From the
The cellular prion protein (PrP
A number of important transmembrane receptors undergo
endocytosis in clathrin-coated pits. Recruitment of receptors into
coated pits depends on specific amino acid motifs in receptor
cytoplasmic tails that bind clathrin-associated adaptor molecules on
the inner surface of the plasma membrane (reviewed by Trowbridge et
al.(1993)). These short amino acid motifs often include a tyrosine
residue and are thought to adopt a
A
diverse group of proteins is attached to the plasma membrane by a
phosphatidylinositol-containing glycolipid (GPI)(
We have been investigating the cellular trafficking
of PrP
We
found previously that chPrP, the chicken homologue of mammalian
PrP
Cell culture reagents were
from the Tissue Culture Support Center at Washington University.
Gold-conjugated secondary antibodies were from Jackson ImmunoResearch.
Osmium tetroxide, uranyl acetate, and Polybed 812 were from
Polysciences, Inc. (Niles, IL).
Phosphatidylinositol-specific
phospholipase C (PI-PLC) was prepared by a modification of the
procedure of Low(1992). Supernatants (500 ml) from cultures of Bacillus subtilis carrying a plasmid encoding PI-PLC from Bacillus thuringiensis were concentrated 20-fold in an Amicon
device and dialyzed against 50 mM Tris acetate (pH 7.4). The
dialysate was then subjected to gel filtration in the same buffer using
two columns (1.5 cm
Internalization was also quantitated by surface
biotinylation. Cells were incubated on ice for 10 min in 250 µg/ml
sulfo-biotin-X-NHS (Calbiochem) in 20 mM HEPES, 150 mM NaCl (pH 7.2). The reaction was quenched by addition of 20 mM glycine in MEM, and the cells rinsed three times in
phosphate-buffered saline. After warming to 37 °C in Opti-MEM for
various times, cells were treated with PI-PLC, and PrP
immunoprecipitated from PI-PLC incubation media and cell lysates, as
described above. Immunoprecipitates were separated by SDS-PAGE and
blotted onto polyvinylidene difluoride membrane. Blots were then
developed with horseradish peroxidase-streptavidin and visualized using
enhanced chemiluminescence (Amersham Corp.). Films were digitized using
an HP ScanJet IIp scanner, and images analyzed using SigmaScan/Image
(Jandel Scientific).
To test the effects of hypertonic treatment,
cells were preincubated for 30 min at 37 °C in Opti-MEM containing
0.45 M sucrose, and 0.45 M sucrose was also included
during the biotinylation and warm-up.
We have shown previously that chPrP constitutively cycles
between the cell surface and an endosomal compartment in N2a cells, and
that endocytosis of this glycolipid-anchored protein is mediated by
clathrin-coated pits (Shyng et al., 1993, 1994). During each
cycle through the cell, a portion of the molecules are proteolytically
cleaved within a highly conserved segment of 24 amino acids near the
center of the polypeptide chain (Harris et al., 1993b). Intact
molecules, as well as membrane-anchored C-terminal fragments, are then
returned to the cell surface. We noticed that, at steady state, the
C-terminal fragment is present almost exclusively on the cell surface,
in contrast to the full-length protein, about half of which is
intracellular. This observation suggested that the C-terminal fragment
might be internalized less efficiently than the full-length protein,
and that sequences in the N-terminal half of the protein might be
important for endocytic targeting.
To test this idea, and to further
define the regions of chPrP that are essential for efficient
endocytosis, we constructed a series of mutant proteins containing
N-terminal deletions, as well as two chimeric proteins consisting of
N-terminal segments of mouse PrP (moPrP) fused to C-terminal portions
of chPrP (Fig. 1). These proteins were expressed in stably
transfected clones of N2a mouse neuroblastoma cells. Metabolic labeling
of each transfected line revealed a protein of the appropriate
molecular weight that was specifically immunoprecipitated by an
anti-PrP antibody (Fig. 2). Each of the proteins was attached to
the plasma membrane by a GPI anchor, as demonstrated by the fact that
>80% of the surface-labeled protein could be released from cells
maintained at 0 °C by incubation with bacterial PI-PLC (see 0-min
time points in Figs. 3, 4, 6, and 7). Endocytosis was quantitated by
iodinating or biotinylating cells at 0 °C, warming them to 37
°C to initiate internalization, and then using PI-PLC to separate
molecules on the cell surface from those that were intracellular.
We chose a 30-min
time point to assess internalization because we had previously shown
that the distribution of wild-type chPrP after surface iodination
approaches a steady state with a t of approximately 10 min
(Shyng et al., 1993). The existence of a steady state results
from recycling of internalized molecules back to the cell surface. To
see if the mutant forms of chPrP behaved similarly, we analyzed the
time course of their internalization (Fig. 4). We found that the
It was not possible to analyze the effect on internalization of a
deletion (
We found that
moPrP, like chPrP, is internalized after surface iodination, and that
the distribution of the protein reaches a steady state, with
approximately half on the cell surface and half intracellular (data not
shown); the rate for approach to this steady state is similar for the
mouse and chicken proteins (t
We have also
analyzed internalization of both moPrP and chPrP using an assay that
involves biotinylation of intact cells with a membrane impermeant
reagent (Fig. 7). Inefficient internalization of deleted forms of
chPrP is observed in the biotinylation assay (data not shown), as it is
in the iodination assay, although the absolute amount of chPrP
endocytosed is approximately 50% greater after biotinylation than after
iodination, an effect resulting from slower recycling of biotinylated
PrP to the cell surface (compare wild-type chPrP in Fig. 3and Fig. 7). Fig. 7shows that internalization of moPrP, like
chPrP (Shyng et al., 1994), is inhibited by incubation of
cells in hypertonic sucrose, a treatment that disrupts clathrin
lattices, suggesting that endocytosis of the murine protein is also
mediated by clathrin-coated pits. We have also found that, following
endocytic uptake, moPrP is proteolytically cleaved within the conserved
central region (Lehmann et al., 1994), a result similar to
that obtained for chPrP.
We have reported previously that chPrP, in contrast to
several other GPI-anchored proteins, is endocytosed via clathrin-coated
pits in neuroblastoma cells, as well as in glia and neurons from
chicken brain (Shyng et al., 1994). In the present study, we
have investigated the structural features of the chPrP molecule that
target it for coated pit-mediated endocytosis. Our results indicate
that sequences in the N-terminal half of the chPrP polypeptide chain
are essential for this process. Deletions within the first 111 amino
acids following the signal sequence reduce internalization of chPrP as
measured by surface iodination and biotinylation. They also decrease
the concentration of the protein in coated pits, as determined by
quantitative EM immunogold labeling. The correlation we observed
between the internalization of the mutant proteins and their
concentration in coated pits further strengthens the conclusion that
coated pits represent the primary pathway for endocytosis of chPrP.
Since the chPrP polypeptide chain is entirely extracellular, our
results sharply distinguish chPrP from transmembrane receptors such as
those for transferrin and low density lipoprotein, whose cytoplasmic
domains contain coated pit targeting information (Trowbridge et
al., 1993). In this way, chPrP, along with other PrP
Endocytosis of chPrP
does not depend on a single, short segment of amino acids, as is the
case for internalization of transmembrane receptors, which display
tyrosine- or dileucine-based motifs encompassing 4-5 residues
(Trowbridge et al., 1993). Progressive N-terminal deletions of
chPrP, ranging from 17 to 67 amino acids, cause graded reductions of
internalization and concentration in coated pits, with no indication
that any single region plays a dominant role. In addition, two
deletions within the hexapeptide repeat region, and one within the
conserved central segment are all about equally effective in reducing
endocytosis.
One possible explanation for the apparent absence of a
dominant targeting signal is that each of the deletions alters the
overall folding pattern of the chPrP polypeptide chain, and thereby
disrupts structural features that are important for endocytosis. Gross
alterations in protein folding seem unlikely, since each of the mutants
was efficiently transported to the cell surface, in contrast to
aberrantly folded proteins, which often accumulate in the endoplasmic
reticulum and are degraded after synthesis (Bonifacino and
Lippincott-Schwartz, 1991). An alternative explanation is that multiple
regions within the N-terminal half of the molecule are involved in
interactions with the endocytic machinery. This might be the case, for
example, if chPrP bound to several different components of coated pits,
or if one of these components bound to several different sites on the
chPrP molecule (see below).
Whatever features of the polypeptide
chain are important for endocytosis, they are likely to be specific for
PrP. DAF, an unrelated GPI-anchored protein, is not internalized when
expressed in N2a cells and is not concentrated in coated pits. Several
other glycolipid-anchored proteins in other cell types are also not
clustered in coated pits (Bretscher et al., 1980; Rothberg et al., 1990; Keller et al., 1992).
The fact that
moPrP, as well as two moPrP/chPrP chimeras, are internalized as
efficiently as chPrP in N2a cells suggests that secondary and/or
tertiary structural features are more important than primary sequence
in endocytic targeting. Although the avian and mammalian proteins are
only 33% identical in amino acid sequence, recent spectroscopic and
computer modeling studies predict that they are likely to adopt the
same conformation, including four
It is
interesting that the tyrosine-containing motifs essential for coated
pit-mediated endocytosis of transmembrane receptors have also been
found to adopt a
Although our results highlight an
important role for the polypeptide chain of PrP in endocytic targeting,
they do not rule out the possibility that the GPI anchor might also be
involved. All of our mutant PrP constructs are attached to the cell
surface by a GPI anchor, indicating that the anchor itself is not
sufficient for normal internalization. It is conceivable, however, that
the anchor might assist in endocytic uptake, for example by increasing
the lateral mobility of PrP and so facilitating its delivery to
clathrin-coated pits. Investigation of the role of the GPI anchor will
require analysis of transmembrane versions of PrP.
To explain the
localization of chPrP in coated pits, we have previously proposed that
its polypeptide chain binds, either directly or indirectly, to the
extracellular domain of a transmembrane protein that contains a
conventional coated pit targeting signal in its cytoplasmic domain
(Shyng et al., 1994). The results reported here strongly
support this model, since deletions in the polypeptide chain of chPrP
would be expected to decrease its affinity for the transmembrane
protein, and thereby reduce the efficiency of endocytosis. Since the
mutations we have analyzed have all been in the N-terminal half of the
protein, we would predict this region to be involved in contacting the
transmembrane protein, although a role for more C-terminal regions
cannot be ruled out. We are currently employing a variety of
biochemical techniques to directly demonstrate the existence of a PrP
binding protein in coated pits.
We observed that although wild-type
and mutant chPrPs were internalized to different extents, the rate at
which a steady state distribution was reached was approximately 10 min
for all forms of the protein (Fig. 4). This effect might be
understood in terms of our model if it is assumed that the rate of
internalization of chPrP depends only on the rate at which the
transmembrane binding protein is internalized, while the steady state
distribution depends on the affinity of chPrP for the binding protein.
If mutated chPrP molecules had reduced affinity, then a smaller
proportion would be bound to the transmembrane protein, although those
that were bound would be internalized at the normal rate.
The model
outlined here is related to one that has been proposed to explain the
function of the glycolipid-anchored receptor for urokinase-type
plasminogen activator (uPA) (Nykjaer et al., 1992; Blasi et al., 1994). This receptor facilitates coated pit-mediated
internalization of uPA bound to its inhibitor (PAI-1) by
``presenting'' the uPA-PAI-1 complex to the low density
lipoprotein receptor-related protein, a large transmembrane protein
with tyrosine-based localization signals in its cytoplasmic domain.
Whether the uPA receptor itself binds to lipoprotein receptor-related
protein, and whether it is internalized along with the ligand, is
unclear. If so, then this mechanism would be analogous to the one we
have proposed here for endocytosis of PrP
The results reported here have
implications for understanding the physiological as well as the
pathological properties of PrP. The normal function of PrP
Neuroblastoma cells transfected with various proteins were incubated
with primary antibodies and gold-conjugated secondary antibodies (see
``Materials and Methods'') after fixation in 4%
paraformaldehyde and were processed for electron microscopy.
We thank Marilyn Levy and Lori LaRose for technical
assistance with immunogold labeling, Doug Lublin for informative
discussions, as well as for DAF antibodies and cDNAs, and Alex
Gorodinsky and Sylvain Lehmann for comments on the manuscript and help
with computer graphics.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) is a
glycolipid-anchored protein that is involved in the pathogenesis of
fatal spongiform encephalopathies. We have shown previously that, in
contrast to several other glycolipid-anchored proteins, chPrP, the
chicken homologue of mammalian PrP
, is endocytosed via
clathrin-coated pits in cultured neuroblastoma cells, as well as in
embryonic neurons and glia (Shyng, S.-L., Heuser, J. E., and Harris, D.
A.(1994) J. Cell Biol. 125, 1239-1250). In this study,
we have determined that the N-terminal half of the chPrP polypeptide
chain is essential for its endocytosis. Deletions within this region
reduce the amount of chPrP internalized, as measured by surface
iodination or biotinylation, and decrease its concentration in
clathrin-coated pits, as determined by quantitative electron
microscopic immunogold labeling. Mouse PrP, as well as two mouse
PrP/chPrP chimeras, are internalized as efficiently as chPrP,
suggesting that conserved features of secondary and tertiary structure
are involved in interaction with the endocytic machinery. Our results
indicate that the ectodomain of a protein can contain endocytic
targeting information, and they strongly support a model in which the
polypeptide chain of PrP
binds to the extracellular domain
of a transmembrane protein that contains a coated pit localization
signal in its cytoplasmic tail.
-turn conformation.
)
anchor (reviewed by Englund(1993)). This group includes
lymphocyte and trypanosome surface antigens, adhesion molecules,
exofacial enzymes, and receptors. Endocytic targeting of these proteins
has been a subject of considerable debate, since they lack cytoplasmic
domains that could interact directly with intracellular adaptors and
clathrin. Some GPI-anchored proteins are not endocytosed at all, and
some are internalized via pathways that do not involve clathrin-coated
pits (Bretscher et al., 1980; Lemansky et al., 1990;
Keller et al., 1992). The vitamin folic acid, for example, is
internalized by binding to a glycolipid-anchored receptor that is
concentrated in non-clathrin-coated invaginations of the plasmalemma
called caveolae (Rothberg et al., 1990; Anderson et
al., 1992).
, a glycolipid-anchored surface protein that is
expressed by neurons, glia, and several peripheral cell types. Although
the physiological function of PrP
is unclear, the protein
is known to be intimately involved in the pathogenesis of an unusual
group of transmissible neurodegenerative diseases called spongiform
encephalopathies or prion diseases (reviewed by Prusiner and DeArmond
(1994)). This group includes Creutzfeldt-Jakob disease, kuru,
Gerstmann-Sträussler syndrome, and fatal familial insomnia in
humans, and scrapie in animals. The infectious agent responsible for
these fatal disorders has been called a prion, and it appears to
consist principally of PrP
, a posttranslationally modified
isoform of PrP
. Conversion of PrP
to PrP
during prion replication appears to take place, at least in part,
along an endocytic pathway in scrapie-infected neuroblastoma cells
(Caughey et al., 1991; Borchelt et al., 1992).
, is endocytosed via clathrin-coated pits in
neuroblastoma cells, and in embryonic neurons and glia (Shyng et
al., 1993, 1994). This surprising result raises the question of
what molecular mechanism accounts for the localization of PrP
in coated pits. To define the structural features of the
PrP
molecule that target it to coated pits, we have
expressed deleted and chimeric forms of the protein in neuroblastoma
cells. Our results indicate that the N-terminal half of the PrP
polypeptide chain is essential for efficient endocytosis assayed
biochemically, and for localization to coated pits analyzed by EM
immunogold labeling. These data are consistent with a model whereby the
polypeptide chain of PrP
binds to the extracellular domain
of a transmembrane protein that itself contains a coated pit
localization signal (Harris et al., 1993a).
Antibodies and Reagents
A rabbit antiserum
raised against a bacterial fusion protein encompassing amino acids
35-96 of chPrP (Harris et al., 1993b) was used to
recognize wild-type chPrP, 25-41,
25-65,
42-65,
66-91,
117-135, and
moPrP(1-39/chPrP(42-267). A rabbit antiserum raised against
a bacterial fusion protein encompassing amino acids 144-220 of
chPrP (Harris et al., 1993b) was used to recognize
25-91 and moPrP(1-88)/chPrP(92-267). A new
rabbit antiserum raised against a synthetic peptide encompassing amino
acids 45-66 of moPrP was used to recognize wild-type moPrP. A
rabbit anti-human decay-accelerating factor (DAF) serum (used for
immunoprecipitations), and a mouse anti-human DAF monoclonal antibody
(1H4) (used for immunogold labeling) were kindly provided by Dr. Doug
Lublin (Coyne et al., 1992).
120 cm) of Sephadex G-75 connected in
series, and fractions containing PI-PLC activity were pooled and
concentrated. The resulting preparations had a specific activity of 150
units/mg and had no detectable protease activity. Cells were treated at
4 °C with PI-PLC at a concentration of 1 unit/ml in Opti-MEM (Life
Technologies).
DNA Expression Constructs
All mutant and chimeric
constructs were created by recombinant PCR (Higuchi, 1989) using
Vent® polymerase (New England Biolabs) and were sequenced in their
entirety. The templates for primary PCR encoded chPrP (Harris et
al., 1991), moPrP (Prn-p allele; Westaway et
al., 1987), and human DAF (Lublin and Atkinson, 1989). Primers for
secondary PCR contained a HindIII site at the 5` end and a BamHI site at the 3` end, each immediately adjacent to the
coding region. Constructs were cloned into the expression vector
pBC12/CMV (Cullen et al., 1986) after cleavage with HindIII and BamHI.
Transfected Cell Lines
N2a mouse neuroblastoma
cells (ATCC CCL131) were grown in minimal essential medium (MEM)
containing 10% fetal calf serum, nonessential amino acids, and
penicillin/streptomycin in an atmosphere of 5% CO, 95% air.
Cells were transfected with a 10:1 mixture of the pBC12/CMV expression
plasmid and pRSVneo (Ulrich and Ley, 1990), using Lipofectin (Life
Technologies, Inc.) according to the manufacturer's directions.
Antibiotic-resistant clones were selected in 700 µg/ml Geneticin
(G418), expanded, and then maintained in 300 µg/ml Geneticin. The
line expressing wild-type chPrP is the clone designated A26 that we
have described previously (Harris et al., 1993b). Even though
N2a cells express endogenous moPrP, stably transfected lines were
prepared that expressed increased levels of the wild-type protein.
Metabolic Labeling
Confluent cultures of N2a cells
were labeled for 5 h with [S]methionine (ICN
Tran
S-label, 250 µCi/ml, 1,000 Ci/mmol) in serum-free
MEM lacking methionine. Cell lysates were treated with 0.01 units/ml N-glycosidase F (Boehringer Mannheim) at 37 °C overnight
to remove N-linked oligosaccharides, and PrP
immunoprecipitated as described previously (Harris et al.,
1993b).
PrP Internalization Assays
Internalization of
cell-surface PrP was measured by surface iodination as described
previously (Shyng et al., 1993). Briefly, cells were incubated
on ice with phosphate-buffered saline containing glucose,
lactoperoxidase, glucose oxidase, and NaI for 20 min, and
the reaction quenched with 1 mM tyrosine and 10 mM sodium metabisulfite. After warming to 37 °C for different
times to allow internalization, cells were treated with PI-PLC for 2 h
at 4 °C prior to lysis. PrP in the PI-PLC incubation media
(surface) and cell lysates (internal) was immunoprecipitated (Harris et al., 1993b), and quantitated by imaging of SDS-PAGE gels
using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Internalization was expressed as the percentage of the total amount of
PrP from each dish that was present in the cell lysate and was
corrected for PI-PLC digestion efficiency (>80% for plates held at 0
°C).
EM Immunogold Labeling
Labeling and morphometric
analysis were carried out exactly as described by Shyng et al. (1994).
Figure 1:
PrP constructs
used in this study. The wild-type and six deletion () mutants are
derived from chPrP. In the two moPrP/chPrP chimeras, the narrow
segments of the schematic represent moPrP and the wide segments chPrP. Numbers refer to amino acid residues. Note that chPrP contains
eight hexapeptide repeats, and moPrP five octapeptide repeats rich in
proline and glycine. In the mature proteins, the GPI anchor is attached
at the position indicated by the arrowhead in the
schematic.
Figure 2:
Stably transfected lines of N2a cells
express mutant PrPs. Cells were metabolically labeled with
[S]methionine for 5 h, after which PrP was
immunoprecipitated from cell lysates, using chPrP-specific antibodies
(see ``Materials and Methods''), and subjected to SDS-PAGE
and fluorography. The specific PrP bands are indicated by arrows and brackets next to each lane; immunoprecipitation of
these bands is blocked by preincubation of antibodies with immunogen
(not shown). Each of the samples was enzymatically deglycosylated prior
to immunoprecipitation to facilitate molecular weight comparisons.
chPrP sometimes migrates as a doublet under these conditions (lanes
with brackets), due either to incomplete deglycosylation, or
to the presence of modifications other than N-linked
oligosaccharides (Shyng et al., 1993). Molecular size markers
are given in kilodaltons. Wild-type chPrP has a molecular mass of 34
kDa, and the deletion mutants have correspondingly lower masses:
25-41, 32.5 kDa;
25-65, 29 kDa;
25-91,
27.5 kDa;
42-65, 31.5 kDa;
66-91, 31.5 kDa;
117-135, 32.5 kDa. The two moPrP/chPrP chimeras migrate at
32.5 kDa.
N-terminal Deletions of chPrP Reduce Internalization
The
C-terminal fragment of chPrP generated by posttranslational cleavage
begins near Lys (Harris et al., 1993b),
suggesting that the 92 amino acids between this site and the end of the
signal peptide may be important in mediating internalization. To
analyze this region in more detail, we constructed three deletion
mutants that were missing 17, 41, or 67 amino acids following the
signal peptide. These deletions include 0, 4, or all 8 of the
proline/glycine-rich hexapeptide repeats. As shown previously (Shyng et al., 1993), approximately 40-50% of wild-type chPrP
is internalized at 30 min following surface iodination (Fig. 3).
The three deletion mutants were each internalized less efficiently than
the wild-type protein, with longer deletions producing a greater
effect: the amount of iodinated protein endocytosed was 25.9%, 12.4%,
and 6.3%, respectively, for
25-41,
25-65, and
25-91. Since
5% of the C-terminal fragment generated
posttranslationally is internalized following surface iodination (data
not shown), removal of additional amino acids between positions 92 and
116 has little further effect on endocytosis.
Figure 3:
N-terminal deletions progressively reduce
the amount of chPrP internalized by N2a cells; DAF is not internalized. Panel A, N2a cells expressing each protein were
surface-iodinated at 0 °C, and then warmed to 37 °C for 0 min
or 30 min. Cells were then treated with PI-PLC for 2 h at 0 °C
prior to lysis. PrP or DAF in the PI-PLC incubation media (lanes marked E, for external) and cell lysates (lanes marked I, for internal) was immunoprecipitated
and subjected to SDS-PAGE. The portions of the autoradiograms
containing the chPrP and DAF bands are shown. In this and subsequent
figures, PI-PLC digestion efficiency (the amount of protein released by
the enzyme from cells maintained at 0 °C) was >80%. The slight
decrease from 0 to 30 min in the total amount of DAF was not consistent
and probably resulted from a small difference in the efficiency of
iodination between the two dishes. Panel B, the amount of
protein internalized at 30 min was quantitated by PhosphorImager
analysis of the gels. Each bar shows the average and S.E. of values
from 4 dishes.
As a negative control,
we also analyzed N2a cells that had been transfected to express human
DAF, a GPI-anchored protein that is unrelated in sequence to chPrP. We
found that <1% of DAF was internalized at 30 min (Fig. 3),
arguing that the glycolipid anchor itself is not sufficient to mediate
efficient endocytic uptake, and that distinctive features of the chPrP
polypeptide chain are critical for this process.
25-41 and
25-91 proteins attained a steady state
distribution, which was maintained for the duration of each experiment
(>90 min). By fitting the kinetic data to an exponential curve, we
calculated that the steady state value for the amount of internalized
protein was 25% for
25-41 and 7% for
25-91,
compared to 50% for the wild-type protein (Shyng et al.,
1993). Surprisingly, although the steady state amounts of internalized
PrP were lower for the two mutants than for the wild-type protein, the t for approach to the steady state was similar for all forms
of the protein (6.6 and 11.5 min for the two mutants, and 10 min for
the wild-type protein). These results indicate that the deletions alter
the final extent of internalization, without significantly affecting
the rate at which a stable distribution is achieved.
Figure 4:
Internalization of the 25-41
and
25-91 mutants approaches a steady state. Upper
panels, N2a cells expressing each protein were surface-iodinated
at 0 °C and then warmed to 37 °C for the indicated periods of
time. Cells were then treated with PI-PLC for 2 h at 0 °C prior to
lysis. chPrP in the PI-PLC incubation media (Surface) and cell
lysates (Internal) was immunoprecipitated and subjected to
SDS-PAGE and autoradiography. The autoradiogram for the
25-91 mutant is overexposed to better visualize the small
amount of internalized protein. Lower panels, the amounts of
surface and internal chPrP at each time point were quantitated by
PhosphorImager analysis of the gels. Each point represents the value
from a single dish. Curves were fit by least squares analysis to the
following equations:
25-41 internal, 0.25(1 - e
);
25-41 surface, 0.75 +
0.25e
;
25-91 internal, 0.07(1
- e
);
25-91 surface, 0.93
+ 0.07e
. These equations assume steady
state values of 0.25 and 0.07 for the fraction of internalized
25-41 and
25-91, respectively. The calculated t values for approach to the steady state are 6.6 and 11.5 min
for the two mutants. These values do not differ markedly from the t of 10 min calculated for wild-type chPrP (Shyng et al.,
1993).
N-terminal Deletions Reduce the Concentration of chPrP in
Coated Pits
We carried out quantitative EM immunogold labeling
to analyze the localization of the deleted proteins at the
ultrastructural level. As reported previously (Shyng et al.,
1994), wild-type chPrP was approximately 4 times more concentrated in
coated pits than over other areas of the plasma membrane (). In contrast, 25-41 was only 1.7 times more
concentrated in coated pits, and
25-91, as well as DAF, were
barely concentrated in these structures at all. Fig. 5shows
representative micrographs illustrating the localization of wild-type
and
25-91 chPrP, as well as DAF. These results demonstrate a
correlation between the efficiency of internalization measured
biochemically and the degree of concentration in clathrin-coated pits
determined morphologically.
Figure 5:
25-91 chPrP and DAF are less
concentrated in clathrin-coated pits than wild-type chPrP. N2a cells
expressing wild-type chPrP (A),
25-91 chPrP (B), or DAF (C) were fixed with
paraformaldehyde and incubated with either rabbit anti-chPrP or mouse
anti-DAF antibodies. Cells were then processed for EM immunogold
detection using goat anti-rabbit antibodies conjugated to 12 nm gold
particles (A and B) or goat anti-mouse antibodies
coupled to 6 nm gold particles (C). For wild-type chPrP, gold
particles (arrowheads) are clustered around clathrin-coated
pits (arrows), as well as over undifferentiated areas of
plasma membrane. In contrast, for
25-91 chPrP and DAF, gold
particles are almost never found near coated pits. Scale bar =
0.1 µm. Morphometric quantitation of these data is presented in
Table I.
Hexapeptide Repeats and the Conserved Region Are
Essential for Internalization
Our results indicated that
sequences within the N-terminal half of the chPrP polypeptide chain are
essential for normal endocytic targeting. To identify which motifs
within this region are important, we constructed three additional
deletions. We first focused on the eight hexapeptide repeats (consensus
HNPGYP) found between residues 42 and 89. Mutants lacking either the
first four (42-65) or last four (
66-91) of these
repeats were internalized with less than half the efficiency of
wild-type chPrP (Fig. 6), indicating that both halves of the
repeat region are essential, and suggesting that normal endocytosis is
likely to require all eight repeats. This conclusion is consistent with
the fact that internalization of the
25-91 mutant, which is
missing all eight repeats, is more severely impaired than that of the
25-65 mutant, which is missing the first four repeats (Fig. 3).
Figure 6:
Deletions of the hexapeptide repeats or
the conserved region reduce internalization of chPrP. PanelA, N2a cells expressing each protein were
surface-iodinated at 0 °C and then warmed to 37 °C for 0 or 30
min. Cells were then treated with PI-PLC for 2 h at 0 °C prior to
lysis. chPrP in the PI-PLC incubation media (lanes marked E, for external) and cell lysates (lanes marked I, for internal) was immunoprecipitated and subjected
to SDS-PAGE. The portions of the autoradiograms containing the chPrP
bands are shown. Panel B, the amount of protein internalized
at 30 min was quantitated by PhosphorImager analysis of the gels. Each
bar shows the average and S.E. of values from 4
dishes.
Farther along the chPrP polypeptide chain is a
segment of 24 largely hydrophobic amino acids (residues 112-135),
which is identical in chPrP and mouse PrP (Prn-p allele)
and is highly conserved in all known PrP species. We found that this
conserved region was also essential for normal internalization, since a
mutant protein (
117-135) in which most of the region was
deleted exhibited significantly impaired endocytosis (Fig. 6).
161-180) in the C-terminal half of the chPrP
molecule because this protein was not expressed on the cell surface,
but was instead retained intracellularly, as judged by lack of surface
immunofluorescence staining and insensitivity to release by PI-PLC
(data not shown). It is possible that C-terminal deletions interfere
with N-glycosylation or disulfide bond formation, which
involve residues in this half of the molecule.
MoPrP and moPrP/chPrP Chimeric Proteins Are Efficiently
Internalized
Our data indicated that endocytosis of chPrP
depends on distinctive features of its polypeptide chain which are not
present in unrelated glycolipid-anchored proteins such as DAF. To
further investigate the sequence specificity of these targeting
signals, we analyzed internalization of a homologous mammalian PrP from
mouse. Although moPrP and chPrP have an overall amino acid sequence
identity of only 33%, they share common structural motifs, including
proline- and glycine-rich peptide repeats, as well as the conserved
central segment. They are therefore likely to adopt similar secondary
and tertiary structures (Huang et al., 1994).
10 min).
Figure 7:
MoPrP
and moPrP/chPrP chimeric proteins are efficiently internalized. PanelA, N2a cells expressing each protein were
surface-biotinylated at 0 °C and then warmed to 37 °C for 0 or
30 min. Cells were then treated with PI-PLC for 2 h at 0 °C prior
to lysis. chPrP in the PI-PLC incubation media (lanes marked E, for external) and cell lysates (lanes marked I, for internal) was immunoprecipitated. Immunoprecipitates
were separated by SDS-PAGE, blotted, and visualized with horseradish
peroxidase-streptavidin and enhanced chemiluminescence. The portions of
the films containing the PrP bands are shown. In one set of
experiments, cells expressing wild-type moPrP were preincubated,
biotinylated, and warmed in the presence of 0.45 M sucrose, to
test the involvement of clathrin-coated pits in endocytosis. Panel
B, the amount of protein internalized at 30 min was quantitated by
densitometric scanning of films. Each bar shows the average and S.E. of
values from 4 dishes.
Two chimeric proteins, consisting of
N-terminal segments of moPrP fused to C-terminal segments of chPrP,
were as efficiently internalized as the wild-type chicken and mouse
proteins (Fig. 7). The moPrP(1-39)/chPrP(42-267)
construct includes 17 amino acids of moPrP between the signal peptide
and the octapeptide repeat region, a segment that is 59% identical to
the corresponding region of chPrP. The
moPrP(1-88)/chPrP(92-267) construct includes 66 amino acids
of moPrP beyond the signal peptide, a segment that contains all five of
the octapeptide repeats, and that is only 27% identical to the
corresponding segment of chPrP. These results indicate that, although
the N-terminal primary amino acid sequences of moPrP and chPrP are
quite divergent, the features responsible for efficient internalization
are conserved.
s,
define a novel mechanism for endocytic uptake.
-helices in the C-terminal half
that may be arranged in the form of an X-bundle (Gasset et
al., 1992; Pan et al., 1993; Huang et al.,
1994). In the N-terminal half of the protein, the octapeptide repeat
unit in the murine protein (consensus PHGG(G/S)WGQ) is chemically
similar to the hexapeptide unit in the chicken protein (consensus
HNPGYP), since both are rich in proline and glycine and both contain an
aromatic amino acid. Although conformational studies of this part of
the PrP molecule have not been carried out, standard secondary
structural algorithms predict that the peptide repeat region will form
a series of
-turns in both proteins (Bazan et al., 1987),
and these could play a role in endocytic targeting.
-turn configuration (Collawn et al.,
1990; Bansal and Gierasch, 1991; Eberle et al., 1991).
Although the PrP repeats are extracellular, and the receptor
tyrosine-containing motifs are cytoplasmic, the
-turn might
represent a common structural element involved in binding to other
membrane-associated molecules.
. Ligand-induced
internalization of the glycolipid-anchored receptor for ciliary
neurotrophic factor might involve a similar process (Curtis and
DiStefano, 1994). This receptor forms a signal-transducing complex with
two transmembrane proteins LIF
and gp130, the second of which has
been shown to contain a di-leucine motif in its cytoplasmic domain that
can mediate endocytic uptake (Dittrich et al., 1994).
Consideration of these two examples prompts the speculation that
binding of an extracellular ligand could play a role in the
internalization of PrP
.
is unknown, although recent electrophysiological studies of mice
in which the PrP gene has been deleted suggest a role in synaptic
transmission within the central nervous system (Collinge et
al., 1994). The identification of molecules in coated pits with
which PrP
interacts is likely to provide additional clues
to the cellular function of this isoform. These putative binding
molecules may also serve as cell surface receptors for PrP
or play some other role in the conversion of PrP
to
PrP
, a process that has been found to take place in part
along an endocytic pathway (Caughey et al., 1991; Borchelt et al., 1992). It is noteworthy that expansion of the
octapeptide repeat region of human PrP is associated with familial
forms of Creutzfeldt-Jakob disease (Poulter et al., 1992).
Since we have found that deletions within this region interfere with
endocytosis, it is possible that the expanded human PrPs are also
internalized abnormally and that this feature plays a role in the
pathogenic process. Finally, our observation that the PrP polypeptide
chain is essential for efficient endocytosis may allow the design of
peptide-based ligands that competitively block internalization of PrP
and thereby inhibit prion replication.
Table: Quantitative analysis of immunogold labeling
experiments in neuroblastoma cells transfected with various proteins
, cellular isoform of the prion protein;
PrP
, scrapie isoform of the prion protein; chPrP, chicken
prion protein; moPrP, mouse prion protein; PI-PLC,
phosphatidylinositol-specific phospholipase C; DAF, decay-accelerating
factor; PCR, polymerase chain reaction; MEM, minimal essential medium;
PAGE, polyacrylamide gel electrophoresis; uPA, urokinase-type
plasminogen activator.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.