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INTRODUCTION |
Prion diseases such as scrapie in sheep, bovine spongiform
encephalopathy in cattle, Creutzfeldt-Jakob disease, and
Gerstmann-Sträussler-Scheinker disease in man are fatal
neurodegenerative diseases that can have an infectious, sporadic, or
familial origin. They are characterized by the intracerebellar
accumulation of
PrPSc,1 an
abnormally folded, aggregated version of the normal, host-encoded cellular prion protein, PrPc (1-3). The molecular
mechanisms leading to the structural changes in PrP are still unclear.
At steady state in normal brain the 35-kDa glycoprotein PrP is anchored
in the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) moiety exposing the polypeptide to the extracellular face of the
plasma membrane (4, 5). During early stages of biogenesis at the ER
membrane, PrP can adopt more than one topological form (5, 6). By using
a cell-free translation system containing ER-derived microsomal
membranes, three different topological forms have been identified (5).
In addition to the fully translocated version (secPrP),
which eventually gives rise to the GPI-anchored form at the plasma
membrane, PrP exists in two transmembrane forms. These span the
membrane with the same hydrophobic region, TM1, but in opposite
orientations. The transmembrane form with the N terminus in the ER
lumen has been designated NtmPrP (N-transmembrane), and the
PrP form integrated in the opposite orientation has been designated the
CtmPrP form (C-transmembrane). The mechanism by which the
two transmembrane forms are generated is not yet known.
NtmPrP is thought to form when translocation is initiated
by the SS and subsequently is stopped by the TM1 segment (6). How CtmPrP is generated is not known. It has been proposed that
different components at the translocation site mediate insertion of
CtmPrP as opposed to secPrP and
NtmPrP (7).
In in vitro translocation assays the three topological PrP
forms were produced in different amounts, with the secPrP
and NtmPrP forms being about equally abundant (40-50%),
and the CtmPrP form representing about 10% of translocated
PrP (5). Disease-associated mutants within PrP affected the proportion
of the three topological forms in vitro but also in
transgenic animals expressing these mutant forms of PrP (5). These
findings suggest that early stages in the biogenesis of PrP may be
important for understanding the generation of some variant forms of PrP
that lead to neurodegeneration.
Formation of the different topological forms of PrP could involve
different signal sequences in PrP. The N-terminal SS obviously mediates
translocation of the N-terminal region in secPrP and
NtmPrP. For generating CtmPrP the SS may not be
engaged, and the TM1 or TM2 may then function as internal topogenic
sequences. To test this hypothesis, we analyzed whether in the absence
of SS other hydrophobic regions in PrP can target the protein to the ER
membrane and mediate membrane insertion. We deleted the SS alone or in
combination with the other hydrophobic regions and tested the resulting
PrP mutants for membrane insertion and translocation in a cell-free assay.
We have found that PrP contains, in addition to the SS, a second
potential targeting signal at its C terminus, TM2. Targeting by TM2
occurs post-translationally and leads to the preferential formation of
the CtmPrP form.
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EXPERIMENTAL PROCEDURES |
Materials--
General chemicals were from Merck or Sigma.
Restriction enzymes were from Roche Molecular Biochemicals;
[35S]methionine was from Amersham Pharmacia Biotech;
pGEM-3Z was from Promega; and protein A-Sepharose was from Amersham
Pharmacia Biotech. The monoclonal antiserum 3F4, which specifically
recognizes amino acid residues 108-111 in PrP (8), was from Senetek
PLC. The polyclonal antiserum directed against preprolactin was made against a peptide encoding amino acid residues 68-80 of preprolactin. The monoclonal antibody 3B5 recognizes residues 51-89 in PrP (9).
Plasmids and Transcription--
The plasmid pGEM-3ZK-PrP3F4,
coding for mouse PrP tagged with the epitope for the monoclonal
antibody 3F4, under the control of the SP6 promoter was generated by
inserting the AatII/SacI fragment harboring the
PrP-coding region derived from pUC-PrP (10) into the
SmaI/SacI sites of pGEM-3Z. The 3F4 epitope
(L108M/V111M) was introduced by using the QuikChangeTM
site-directed mutagenesis kit (Stratagene). Primers and reaction conditions were selected as recommended in the manufacturers' instructions.
By using pGEM-3ZK-PrP3F4, codons 3-22 were removed to yield
pGEM-3ZK-PrP3F4-
SS. This was achieved by cleavage of pGEM-3ZK-PrP3F4 with BamHI and SgrAI and re-ligation in the
presence of a double-stranded oligonucleotide encompassing the deletion.
The deletion of the TM1 region (codons 112-121) was generated by using
the ExSiteTM polymerase chain reaction-based site-directed
mutagenesis kit (Stratagene) yielding plasmid
pGEM-3ZK-PrP3F4-
SS-
TM1. All plasmids were amplified in
Escherichia coli and isolated with the
NucleobondTM plasmid purification kit (Machery Nagel). The
complete PrP-coding regions of all constructs were checked by DNA sequencing.
To obtain mRNAs encoding the respective PrP chains, the coding
regions of the relevant plasmids were amplified by polymerase chain
reaction using Pfu DNA polymerase (Stratagene) and
transcribed with SP6 RNA polymerase (11, 12). To express PrP mutants
with a deleted TM2 region, a 3' primer encoding an artificial stop codon at codon 231 was used. In all other cases, the 3' primer used
encoded the endogenous PrP stop codon. The mRNA encoding preprolactin was synthesized by in vitro transcription using
the pGEM-3Z vector and SP6 RNA polymerase. The preprolactin cDNA
was linearized with EcoRI.
Translation--
mRNAs were translated in vitro
in 10 µl of nuclease-treated reticulocyte lysate (Promega) in the
presence or absence of microsomal membranes (RM) (13) prepared from dog
pancreas and [35S]methionine as described previously
(14). Translations were incubated at 32 °C for 60 min. Translocation
reactions that were not incubated with proteinase K were centrifuged at
27,000 × g for 15 min at 4 °C. Alternatively,
protease accessibility assays were performed as described below. The
pelleted rough microsomes were solubilized in Tris-HCl (100 mM, pH 7.5) containing 1% SDS and then processed for
immunoprecipitation (see below).
Assay for Post-translational Translocation--
mRNAs were
translated in vitro in 10 µl of nuclease-treated
reticulocyte lysate at 32 °C for 30 min. Puromycin (final
concentration 1.25 mM) was added, and the reaction was
further incubated for 15 min at 32 °C. Where indicated, a further
incubation with apyrase (Sigma, final concentration 50 milliunits/µl)
was performed for 10 min at 32 °C. To test for post-translational
translocation activity, RM were added, and the incubation was continued
for 15 min. When a cotranslational assay was performed in parallel to
the post-translational assay, translation was also carried out for 30 min at 32 °C. The resulting translocation products were analyzed by
protease accessibility assays followed by immunoprecipitation analysis
with the monoclonal antibody 3F4 as described below.
Protease Accessibility Assays--
When protease accessibility
of translocation products was tested, proteinase K (500 µg/ml) and
Triton X-100 (1%, w/v) were added as indicated. After incubation for
60 min on ice, phenylmethylsulfonyl fluoride was added to 1 mg/ml, and
membranes were further incubated on ice for 5 min prior to analysis by
immunoprecipitation as described below.
After quantification of the PhosphorImager results, the numbers
obtained for each of the three protein fragments representing the three
topological forms of PrP were corrected for methionine content and then
normalized to the amount of precursor produced in the absence of RM.
Immunoprecipitation and Endo H Treatment--
For
immunoprecipitation, proteins were denatured in 78 µl of Tris-HCl
(100 mM, pH 7.5) containing 1% SDS for 5 min at 95 °C. The proteins were then solubilized in 500 µl of buffer containing 20 mM HepesKOH, pH 7.6, 100 mM NaCl, 5 mM MgCl2, 1% (w/v) Triton X-100, 0.2 mg/ml
phenylmethylsulfonyl fluoride, and 10 µg/ml chymostatin, leupeptin,
aprotinin, and pepstatin A. A preclearing step was performed by
incubating the samples for 1 h at 4 °C with 30 µl of protein
A-Sepharose (Amersham Pharmacia Biotech) equilibrated in IP buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.2% (w/v) Triton X-100). After removal of the
protein A-Sepharose beads by centrifugation, the supernatants were
supplemented with the relevant antibodies and incubated overnight at
4 °C. Antigen-antibody complexes were adsorbed to protein
A-Sepharose and recovered by centrifugation. The Sepharose beads were
washed twice with 1 ml of IP buffer, twice with 1 ml of IP buffer
containing 500 mM NaCl, and twice with 1 ml of 10 mM Tris-HCl, pH 7.5 (15). For Endo H treatment, washed
Sepharose beads were resuspended in 50 µl of 50 mM sodium
citrate, pH 5.5, supplemented with 2 µl (2000 units) of Endo H (New
England Biolabs). After incubation for 1 h at 37 °C, the beads
were washed with 1 ml of 10 mM Tris-HCl, pH 7.5. The washed
protein A-Sepharose beads were resuspended in 40 µl of SDS-PAGE
sample buffer (125 mM Tris-HCl, pH 6.8, 5 mM
EDTA, 2% 2-mercaptoethanol, 5% glycerol and 2% SDS), incubated for 10 min at 95 °C, and analyzed on 13.5% acrylamide gels (16). [35S]Methionine-labeled proteins were visualized by using
a Fuji PhosphorImager BAS1000 and quantified with Fuji MacBAS Ver 2.0 software.
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RESULTS |
Membrane Insertion of PrP Lacking Its Signal Sequence
(PrP-
SS)--
To investigate whether there are other ER targeting
signals in PrP in addition to the N-terminal SS, we deleted amino acids 3-22 comprising essentially the SS. Membrane insertion of the resulting PrP-
SS (outlined in Fig.
1A) was then studied using an
in vitro translation system (reticulocyte lysate)
supplemented with pancreatic, ER-derived rough microsomes (RM). To
determine the topologies of the resulting translocation products,
nontranslocated portions of the proteins were digested by the addition
of protease. The fragments remaining after proteolysis were identified
by immunoprecipitation using antibodies to two PrP epitopes. Proteins
were then separated by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and 35S-labeled proteins visualized by
phosphorimaging.

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Fig. 1.
Translocation of pPrP and
PrP- SS. A, outline of
precursor PrP (pPrP) and of PrP- SS which lacks the
N-terminal signal sequence. The three hydrophobic regions are depicted
as gray boxes, representing the signal sequence
(SS), the internal transmembrane region (TM1),
and the C-terminal hydrophobic region (TM2) that is cleaved
before attachment of the GPI anchor. The stop transfer effector
(STE) region just N-terminal to TM1 is depicted as a
white box. The two N-glycosylation sites at
positions 180 and 196 are drawn as forked structures. The
epitopes recognized by the monoclonal antibodies (Ab) 3B5
(B5) and 3F4 (F4) are indicated by black
lines. PrP- SS contains a deletion of the N-terminal SS (amino
acids 3-22). B and C, analysis of membrane
translocation of pPrP and PrP- SS. Messenger RNA coding for pPrP and
PrP- SS was translated in the presence or absence of rough microsomes
(RM). Where indicated, samples were treated with proteinase
K (PK). In addition, proteinase K digestions in the presence
of Triton X-100 (TX) were performed. Immunoprecipitations
were performed with either 3F4 (F4) or 3B5 (B5).
Where indicated, N-linked carbohydrates were removed by Endo
H treatment. Proteins were separated by SDS-PAGE and visualized by
phosphorimaging. pPrP/PrP- SS indicates
unmodified proteins. PrP-2g/PrP- SS-2g
indicates doubly glycosylated forms. PrP, PrP lacking SS;
Ctm-2g, PK-protected C-terminal part of PrP; Ctm,
de-glycosylated, PK-protected C-terminal part of PrP (marked by a
black dot); Ntm, PK-protected N-terminal part of
PrP; sec, fully translocated, completely PK-protected PrP
after de-glycosylation, including proteins showing a slightly slower
migration due to the cleavage specificity of Endo H leaving one
N-acetylglucosamine attached to each asparagine.
D, schematic representation of the three topological forms
of PrP at the ER membrane. The protein fragments protected against
proteinase K digestion are indicated by Ntm and Ctm. E,
quantification of the amounts of the three topological forms of pPrP
and PrP- SS. Translocation reactions shown in B,
lane 7, and C, lane 8, were used for
quantification. The amount of precursor produced in the absence of RM
(B and C, lanes 1) was taken as 100%
(RM ). The white bar represents the amount of
CtmPrP (Ctm), the gray bar of
NtmPrP (Ntm), and the black bar of
the fully translocated secPrP (sec). The
numbers denote the respective percentages of each
topological variant.
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Translation of wild-type PrP in the absence of RM led to the formation
of one major band (~27 kDa) representing the uncleaved precursor form of PrP (pPrP) (Fig. 1B, lane 1). In the
presence of RM the SS of pPrP was cleaved and glycosylation of PrP at
both N-glycosylation sites occurred, resulting in the
formation of PrP with two glycans (PrP-2g) (Fig. 1B, lane
2), as has been demonstrated previously (5). Proteolysis and Endo
H treatment of translocation reactions confirmed the production of
three different topological forms characterized by the protection of
the glycosylated, full-length PrP (secPrP), the
glycosylated Ctm fragment (CtmPrP), and the unglycosylated
Ntm fragment (NtmPrP) (Fig. 1B, lane 3)
schematically depicted in Fig. 1D. Endo H treatment
confirmed that the Ctm fragment and the fully translocated PrP were
glycosylated, whereas the Ntm fragment was not (Fig. 1B,
compare lanes 3 and 7). The slightly slower
migration of PrP after Endo H treatment as compared with the
unglycosylated protein (Fig. 1B, lanes 6 and 7)
is due to the cleavage specificity of the glycosidase, leaving one
N-acetylglucosamine attached to each asparagine. The
identities of the Ntm and Ctm fragments were confirmed by differential
immunoprecipitations with the 3F4 and 3B5 antibodies (Fig. 1B,
lanes 3 and 4 and 7 and 8). The
monoclonal antibody 3F4 is directed against an epitope located
N-terminal to TM1 (Fig. 1A) and recognizes PrP fragments of
all three topological forms after translocation/proteolysis assays (5).
The monoclonal antibody 3B5 recognizes an epitope in the N-terminal
part of PrP (amino acid residues 51-89) (Fig. 1A) (9).
Proteinase K treatment after solubilization of the membranes with
Triton X-100 resulted in complete digestion of all proteins,
demonstrating that protease protection was indeed due to translocation
into ER microsomes (Fig. 1B, lanes 5 and 9).
Translation of mRNA coding for PrP-
SS in the absence of RM led
to the production of one major product with a mass of ~26 kDa,
PrP-
SS (Fig. 1C, lane 1). Translation in the presence of RM resulted in the formation of an additional product with a mass of
~31 kDa. This form disappeared after Endo H digestion suggesting it
to be the doubly glycosylated form, PrP-
SS-2 g (Fig. 1C, lane 2 and 6). As expected, no SS-cleaved form appeared
(Fig. 1C, compare lanes 1 and 2).
After proteinase K treatment and subsequent immunoprecipitation with
antibody 3F4, two major fragments were immunoprecipitated, the
C-terminal, glycosylated PrP fragment (Ctm-2g, ~25 kDa) and the
N-terminal fragment (Ntm, ~13 kDa) (Fig. 1C, lane 3). A
small amount of fully translocated, di-glycosylated PrP-
SS-2g was
also produced, as is evident from the presence of a fragment that is not reduced in size after proteinase K digestion (Fig. 1C, lane 3). The Ntm fragment was also immunoprecipitated with the 3B5 antibody (Fig. 1C, lanes 3 and 4 and 8 and 9). Endo H treatment resulted in a 6-kDa reduction in
size of the Ctm-2g fragment confirming its glycosylation (Fig.
1C, lanes 3 and 8).
Thus the same translocated fragments are seen with pPrP and PrP-
SS,
although with different efficiencies and different proportions. The
overall translocation efficiency of PrP-
SS was reduced as compared
with pPrP. 7.1% of total PrP-
SS synthesized became
membrane-inserted or translocated as compared with 84.2% of pPrP (Fig.
1E). Differences are also seen with respect to the
proportion of membrane spanning and translocated forms. The absolute
amount of CtmPrP was almost identical in translocation
reactions with pPrP and PrP-
SS (pPrP = 5.1%, PrP-
SS = 4.8%, Fig. 1E). In contrast, the efficiency of formation of
the other two forms was drastically reduced (for NtmPrP,
pPrP = 37.7%, PrP-
SS = 1.6%; and for secPrP,
pPrP = 41.4%, PrP-
SS = 0.7%).
TM2 Can Function as a Second ER Targeting Signal--
The fact
that PrP-
SS was membrane-inserted suggested that a second ER
targeting signal must exist in PrP. Both the internal TM1 region and
the C-terminal TM2 region are of hydrophobic nature and therefore could
be potential targeting signals (17, 18). To identify the second
targeting signal sequence, we deleted the core region of TM1 (amino
acid residues 112-121) in addition to the SS resulting in
PrP-
SS-
TM1 (Fig. 2A).
Translocation assays followed by protease digestion and
immunoprecipitation with the 3F4-antibody were performed as described
above. In the presence of RM a glycosylated form, PrP-
SS-
TM1-2g,
accumulated, that was protected against protease digestion (Fig.
2B, lanes 7 and 8). Glycosylation of this protein
was demonstrated by the reduction in size after treatment with Endo H
(Fig. 2B, lane 9). Thus deletion of TM1 did not abolish
translocation. No accumulation of either Ntm or Ctm fragments occurred
as in the case of PrP-
SS (Fig. 2B, lanes 3 and
4) consistent with TM1 being required for the generation of
membrane-spanning PrP forms.

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Fig. 2.
Membrane translocation of
PrP- SS,
PrP- SS- TM1, and
PrP- SS- TM2.
A, outline of constructs containing a deletion of the
N-terminal SS and in addition either a deletion in TM1 (residues
112-121) or a deletion of TM2 (residues 231-254). B and
C, translocation assay. PrP- SS, PrP- SS- TM1, and
PrP- SS- TM2 were synthesized in the absence and presence of RM,
and translocation was tested by proteinase K (PK) treatment.
In addition, proteinase K digestions in the presence of Triton X-100
(TX) were performed. All immunoprecipitations were performed
with antibody 3F4. Where indicated, N-linked carbohydrates
were removed by Endo H treatment. Proteins were separated by SDS-PAGE
and visualized by phosphorimaging.
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To see whether the second targeting signal is localized to the
C-terminal hydrophobic region TM2, translocation of PrP-
SS-
TM2 (outlined in Fig. 2A) was analyzed by protease
protection experiments followed by immunoprecipitations.
PrP-
SS-
TM2 did not result in any glycosylated forms nor were
protease-protected fragments produced (Fig. 2C, lane 3).
This indicates that in the absence of an N-terminal signal sequence the
TM2 is required for ER targeting and translocation.
Membrane Insertion/Translocation of pPrP-
TM2--
To test
whether TM2 also contributes to membrane insertion when an SS is
present, we deleted TM2 from pPrP (outlined in Fig. 3A). Translation of mRNA
coding for pPrP-
TM2 resulted in the production of one major protein
species, pPrP-
TM2 (Fig. 3B, lane 1). When the translation
was performed in the presence of RM, three protein species could be
observed. These are the signal sequence cleaved forms with different
degrees of glycosylation, namely non- (PrP-
TM2), mono-
(PrP-
TM2-1g), and di-glycosylated (PrP-
TM2-2g) (Fig.
3B, lane 2). Endo H digestion confirmed that the increase in
molecular weight was indeed due to glycosylation (Fig. 3B,
compare lane 2 and 7). The glycosylation
efficiency of pPrP-
TM2 was significantly reduced compared with pPrP,
leading to the formation of about equal amounts of unglycosylated,
mono-glycosylated, and di-glycosylated forms. Most of the glycosylated
PrP-
TM2 resisted proteinase K digestion indicating complete
translocation. Some NtmPrP was produced, as is evident from
the presence of the Ntm fragment after proteinase K digestion (Fig.
3B, lanes 4 and 5 and 9 and 10). After Endo H treatment, a very small amount of the Ctm
fragment representing CtmPrP could be observed (Fig.
3B, lane 9). When compared with pPrP, the amount of
pPrP-
TM2 inserted in the CtmPrP orientation was
significantly reduced by about 75% (see also Fig. 3C for
the absolute values). A less pronounced reduction of about 50% was
observed for NtmPrP. In contrast, the efficiency of full
translocation was slightly increased (pPrP-
TM2 = 50.0%,
pPrP = 41.4%).

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Fig. 3.
Translocation and protease protection assay
with pPrP- TM2. A, outline of
pPrP- TM2, lacking the C-terminal TM2 region (residues 231-254).
B, translocation assay. pPrP- TM2 was translated in the
absence and presence of RM, and translocation was tested by proteinase
K (PK) treatment, as indicated. In addition, proteinase K
digestions in the presence of Triton X-100 (TX) were
performed. Immunoprecipitations were performed with either 3F4
(F4) or 3B5 (B5). Where indicated,
N-linked carbohydrates were removed by Endo H treatment.
Proteins were separated by SDS-PAGE and visualized by phosphorimaging.
PrP- TM2-2g, di-glycosylated form;
PrP- TM2-1g, mono-glycosylated form;
PrP- TM2, PrP lacking both the N-terminal
signal sequence and the C-terminal TM2 region; Ctm, Ntm; C-
and N-terminal PK-protected PrP fragments; sec, fully
translocated, completely PK-protected PrP after de-glycosylation.
C, quantification of the amounts of the three topological
PrP forms of pPrP and pPrP- TM2. Translocation reactions shown in
Figs. 1B, lane 7, and Fig. 3B, lane 9, were used
for quantification. The amount of precursor produced in the absence of
RM (Fig. 1B and Fig. 3B, lane 1) was taken as
100% (RM ). The white bar represents the
amount of CtmPrP, the gray bar of
NtmPrP, and the black bar of fully translocated
secPrP. The numbers denote the respective
percentages of each topological variant.
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Thus the absence of TM2 in pPrP affects the proportion of
CtmPrP and, to a lesser extent, also of
NtmPrP.
Post-translational Targeting by TM2--
The fact that the second
ER targeting signal is located at the extreme C terminus of PrP makes
it likely to function post-translationally because it would not emerge
from the ribosome before translation is terminated. To test this we
added RM after PrP-
SS-
TM1 had been synthesized and any nascent
chains had been released from ribosomes with puromycin. A proportion of
PrP-
SS-
TM1 became fully translocated and twice glycosylated
(PrP-
SS-
TM1-2g) as evidenced by proteinase K (Fig.
4A, lane 6) and Endo H
treatment (Fig. 4A, lane 9), respectively. The efficiency of
post-translational translocation of PrP-
SS-
TM1 was 20.8% of its
cotranslational translocation efficiency (Fig. 4A, lane 3 and lane 6). Post-translational translocation was inhibited
by apyrase treatment, as shown by the lack of protease-protected
PrP-
SS-
TM1-2g (Fig. 4A, lane 12), strongly indicating
that it is ATP-dependent. ATP-dependent post-translational translocation was also observed for PrP-
SS (data
not shown). When the same post-translational assay was performed with
preprolactin, no translocation occurred in contrast to efficient cotranslational translocation of preprolactin (Fig. 4B,
compare lanes 3 and 6). When pPrP and pPrP-
TM2
were tested for post-translational translocation, only very small
amounts of translocated, proteinase K-protected and -glycosylated
PrP could be detected (as shown for pPrP-
TM2 in Fig.
5, lane 4, post-translational
translocation efficiency: 3.3% of its cotranslational translocation
efficiency, lane 2). In contrast to the SS-deleted PrP
forms, translocation of pPrP and pPrP-
TM2 was not inhibited by
apyrase treatment (shown for pPrP-
TM2 in Fig. 5, lane 6).
Post-translational translocation assays performed with both
PrP-
SS-
TM1 and pPrP-
TM2 showed the production of protein
fragments of low molecular weight that were resistant to proteinase K
digestion even in the presence of Triton X-100. These could potentially
be derived from abnormally aggregated PrP.

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Fig. 4.
Post-translational translocation of
PrP- SS- TM1.
A, for cotranslational translocation (co)
PrP- SS- TM1 was translated in the presence of RM. For
post-translational translocation (post) PrP- SS- TM1 was
translated; puromycin was added to ensure that all chains are released
from the ribosomes and then RM were added. Glycosylation was tested by
Endo H treatment. Apyrase (Apy) was added after translation
to deplete the reaction of ATP. Translocation was analyzed by
proteinase K (PK) treatment. In addition, proteinase K
digestions in the presence of Triton X-100 (TX) were
performed. Immunoprecipitations were performed with the antibody 3F4.
B, the same co- and post-translational translocation assay
as described in A was performed with preprolactin
(pPL). Immunoprecipitations were performed with a polyclonal
antibody directed against amino acid residues 68-80 of pPL.
PPL, unmodified protein; PL, prolactin lacking
the N-terminal signal sequence.
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Fig. 5.
Post-translational translocation of
pPrP- TM2. pPrP- TM2 translocation was
performed in parallel in the cotranslational (co) and the
post-translational (post) translocation assay in the absence
or presence of apyrase (Apy) as described in Fig. 4.
Translocation was determined by proteinase K treatment. In addition,
proteinase K digestions in the presence of Triton X-100 (TX)
were performed. The proteins were immunoprecipitated with the antibody
3F4.
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DISCUSSION |
The generation of different topological forms of PrP in the ER
suggests the presence of different signal sequences in PrP and the
alternative use of these signals. To identify additional signal
sequences in PrP, we have deleted the N-terminal signal sequence and
analyzed the resulting mutant protein for the presence of a second
signal sequence. We have identified the C-terminal hydrophobic region,
TM2, as a signal sequence that can mediate post-translational membrane
insertion. Membrane insertion mainly occurred in the Ctm orientation.
When the TM2 was deleted from PrP the amount of CtmPrP was
drastically reduced. We conclude from our data that the TM2 of PrP
contributes significantly to the formation of the Ctm form of PrP.
CtmPrP exposes the N terminus on the cytoplasmic side and
spans the membrane at the internal hydrophobic region, TM1. During biosynthesis, the C-terminal TM2 region is inserted into the membrane and then cleaved and replaced by a glycolipid membrane anchor (19). A
C-terminal hydrophobic region is also found in the so-called "tail-anchored proteins." However, in this case the entire
N-terminal part of these proteins is exposed to the cytoplasmic side,
and only the small C-terminal tail is inserted into the membrane (20, 21). It remains to be seen what sequence characteristics determine the
orientation of proteins that are inserted into the membrane by
C-terminal hydrophobic regions.
TM2 can mediate post-translational translocation of PrP, and this is
dependent on ATP. ATP requirement has also been demonstrated for the
membrane insertion of tail-anchored proteins (Vamp1, Vamp2, and Vamp8)
(22), and for post-translational translocation of secretory proteins in
Saccharomyces cerevisiae (23-26). For some proteins it has
been shown that members of the Hsp70 family of ATPases are required for
membrane insertion (27, 28). Binding of cytosolic Hsp70 may prevent
premature folding of post-translational substrates, whereas lumenal BiP
may facilitate the actual translocation process (29, 30).
How could an N- and C-terminal signal sequence cooperate in the
generation of alternative topologies? Our data suggest that the
N-terminal (cotranslational) signal sequence of PrP is
"inefficient," allowing some molecules to be fully synthesized and
then post-translationally translocated by using the TM2 as signal
sequence. Cotranslational translocation requires that a cytoplasmic
ribonucleoprotein particle, the signal recognition particle (SRP),
efficiently interacts with the signal sequence of the nascent
polypeptide resulting in an arrest of translation. Subsequently, the
complex of ribosome, nascent polypeptide and SRP contacts the SRP
receptor at the ER membrane leading to the insertion of the nascent
polypeptide into the membrane (31-33). We propose that multiple
topologies of PrP are generated by the differential use of the two PrP
signal sequences. Inefficient use of a signal sequence has been
demonstrated previously for both membrane as well as secretory
proteins. Functional consequence is the expression of topologically
different proteins produced from the same mRNA. In the case of
polytopic membrane proteins, the facultative insertion of a
transmembrane region can lead to the formation of alternative membrane
topologies of a single protein. One such example is the large envelope
protein (L protein) of duck hepatitis B virus. Its transmembrane region
I is inserted into the membrane with an efficiency of about 50% (34).
This results in cytosolically or lumenally located preS domain. On the
cytosolic side preS has a role in capsid binding during virus budding,
whereas in the opposite orientation it is required for receptor binding
during virus infection. Another example is the generation of a secreted
and a cytosolic form of the plasminogen activator inhibitor-2. In this
case, the signal sequence functions inefficiently at two steps as
follows: SRP-mediated targeting to the ER membrane and the subsequent
formation of a committed translocation complex (35). In contrast to
these proteins, where the differential utilization of a single signal
sequence leads to the formation of two different topologies, we propose
a model for PrP translocation where not only the N-terminal signal
sequence is facultatively used but, in addition, another signal
sequence at the C terminus is used post-translationally.
In pPrP, about 80% of polypeptides are targeted
cotranslationally by the N-terminal SS yielding secPrP and
NtmPrP. According to our model, the 5% polypeptides
integrated in the CtmPrP orientation would be derived from
the 20% of chains not translocated cotranslationally. This implies
that the translocation efficiency of TM2 in pPrP is about 25%, being
5-fold higher than in PrP-
SS where only 5% translocation efficiency
by TM2 was achieved. Efficient post-translational translocation by TM2
therefore seems to be promoted by the presence of the N-terminal SS.
Following successful SRP-mediated targeting to the ER, a proportion of
PrP nascent chains may not engage in productive cotranslational
translocation due to inefficient recognition of the SS by components of
the translocon. PrP polypeptides that failed to translocate
cotranslationally would then be a substrate for post-translational
translocation by TM2.
It is very likely that the synthesis of the different topological forms
of PrP varies in different cell types. In addition to regulation at the
level of SRP binding to SS, changes of the translocation machinery,
either by expression of different regulatory factors (36, 37) or by
differential modification of certain translocon components (38), could
influence the ratio of the different PrP forms. As this could be a
mechanism for the functional regulation of PrP in different cell types
or under varying physiological conditions, it is possible that
incorrect regulation of the translocation machinery leading to the
perturbance of the delicate balance of the different forms of PrP could
lead to disease. Indeed, it has already been shown that a shift of the
relative ratios toward increased expression of CtmPrP
resulting from changes in the PrP sequence itself can lead to
neurodegenerative disease (5, 39). Transgenic mice expressing mutant
forms of PrP showed increased levels of CtmPrP, the levels
of which could be correlated with the propensity of each mutant form to
induce neurodegeneration (39). However, it remains unclear what
proportion of the CtmPrP production occurred co- or
post-translationally.
The mechanism by which increased expression of CtmPrP leads
to neurodegeneration is not yet known. In analogy to processing of the
amyloid precursor protein in Alzheimer's disease (40, 41), different
cleavage products of PrP, either destroying or leaving intact the
amyloidogenic region in TM1, have been identified (42). Very likely, if
PrP is processed by potential secretases located at the ER membrane,
then cleavage products could vary with different PrP topologies.
It will be interesting to see if a perturbation of the balance between
SS- and TM2-mediated membrane insertion of PrP is related to
neurodegeneration in certain forms of prion disease.