 3F4. Lanes 1 and 2,
detergent-insoluble pellet (P) or supernatant
(S), respectively, upon ultracentrifugation; lane
3, fraction treated with 20 µg/ml PK; lane 4, aliquot
digested with PNGase. C, upper panel, transiently
transfected N2a cells expressing chimeric PrP Xen(23-69) were
incubated for 0, 45, or 60 min at 37 °C after surface biotinylation
on ice for 15 min. Cells were lysed immediately ( ) or subjected to
trypsin digestion (+) for 10 min on ice before lysis and were then
precipitated with antibody 3F4. Samples were subjected to SDS-PAGE, and
signals were detected by streptavidin. Molecular mass markers are shown
in kDa on the left. Lower panel, mean values from
two independent experiments represent the amount of internalized
protein expressed as a percentage of total labeled protein without
trypsin digestion (at the same chase time) and plotted as a function of
different time points. D, upper panel,
Xen(23-69) was transiently transfected into N2a cells, metabolically
labeled with [35S]Met/Cys for 5 min on ice, and then
chased in 35S-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 ( ) and after
(+) 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.
|
|
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 PrPc 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 wtPrPc,
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 wtPrPc with Endo-H
immediately after labeling resulted in a shift of the 33 and 28 kDa
bands to a band of ~25 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 PrPc do not significantly affect kinetics of
glycosylation and transport of PrPc to the Golgi.

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Fig. 8.
Endo-H digestion of wtPrPc and
N-terminal deletion constructs. Confluent N2a cells transfected
with wtPrPc, PrP (23-90), or PrP (48-93) were
metabolically labeled with [35S]Met/Cys for 5 min on ice
and then either harvested immediately or incubated in culture medium
without 35S at 37 °C for different chase periods before
lysis. Proteins were then immunoprecipitated with antibody 3F4 and
treated with (+) or without ( ) Endo-H overnight at 37 °C before
analyzing by SDS-PAGE. Bars on the right indicate
unglycosylated PrP-specific bands. Molecular mass markers are shown in
kDa on the left.
|
|
 |
DISCUSSION |
The structure of the PrPc 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 PrPc into
PrPSc, residues 23-89 of PrPc remain entirely
susceptible to PK digestion, whereas the rest of the protein becomes
partly protease-resistant (1). Regions within the N-terminal part of
PrPc have been suggested to influence interactions required
for PrPSc formation and to stabilize the conformation of
PrPc (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
PrPc 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 PrPc
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 wtPrPc 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 wtPrPc 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 PrPc (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 (~30%) 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
PrPc 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
PrPc 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
(23-90). 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 wtPrPc 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 PrPc. 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
PrPc 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 plasma membrane domains
(31, 32). Clustering of PrPc 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 PrPc 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 ~2.6 h for
wtPrPc, confirming published data on PrPc
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 wtPrPc. To our knowledge, these findings
are new because we show that inefficient internalization of
PrPc 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 PrPc (35), but prolonged
half-life was not reported.
The N Terminus of PrPc and the Secretory
Pathway--
Given the importance of the N-terminal part of
PrPc for internalization and rate of degradation, we next
focused on the transport of PrPc 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 ~50 min. Our experiments also
evidenced that wtPrPc 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 phospholipid-binding 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
PrPSc 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 PrPc, where a truncated form of
PrPc lacking amino acids 32-94 influenced the quantity and
the conformation of generated PrPSc (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 PrPc 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 wtPrPc.
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 PrPc. 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
PrPc 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.
 |
ACKNOWLEDGEMENTS |
We are grateful to Ulrich Koszinowski for
continuous support and to Christian Spielhaupter and Jenna Lemerise for
editorial supervision.
 |
FOOTNOTES |
*
This work was supported in part by Deutsche
Forschungsgemeinschaft Grant SCHA 594/3-3, Sonder Forschungsbereich
596, and by European Union BIOMED Program Contract
BMH4-CT98-6040).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Virology, Max
von Pettenkofer Institute, Gene Center Munich, Feodor-Lynen-Str. 25, Munich D-81377, Germany. Tel.: 49-89-2180-6855; Fax:
49-89-2180-6898; E-mail: schaetzl@lmb.uni-muenchen.de.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M206313200
 |
ABBREVIATIONS |
The abbreviations used are:
PrPc, cellular prion protein;
biotin sulfo-NHS, biotin
N-hydroxysuccinimide ester;
EGFP, enhanced green
fluorescent protein;
Endo-H, endoglycosidase H;
GPI, glycosylphosphatidylinositol;
PBS, phosphate-buffered saline;
PK, proteinase K;
PNGase F, peptide N-glycosidase F;
wtPrPc, wild type cellular prion protein.
 |
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