(Received for publication, May 30, 1996, and in revised form, October 1, 1996)
From the Presenilins 1 and 2 are unglycosylated proteins
with apparent molecular mass of 45 and 50 kDa, respectively, in
transfected COS-1 and Chinese hamster ovary cells. They colocalize with
proteins from the endoplasmic reticulum and the Golgi apparatus in
transfected and untransfected cells. In COS-1 cells low amounts of
intact endogeneous presenilin 1 migrating at 45 kDa are detected
together with relative larger amounts of presenilin 1 fragments
migrating between 18 and 30 kDa. The presenilins have a strong tendency to form aggregates (mass of 100-250 kDa) in SDS-polyacrylamide gel
electrophoresis, which can be partially resolved when denatured by SDS
at 37 °C instead of 95 °C. Sulfation, glycosaminoglycan modification, or acylation of the presenilins was not observed, but
both proteins are posttranslationally phosphorylated on serine residues. The mutations Ala-246 Alzheimer's disease is a major health problem. Patients suffer
from a progressive dementia caused by massive neuronal loss in cortical
and hippocampal areas of the brain (1-6). Neuropathological signs of
the disease are tangles and amyloid deposits in the brain parenchyma,
and amyloid deposits in the brain vasculature. The cause of the
sporadic form of the disease is still unknown, although an increased
risk is associated with the presence of apolipoprotein allele E4 (6,
7). On the other hand, familial early onset Alzheimer's disease is
caused by point mutations in the amyloid precursor protein gene on
chromosome 21 (8), in the presenilin 2 (PS2)1 gene on chromosome 1 (9-11), or,
most frequently, in the presenilin 1 (PS1) gene on chromosome 14 (12-15). Amyloid precursor protein (APP) is a type I integral membrane
protein and is the precursor of the amyloid peptide, the main component
of the senile plaques (1-3). Point mutations in exons 16 and 17 of the
APP gene cause alterations in the metabolism of APP. This results in an
increased production of intracellular A hypothetical final common pathway in the pathogenesis of the genetic
and sporadic forms of Alzheimer's disease has been postulated, based
on the invariable occurrence of the amyloid deposits, the
neurofibrillar tangles and the neurodegeneration in all affected
brains. The central question is thus how mutations in the presenilin
genes can cause this typical neuropathology. Studies indicating an
increased production of more amyloidogenic We used COS-1 cells and CHO cells to express PS1 and PS2, as well as
Myc-tagged PS1 and PS1-containing mutations that cause familial
Alzheimer's disease. The biosynthesis of transfected and untransfected
presenilins was studied using immunoblotting or metabolical labeling
and immunoprecipitation assays. We demonstrate that transfected
presenilins are phosphorylated on serine residues. Using
immunofluorescence microscopy, we document the association of
presenilin 1 with the early compartments of the biosynthetic pathway
and demonstrate the cytoplasmic orientation of the two major
hydrophilic domains.
The cDNA coding for mouse PS1 and human PS1,
PS2, and PS1 containing Ala-246 Polyclonal rabbit antisera B13, B14, and B15
were raised against peptide p45 (NDNRERQEHNDRRSLC), which is in the
amino-terminal domain of PS1 (12). Polyclonal rabbit sera B16 and B17
were raised against peptide p46 (EGDPEAQRRVSKNSKC) situated in the hydrophilic loop domain of PS1. Polyclonal rabbit antiserum 519 was
raised against residues KDGQLIYTPFTEDTE(C). This antiserum reacts with
a sequence in the second loop domain of PS1 and PS2. The synthetic
peptides p45 and p46 were manufactured by Eurogentec (Liège,
Belgium), while peptide p519 was synthesized by solid-phase techniques
and purified by reverse phase high pressure liquid chromatography in
our laboratories.2 Rabbits were injected
every 2 weeks with 100, 200, or 300 µg of peptide in complete
Freund's adjuvant, coupled to keyhole limpet hemocyanin or bovine
serum albumin (Pierce) and solubilized in PBS mixed with complete
Freund's adjuvant. Monoclonal antibody (mAb) PS1-3, reacting with the
peptide RRVSKNSKYNAESTERESQDTVAEN in the hydrophilic loop domain of PS1
mAb 9E10 against the Myc tag (26), was kindly provided by Dr. J. Creemers. mAbs MON160, 161 and 162 and MON148 and 152 against
NSP/reticulon and furin have been described (22-25). mAbs against the
immunoglobulin-binding protein (BiP) and against the calcium pump Serca
2a (IID8) were purchased from, respectively, StressGen (Victoria,
Canada) and Affinity BioReagents (Neshanic Station, NJ).
The fibroblast cell line (designation
AG07657, Coriell Institute) from an unaffected individual of the FAD1
lineage was cultured in Dulbecco's modified Eagle's medium
supplemented with 20% fetal bovine serum (Life Technologies, Inc.).
COS1 cells were cultured as described previously in Dulbecco's
modified Eagle's medium/Ham's F-12 with 10% fetal bovine serum (27).
CHO cells stably transfected with APP770 were kindly provided by Dr. B. Greenberg (Cephalon, West Chester, PA) and cultured in high glucose
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 0.1 mM minimal essential medium
(nonessential amino acids), 3% fetal calf serum, and 250 nM methotrexate.
COS cells were transfected using DEAE/dextran (27), while CHO cells
were transfected using LipofectAMINE according to the instructions of
the manufacturer (Life Technologies, Inc.).
Metabolic labeling was done with 100 µCi of
[35S]methionine, 250 µCi of [35S]sulfate,
60 µCi of [3H]glucosamine, 500 µCi of
[3H]palmitic acid or [3H]myristic acid, or
500 µCi of [32P]orthophosphate/ml of the appropriate
culture medium. All radiolabeled precursors were from Amersham.
Cellular labeling was done for 4 h unless otherwise specified, and
cell extracts were made as described previously (27). For analysis of
phosphorylation, a postnuclear extract was prepared using 0.5% (v/v)
Triton X-100 in Tris-buffered saline (TBS) containing proteinase
inhibitors (100 units/ml aprotinin, 1 µg/ml pepstatin) and tyrosine
and serine/threonine phosphatase inhibitors (1 mM sodium
orthovanadate, 5 mM EDTA, 5 mM EGTA, 20 mM NaF). The nuclei were pelleted by centrifugation at
14,000 rpm (15 min) in a cooled Eppendorf centrifuge. Antisera were
added to the cell extracts at a 1/250 dilution, and antibody-antigen complexes were collected by incubation with immobilized protein G
(Pierce) overnight at 4 °C. The immunoprecipitates were washed using
Tris-buffered saline containing 1% (v/v) Triton X-100, 1% (w/v)
deoxycholate, and 0.1% (w/v) SDS (27). For analysis of phosphorylation, phosphatase inhibitors as detailed above, were added
to all buffers.
Phorbol myristic acid, phorbol dibutyric acid, forskolin, okadaic acid
and staurosporin (all from Sigma) were added to the cell cultures during the last 30 min of the metabolic labeling at the
indicated final concentrations. For in vitro labeling
assays, immunoprecipitated PS1 or PS2 bound on protein G beads were
incubated with purified protein kinase A or C in Tris-buffered saline,
in the presence of 0.1 mM [ For immunoblotting experiments, cells were scraped in PBS, centrifuged
(10 min, 1000 rpm), and solubilized in Laemmli sample buffer. Blots
were stained with mAb 9E10 (Myc tag) ascites fluid (1/1000 dilution) or
mAb PS1-3 hybridoma supernatant (1/100 dilution) and affinity-purified
goat anti-mouse peroxidase-conjugated antibodies (1/10,000; Bio-Rad),
using the sensitive ECL system (Amersham).
Immunoprecipitated
32P-labeled PS were size-fractionated in SDS-PAGE, and
labeled bands were localized by autoradiography. The proteins were
hydrolyzed in 6 N HCl (110 °C, 90 min). The hydrolysate was supplemented with phosphoamino acid standards and analyzed by
two-dimensional thin-layer electrophoresis at pH 1.9 and 3.5 in the
respective dimensions (28). Phosphoserine, phosphothreonine, and
phosphotyrosine were localized using ninhydrin staining, and the
radioactive phosphoamino acid residues were visualized by autoradiography.
Fibroblasts were grown on coverslips
coated with mouse collagen IV (Collaborative Biomedical Products; 1 µg/cm2). Transfected COS-1 or CHO cells were cultured in
Lab-TEK chamber slides (Nunc). Cells were washed twice in PBS, fixed in
4% formaldehyde in PBS for 10 min at room temperature, and washed
three times in PBS and once in TBS. Cells were permeabilized with
0.02% (v/v) Triton X-100 in TBS for 20 min or with 0.2% (w/v) saponin
for 10 min and washed with 0.1% Tween 20 in TBS. Nonspecific binding was blocked with 0.2% cold water fish gelatin, 2% bovine serum albumin, and 2% fetal calf serum (blocking buffer). Cells were probed
with affinity-purified primary antibody 519 (1:25) or with immune serum
(1:400). Appropriate FITC- and TRITC-conjugated anti-mouse, anti-rat,
and anti-rabbit antibodies (Sigma) were used at 1/400 dilution. Preparations were viewed on a Nikon Diaphot 300 or a Zeiss
Axiophot UV microscope. Digitized immunofluorescence images were
obtained using an LSM419-inverted laser-scanning confocal microscope
(Zeiss Inc.) and processed using NIH Image software.
Selective permeabilization of the plasma membrane was obtained by
incubating fixed cells in 10 mM Pipes buffer (pH 6.8)
containing 0.3 M sucrose, 0.1 M KCl, 2.5 mM MgCl2, 1 mM EDTA, 5 µg/ml
digitonin during 15 min at 4 °C (29). Cells were washed with PBS and
further processed. A rat monoclonal antibody against the KDEL sequence (30, 31) was used to demonstrate permeabilization of the endoplasmic reticulum membrane.
The subcellular localization of
the presenilins was investigated in permeabilized and fixed fibroblasts
using affinity-purified polyclonal antibody 519 raised against the
peptide KDGQLIYTPFTEDTE, which is a conserved sequence in the second
loop domain of PS1 and PS2 (9, 12). A fine reticular staining in the
cytoplasm and a more pronounced perinuclear staining was observed in
the cells (Fig. 1A), which partially
colocalized with the staining obtained with a mAb against BiP, an
endoplasmic reticulum marker. Preabsorption of antibody 519 with the
peptide antigen resulted in loss of staining, demonstrating the
specificity of the observed signals (Fig. 1C).
The same, lacelike network was seen in COS-1 cells transfected with
plasmids containing the cDNA for PS1 and using antiserum B14
against peptide NDNRERQEHNDRRS in the amino-terminal domain of PS1 or
antiserum B16 against peptide EGDPEAQRRVSKNSKY in the hydrophilic loop
domain of PS1 (Fig. 2, a and c).
Untransfected COS-1 cells remained negative under the experimental
conditions used, probably because of the very low levels of endogeneous
PS present in these cells (see below). The same pattern of staining was
observed with monoclonal antibody IID8 (32) against Serca 2a
Ca2+-ATPase (results not shown), and with antibodies
against transfected reticulon/NSP (Fig. 2b). Both proteins
are located in the endoplasmic reticulum and the Golgi apparatus (24).
The distribution of PS1 and reticulon/NSP remained identical in double
transfected cells (compare panels a and b in Fig.
2). Transfected APP (Fig. 2e) and transfected furin (Fig.
2d), in contrast, were mainly found in the Golgi apparatus,
as shown previously (33, 34). Similar results were obtained in CHO
cells (results not shown).
Metabolic labeling of
untransfected or "mock" transfected COS-1 or CHO cells using
[35S]methionine, followed by immunoprecipitation of the
cell extracts using antibodies 519, B14, or B17 and resolution of the
immunoprecipitates in SDS-PAGE, yielded no signals (Fig.
3A, lane 4) or, after prolonged exposures (2 weeks and more), only nonspecific signals in
autoradiography (results not shown). Immunoprecipitation of detergent
extracts of COS-1 cells transfected with plasmids containing the
cDNA of wild type PS1, in contrast, yielded strong specific signals
of radiolabeled protein migrating with an apparent molecular mass of 45 kDa (Fig. 3A, lanes 1-3). Diffuse protein bands
with masses between 100 and 250 kDa were observed to a variable extent
(Fig. 3A). Similar results were obtained with PS2 cDNA.
The main PS2 species migrated, however, slightly more slowly than PS1,
resulting in an apparent mass of 50 kDa (see below). Unrelated
polyclonal antibodies, or untransfected cells did not yield these
bands, while immunoblots of COS-1 cells transfected with Myc-tagged PS1 and stained with the Myc tag-specific mAb 9E10 revealed again the
pronounced smearing (Fig. 3B). This result clearly
demonstrated that the 100-250-kDa protein smears consisted of PS
protein, either associated or not associated with other proteins.
Essential similar patterns were observed when PS1 FAD1 (Ala-246
Since the levels of endogeneous PS1 were not detectable using classical
immunoprecipitation, a sensitive immunological "sandwich" type
assay was developed. PS1 was immunoprecipitated from detergent extracts
of 10 × 106 untransfected COS-1 cells using
polyclonal PS1 antisera. A small amount of transfected cells (0.5 × 105) was added to the positive control samples. The
immunoprecipitates were then resolved in SDS-PAGE electrophoresis and
transferred to a nitrocellulose filter. Immunoprecipitated PS1 was
finally detected with mAb PS1-3 raised against peptide
RRVSKNSKYNAESTERESQDTVAE in the loop domain of PS1. Weak signals
representing intact endogeneous PS1 were revealed in untransfected
COS-1 cells (Fig. 3C, lanes 2 and 4).
Endogeneous PS1 migrated with the same mobility (45 kDa) as transfected
PS1, as is best seen in the experiments using the amino-terminal
domain-specific antiserum B13 (Fig. 3C, lane 2).
Since mAb PS1-3 recognizes an epitope in the carboxyl-terminally located hydrophilic loop, the combination of these antibodies is
expected to detect mainly intact PS1 (Fig. 3C, lane
2). With hydrophilic loop-specific antiserum B17, in contrast,
relatively pronounced 18-30-kDa fragments were visualized together
with intact PS1. These fragments represent most likely
carboxyl-terminal fragments of presenilin (35). Remarkably, the
observed fragments were not, or only marginally, increased in COS-1
cells overexpressing PS1 (compare lanes 3 and lane
4 in Fig. 3C). Pulse-chase experiments on transfected
CHO-cells (Fig. 4, A and C) and
COS-1 cells (results not shown) further showed that the transfected
45-kDa PS1 species has a half-life of about 4 h. No fragments were
seen at any time point of this assay, either with amino-terminal (B13)
or hydrophilic loop-specific (B17) antisera. APP, immunoprecipitated
from the same cell extracts (Fig. 4B), displayed a much
faster turnover (half-life: 2 h), indicating that the relative
high expression of PS1 did not interfere with the normal turnover of
APP.
The problem of the presenilin smears in SDS-PAGE was
further investigated. Enzymatic digestion of immunoprecipitated PS1 and PS2 protein with glycosidase F (3 milliunits/µl),
O-glycanase (60 nanounits/µl), endoglycosidase H (60 nanounits/µl), sialidase (100 nanounits/µl), or combinations of
these enzymes did not affect the mobility of the proteins, indicating
that no glycosylation of PS1 and PS2 occurred (results not shown).
Digestions with heparinase (0.1 milliunits/µl), heparitinase (0.1 milliunits/µl), chondroitinase AC (5 milliunits/µl), and
chondroitinase ABC (5 milliunits/µl) also had no effect on the
mobility of the protein smears, indicating that PS1 is not modified by
glycosaminoglycan chains (Fig. 5). These negative
results were independently confirmed by metabolic labeling experiments
using [3H]glucosamine or
[35S]O4. While both radioactive precursors
were incorporated in proteins in the cell extracts of the labeled
cells, no signal was obtained when transfected PS1 was
immunoprecipitated (results not shown). Experiments using
[3H]palmitic or [3H]myristic acid
demonstrated also that the presenilins did not incorporate fatty acids.
Sonication or boiling of the samples in SDS and 6 M urea
(data not shown) or extraction of the cells in the presence of 5 mM dithiothreitol did not resolve the aggregates (Fig.
6). Denaturation of the immunoprecipitates at 37 °C
instead of at 95 °C reduced the aggregates considerably but not
completely (Fig. 6, lane 2). This demonstrated that the
aggregates consisted mainly, if not exclusively, of PS1 as the
radiolabeled species (Fig. 6) and also suggested that the PS aggregates
are at least partially produced during the processing of the samples
for electrophoresis. It should be noticed, however, that even when
freshly prepared material was used and heating of the samples was
avoided, protein smears in the 100-250-kDa region remained visible in
SDS-PAGE (Fig. 6).
The high level of serines,
threonines, and tyrosines in the primary amino acid sequences of both
PS1 and PS2 suggested the possibility that the PS proteins are
phosphoproteins (12, 13). Transfected COS-1 cells (Fig.
7) and CHO cells (Fig. 8B)
incorporated 32P in presenilins. Acid hydrolysis of
immunoprecipitated 32P-phosphorylated PS1 and PS2 yielded
mainly phosphoserine (Fig. 7B). Longer exposures revealed
very weak signals for phosphothreonine, while phosphotyrosine was never
observed. The phosphorylation of PS1 was strongly increased, and that
of PS2 slightly or not increased, by treating the cells with the
phosphatase inhibitor okadaic acid (300 nM, Fig.
8A). Okadaic acid inhibits the two major classes of
serine/threonine phosphatases (49). The protein kinase C inhibitor
staurosporin (300 nM) or the agonists phorbol myristic acid
(1 µM) and phorbol dibutyric acid (1 µM)
and the protein kinase A agonist forskolin (100 µM) had
no effect on the phosphorylation extent or pattern (Fig.
8A). Attempts to phosphorylate immunoprecipitated PS1
in vitro using protein kinase A or protein kinase C remained
also negative (results not shown).
We finally addressed the
issue whether the two major hydrophilic domains in presenilin 1, i.e. the amino-terminal domain and the hydrophilic loop
domain (12) are oriented toward the cytoplasmic or the luminal side of
the endoplasmic reticulum. Current models for the orientation of the
presenilins in membranes are based on theoretical predictions (see
"Discussion"). Both domains are candidate regions for interactions
with other proteins, and their orientation determines whether this
interaction occurs with cytoplasmic or endoplasmic reticulum proteins.
To analyze this question, we used low concentrations of digitonin to
selectively permeabilize the plasma membrane (see "Materials and
Methods") and antibodies against the amino-terminal domain (antiserum
B14), against the Myc tag introduced at the amino terminus (mAb 9E10),
against the second loop domain (antibody 519), and, finally, against
the hydrophilic loop domain (antiserum B16) of PS1. To monitor the
permeabilization procedure, rat monoclonal antibody against the luminal
endoplasmic reticulum retention signal KDEL was used (30, 31).
Antibodies against the amino-terminal domain of PS1, against the
amino-terminally inserted Myc tag (Fig. 9a)
or against the hydrophilic loop (Fig. 9e) reacted with PS1
in digitonin-permeabilized cells under conditions in which the
KDEL-specific monoclonal antibody did not result in labeling (Fig. 9,
a and e). On the other hand, antibodies against the second hydrophilic loop domain (519) reacted with PS1 when saponin
(Fig. 9d), but not when digitonin (Fig. 9c) was
used to permeabilize the cells, suggesting a luminal localization of
this domain.
The current investigation provides a detailed characterization of
the biosynthesis of presenilins overexpressed in COS-1 cells and CHO
cells. The results show that the PS proteins are unglycosylated phosphoproteins with apparent molecular masses of about 45 and 50 kDa
in SDS-PAGE. They have a strong tendency to form SDS-resistant complexes with apparent molecular masses between 100 and 250 kDa. Double immunofluorescence studies showed that PS1 is mainly located in
the endoplasmic reticulum and the Golgi apparatus and that its
amino-terminal domain and the hydrophilic loop domain are oriented to
the cytoplasmic side.
The localization of transfected PS1 in the endoplasmic reticulum of
COS-1 cells or CHO cells is not a simple consequence of overexpression
of the protein or nonspecific general effects on the biosynthetic
pathway in these cells. First, endogeneous presenilins are located in
the endoplasmic reticulum of untransfected cells as demonstrated in
Fig. 1A using confocal laser microscopy. Second, using
identical transfection conditions, APP and furin were found to
accumulate in the Golgi apparatus, as demonstrated before (33, 34).
Third, the turnover (Fig. 4) and the secretory processing of
APP3 in CHO cells stably expressing human
APP770 was not affected by transfection of PS1, ruling out general
inhibitory effects on protein transport and processing. On the other
hand, the levels of endogeneous presenilin in COS-1 cells are
apparently very low. Only the combination of immunoprecipitation to
concentrate PS1 from detergent extracts of 10 × 106
cells, followed by immunoblotting using mAb PS1-3, allowed us to
detect endogeneous PS1 migrating at the same molecular weight as
transfected PS1. The disadvantage of this approach is that the
antibodies from the immunoprecipitation step interfere with the
consecutive detection of possible PS-aggregates in the immunoblotting step (Fig. 3C). This assay also precludes a dynamic analysis
of the metabolism of the presenilins using metabolic labeling and pulse-chase experiments. On the other hand, it allowed us to
demonstrate the presence or absence of particular antibody epitopes on
PS1 fragments. Importantly, the use of hydrophilic loop-specific
antibodies in the immunoprecipitation step resulted in the detection of
relative large amounts of, presumably proteolytic, fragments of PS1 in the 18-30-kDa range (Fig. 3C). The level of these fragments
was not, or only slightly, increased in COS-1 cells that overexpressed PS1, which explains why they were not readily detected in the transfection experiments. The proteolytic process involved is probably
easily saturated or even inhibited by PS overexpression (35, 36). Our
data therefore do not allow us to speculate any further on the exact
nature or the biological significance of this process. We can only
conclude that overexpression studies of the type used here are not
suitable to study this particular aspect of presenilin metabolism. On
the other hand, it is clear that untransfected cells (Fig. 3) and brain
tissue in vivo (36, 37) contain detectable amounts of intact
presenilin. Since this endogeneously expressed PS1 has the same
mobility as transfected PS1, the conclusion that transfected PS1 (and
PS2) are not subject to glycosylation, sulfation, glycosaminoglycan
modification, palmitoylation, or myristoylation also holds apparently
true for the endogeneous protein. These posttranslational modifications
are thereby also excluded as contributing to the formation of the high
molecular mass, SDS-resistant aggregates (100-250 kDa) in SDS-PAGE
electrophoresis. These aggregates were observed to a variable extent in
all our experiments, and were noticed by others using other cell types or brain tissue (36, 38, 39). Since the smears were detected with three
different antibodies recognizing three different epitopes, and by a
Myc-directed mAb using a Myc-tagged PS1 construct, the aggregates must
contain presenilin core proteins, alone or associated with unidentified
components. Denaturation of immunoprecipitates at 37 °C instead of
95 °C, resulted in less aggregates and increased amounts of the
45-kDa PS1 band (Fig. 3C). The aggregates therefore probably
consist mainly of oligomers of presenilins. It is unclear whether this
property to form SDS-resistant aggregates in vitro has any
physiological significance in vivo, but immuno-electron microscope observations suggest that clustering of presenilins also
occurs in the endoplasmic reticulum of untransfected
cells.2 It should be envisaged that in pathological
conditions exacerbated aggregation of presenilins could become a
problem. In this context, it should be mentioned that antibodies
against PS1 stain amyloid plaques in the brains of Alzheimer's disease
patients (40).
Importantly, the two point mutations causing Alzheimer's disease that
were studied here, i.e. PS1 Ala-246 Our study demonstrates furthermore that the presenilins are
phosphorylated on serine residues. Phosphorylation of PS1 and PS2 was
evident both in COS-1 cells and CHO cells, but the phosphorylation of
PS1 was less intense and more variable than of PS2. The observation that okadaic acid enhanced the PS1 phosphorylation suggests that PS1 is
more prone to phosphatase activity than PS2 (Fig. 8A). Protein kinase C is not responsible for PS phosphorylation, since neither stimulation of cells by phorbol esters nor purified kinase added to immunoprecipitated PS1 or PS2 resulted in increased labeling. It is therefore unlikely that the presenilins are directly involved in
the regulation of APP secretion by protein kinase C (46, 47).
Finally, we showed that the hydrophilic NH2-terminal and
the major loop domain of PS1 are exposed to the cytoplasmic side of the
endoplasmic reticulum membrane. Digitonin at low concentrations selectively permeabilizes the cell membrane, but not the endoplasmic reticulum membrane (29). Antibodies directed toward the amino-terminal domain of PS1 (Fig. 9) displayed similar immunofluorescent staining patterns in digitonin and saponin permeabilized cells. Control experiments with antibodies against a luminal epitope, i.e.
the KDEL endoplasmic reticulum retention signal (30, 31), showed that
digitonin did not permeabilize the endoplasmic reticulum membrane. For
the major hydrophilic loop domain, essentially identical results were
obtained. The conclusion therefore is that the two major hydrophilic
domains in PS1 are oriented to the cytoplasmic side of the endoplasmic
reticulum, which directs the search for candidate proteins interacting
with these domains toward the cytoplasm. Consistent with this
conclusion, antibodies against the second loop domain reacted with PS1
after saponin, but not after digitonin permeabilization. This not only
confirms the luminal localization of the recognized epitope, but also
validates the approach as it was used.
While more detailed studies, including electron microscopy and
enzymatic digestion protection assays, are required to settle definitively the orientation of the presenilins in the endoplasmic reticulum membrane, our results clarify already two important issues.
The first problem is the orientation of the first transmembrane domain
in PS1, which determines in fact the topology of all the following
transmembrane domains (46). The positive charge difference between the
15 carboxyl-terminal and the 15 amino-terminal residues flanking the
first membrane spanning domain suggests a luminal orientation of its
amino terminus (48). However, several exceptions to this rule are
known, mainly of proteins of which the amino-terminal domain contains
more then 17 charged amino acid residues (48). In PS1, this domain
contains 29 charged residues, which would explain its cytoplasmic
orientation. The second issue, which has been pointed out by others
before (9, 13, 17), is whether two putative hydrophobic stretches in
the PS amino acid sequence flanking the hydrophilic loop, can
additionally span the membrane, which would make the presenilins nine
transmembrane domain proteins. Our data are compatible with a
seven-transmembrane domain model with the amino-terminal domain and the
hydrophilic loop located in the cytoplasm (Fig. 10; see
also Note Added in Proof). In conclusion, the
current study has analyzed the biosynthesis and the subcellular localization of the presenilins and provides a basis for the further study of their cell biology and their possible interactions with integral membrane and cytoplasmic proteins such as APP or Tau, both
implicated in the pathogenesis of Alzheimer's disease (4).
We gratefully acknowledge the contribution of
expertise or materials of the following scientists: B. Greenberg; A. Roebroek, J. Creemers, and W. Van De Ven; and G. Schellenberg
(Geriatric Research, Education and Clinical Center, Seattle, WA).
An eight-transmembrane model, with
hydrophobic region VIII in the loop domain spanning the endoplasmic
reticulum membrane, is also compatible with these data. This would
result in a cytoplasmic orientation of the carboxyl-terminal domain of
PS1.
Experimental Genetics Group and
Molecular Oncology Group,
Scios Inc.,
Sunnyvale, California 94043
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
Note Added in Proof
REFERENCES
Glu or Cys-410
Tyr that cause Alzheimer's disease do not interfere with the biosynthesis or phosphorylation of presenilin 1. Finally, using low concentrations of
digitonin to selectively permeabilize the cell membrane but not the
endoplasmic reticulum membrane, it is demonstrated that the two major
hydrophilic domains of presenilin 1 are oriented to the cytoplasm. The
current investigation documents the posttranslational modifications and
subcellular localization of the presenilins and indicates that
postulated interactions with amyloid precursor protein metabolism
should occur in the early compartments of the biosynthetic pathway.
A4 amyloid peptide containing
carboxyl-terminal APP fragments and in an increased secretion of the
potentially neurotoxic
A4 peptide (1-3, 16). 63% of the amino acid
residues in the sequences of the two presenilins are conserved, which
strongly suggests that both proteins are involved in similar functions and have a similar pathogenic role in Alzheimer's disease. Based on
computer algorithms, seven membrane spanning domains have been defined
(9-15), although the possibility of nine transmembrane domains cannot
be ruled out at this moment (17). The amino-terminal domain and the
acidic loop domain, located between transmembrane domains six and
seven, are hydrophilic and can be alternatively spliced (9, 13). The
mutations that cause familial Alzheimer's disease are found all over
the protein, but the hydrophilic loop constitutes a "hot spot" with
nine different mutations described to date (5, 15). The biological
function of the presenilins remains essentially unknown, but,
interestingly, the 103 carboxyl-terminal amino acid residues of PS2 can
inhibit apoptosis in a "deathtrap" assay (18). Based on the
homology with Caenorhabditis elegans proteins, roles in
intracellular protein sorting and/or intercellular signal transmission
have been proposed as well (19, 20).
A4-(1-42) peptide in
fibroblasts obtained from patients with presenilin mutations would
support the amyloid hypothesis that postulates abnormal
A4 amyloid
peptide production and plaque formation as the pivotal event in the
pathogenesis of the disease (2, 21). However, the observed increases in
A4-(1-42) peptide production were relatively small, and the
suggested relationship between presenilin mutations and APP metabolism
should be further corroborated by experiments showing directly the
effect of presenilin mutations on APP processing in transfected cells
or in brains of transgenic animals. Moreover, other aspects of APP
metabolism such as the production of carboxyl-terminal APP fragments
(16) should be investigated in greater detail. The possibility that presenilins interact with the cytoskeleton or exert their effect via
apoptotic pathways should not be disregarded at this time (18).
In addition to addressing these questions, basic information is
needed on the subcellular localization, the posttranslational modifications, and the membrane orientation of the presenilins.
Constructs
Glu (FAD1) or Cys-410
Tyr
(NIH2) mutations have been described (9, 12). A Myc-tagged PS1 fragment
was generated by PCR using primers
5
-CGGGATCCATTATG
ACAGAGTTACCTGCACCG-3
and 5
-GATCACATGCTTGGCGCCATAT-3
(the sequence coding for the Myc
tag is underlined). This fragment was used to replace the NarI/BamHI restriction fragment of PS1 in pSG5.
The resulting cDNA codes for PS1 with the Myc tag (EQKLISEEDL)
immediately after the initiator methionine, as confirmed by cDNA
sequencing. Plasmids containing the cDNA for furin (22, 23) or
reticulon/NSP (24, 25) were kindly provided by Dr. J. Creemers, Dr. A. Roebroek, and Dr. W. Van De Ven (Center for Human Genetics, Leuven,
Belgium).
-32P]ATP, 2 mM Mg2+ during 60 min at 30 °C. The
precipitates were washed and analyzed in SDS-PAGE. Gels were
quantitatively analyzed using a PhosphorImager (Molecular
Dynamics).
Immunocytochemical Localization of Presenilin 1 in the Endoplasmic
Reticulum and the Golgi Apparatus
Fig. 1.
Immunofluorescent staining of PS1/2 in
untransfected fibroblasts. Fibroblasts were fixed with
formaldehyde and permeabilized with Triton X-100. Cells were incubated
with affinity-purified rabbit antibody 519 against the loop 2 domain of
PS1/2 (panel A) and mouse mAb against BiP (panel
B), followed by appropriate TRITC- and FITC-conjugated secondary
antibodies. Presenilin staining is observed in the cytoplasm as a
reticular pattern (A) that partially colocalizes with the
staining obtained with the mAb against the endoplasmic reticulum marker
BiP (B). Panel C, fibroblasts stained with
antibody 519 preabsorbed with peptide revealed only weak background
fluorescence.
[View Larger Version of this Image (32K GIF file)]
Fig. 2.
Immunofluorescent staining of PS1 in
transfected COS cells. COS-1 cells were double transfected with
plasmids coding for PS1 and reticulon/NSP (a and
b) or single transfected with plasmids coding for PS1,
amyloid precursor protein, or furin (c, d, and
e). Cells were fixed with formaldehyde and permeabilized with saponin. Immunostaining was done with antiserum B14 against the
amino-terminal domain of PS1 (a) or B16 against the loop
domain of PS1 (c), with a mix of monoclonal antibodies
MON160-162 against reticulon/NSP (b), or MON148 and 152 against furin (d), or mAb 22C11 against amyloid precursor
protein (e), followed by the appropriate TRITC-conjugated
(red) or FITC-conjugated (green) secondary
antibodies. Notice the fine reticular pattern obtained with PS1
antibodies in panels a and c, which distributes
with reticulon/NSP (panel b) in double transfected cells.
Panel e demonstrates that amyloid precursor protein is
located mainly in the Golgi apparatus, similar to furin (panel
d).
[View Larger Version of this Image (128K GIF file)]
Glu) or PS1 NIH2 (Cys-410
Tyr) were expressed (Fig. 3A).
These clinical mutations (12) therefore do not cause major alterations
in the biosynthesis of PS1 protein when overexpressed in COS-1 cells
(Fig. 3A). Independent experiments performed in CHO cells
confirmed completely these results (results not shown).
Fig. 3.
Biosynthesis of PS1 protein in COS-1 cells.
Panel A, COS-1 cells were transfected with constructs
containing the cDNA coding for wild type PS1 (PS1 WT),
PS1 FAD1 (Ala-246 Glu), PS1 NIH2 (Cys-410
Tyr), or with
expression vector alone (pSG5 control). Cells were
metabolically labeled with [35S]methionine for 4 h.
Cells were solubilized, and PS1 was immunoprecipitated with antiserum
B17 (1/250). Immunoprecipitates were resolved by 4-20% gradient
SDS-PAGE. Molecular size markers are indicated at the left
in kDa. Panel B, Western blotting of COS-1cells transfected with PS1 containing amino-terminally inserted Myc epitope. Cell extracts were electophoresed on a 4-20% gradient acrylamide gel and
transferred to a nitrocellulose membrane. mAb 9E10 against the Myc
epitope was used to detect PS1. Panel C, combined
immunoprecipitation and Western blotting of PS1 in untransfected COS
cells. Detergent extracts of 10 × 106 untransfected
COS-1 cells were made, and PS1 was immunoprecipitated using antiserum
B13 (N-term) or B17 (Loop). In lanes
1, 3, 5, and 7, 0.5 × 105 COS-1 cells transfected with wild type PS1 were added
to the untransfected cells. Immunoprecipitated material was resolved in
4-20% gradient SDS-PAGE and transferred to a nitrocellulose filter.
Filters were reacted with mAb PS1-3 and goat anti-mouse peroxidase-conjugated antibodies (lanes 1-4, indicated by
PS1-3) or with goat anti-mouse peroxidase conjugated
antibodies alone (lanes 5-8,
Co). The mobility
of intact PS1 is indicated by an arrow at the
right.
[View Larger Version of this Image (24K GIF file)]
Fig. 4.
Pulse-chase metabolic labeling of PS1 in
transfected CHO cells. CHO cells expressing stably human APP770
were transfected with PS1 and metabolically labeled with
[35S]methionine during 10 min in methionine-free medium.
Incorporated label was chased by incubation of the cells in complete
medium for the indicated time. PS1 was immunoprecipitated from the cell extracts using B13 (N-term) or B17 (Loop)
antiserum and resolved by 4-20% gradient SDS-PAGE (panel
A). APP was consecutively immunoprecipitated from the same cell
extracts using antibody 207 (45, 47) and analyzed in 6% homogeneous
SDS-PAGE. Notice the difference in turnover of transfected presenilin 1 and transfected APP. Panel C displays a quantitative
analysis (mean ± S.E.) of three independent pulse-chase
experiments of PS1. Signals were quantitated using a PhosphorImager.
The points represent the fraction of PS1 synthesized during a 10-min
pulse and remaining after the period of chase as indicated. The results
obtained for APP in two experiments are also displayed.
[View Larger Version of this Image (14K GIF file)]
Fig. 5.
Presenilins are not modified by
glycosaminoglycan chains. PS1 was immunoprecipitated using serum
B16 from metabolically labeled COS-1 cells transfected with wild type
PS1 and digested with heparinase, heparitinase, chondroitinase ABC, or
chondroitinase AC. Digested material was resolved in 12% homogeneous
SDS-PAGE. The activity of the heparinase and heparitinase was confirmed using proteoglycans immunopurified from fibroblasts (50). The activity
of chondroitinase ABC and AC was demonstrated using a test solution of
chondroitinesulfate which was clarified after addition of the enzymes.
Molecular size markers are indicated at the left in
kDa.
[View Larger Version of this Image (39K GIF file)]
Fig. 6.
The presenilin aggregates are
temperature-sensitive. Immunoprecipitated PS1 (antiserum B16) from
transfected COS cells was denatured for 10 min with SDS and
-mercapthoethanol at 95 °C (lanes 1 and 3)
or at 37 °C (lane 2). In lane 3 cell extracts were prepared in the presence of 5 mM dithiothreitol
(DTT). Notice the decrease of the 100-250-kDa aggregates
and the slightly increased intensity of the 45-kDa band in lane
2 as compared to lanes 1 and 3. Samples were
resolved on a 4-20% SDS-PAGE.
[View Larger Version of this Image (28K GIF file)]
Fig. 7.
Phosphorylation of PS1 and PS2 on serine
residues. Panel a, transfected COS-1 cells were
metabolically labeled using [32P]orthophosphate and
post-nuclear cell extracts were prepared (see "Materials and
Methods"). PS1 and PS2 were immunoprecipitated with antiserum B16
(PS1) or 519 (PS2). COS-1 cells transfected with pSG5 were labeled,
extracted, and immunoprecipitated with the same antibodies ().
Panel b, thin layer chromatography of hydrolyzed PS1 (45 kDa) and PS2 (50 kDa) identifying serine as the phosphorylated
residues. The mobility of phosphoserine (S), phosphothreonine (T), or phosphotyrosine (Y), as
detected by ninhydrin staining, is indicated by the dotted
lines.
[View Larger Version of this Image (31K GIF file)]
Fig. 8.
Analysis of PS1 and PS2 phosphorylation.
Panel A, staurosporin (300 nM) or okadaic acid
(300 nM) were added to the medium of transfected COS cells
after a 3.5-h incubation with [32P]orthophosphate. 30 min
later, cell extracts were made as in Fig. 4. PS1 or PS2 were
immunoprecipitated using, respectively, antiserum B17 or 519 and
resolved by a 4-20% gradient gel. Panel B, transfected CHO
cells were labeled with [32P]orthophosphate and PS was
immunoprecipitated using antiserum 519 (PS2 WT) or B16 (all
other lanes) and resolved by a 4-20% gradient gel.
[View Larger Version of this Image (30K GIF file)]
Fig. 9.
Cytoplasmic orientation of the amino-terminal
domain and the hydrophilic loop of PS1. COS-1 cells transfected
with PS1Myc (panels a and b) or with PS1 WT
(panels c-f) were permeabilized using
digitonin (panels a, c, and e) or
saponin (panels b, d, and
f) and immunostained with anti-Myc mAb 9E10
(panels a and b), with polyclonal antibody
519 against the second loop domain (panels c and
d), with polyclonal antibody B16 (panels e
and f) and with rat monoclonal antibody against the
KDEL sequence (panels a-f). PS1 immunoreactivity is
observed as red/yellow staining, while KDEL immunoreactivity
is green. Panels a and b show that the
amino terminus of PS1 is oriented to the cytoplasmic side of the
endoplasmic reticulum membrane. Cells that express PS1Myc stain
strongly with the amino-terminal domain directed 9E10 mAb both in
digitonin (a) or saponin (b) permeabilized cells.
When saponin is used, the untransfected cells are stained with the KDEL
antibody (green) since it reacts with endogenous
proteins (b). When digitonin is used, no staining is
observed (a), indicating that the endoplasmic reticulum
membrane is not permeable for antibodies under the conditions used. In
panels c and d, similar experiments are displayed
but using antiserum 519 against the second loop domain. Staining is
only observed when saponin is used, indicating a luminal orientation of
this domain (panel d). Panels e and
f demonstrate that antiserum B16 against the hydrophilic
loop domain of PS1 react both with digitonin (e) or saponin
(f) permeabilized cells, indicating a
cytoplasmic orientation of the hydrophilic loop.
[View Larger Version of this Image (91K GIF file)]
Glu (FAD1 kindred) and PS1 Cys-410
Tyr (NIH2 kindred), did not alter the biosynthesis or level of expression of the PS1 protein in COS-1 cells and CHO cells.
These mutations must cause therefore more subtle effects. The
possibility that they interfere with the fragmentation of PS1, as was
shown recently for two other mutations (35, 36), cannot be excluded
from the current data. Analysis of brains of transgenic mice expressing
the PS1 Ala-246
Glu mutant indicate, however, that at least this
mutation does not interfere with the proteolytic processing of
PS1.4 Since all PS mutations known to date
act in a dominant way in the pathogenesis of Alzheimer's disease, a
gain of function is most likely. Evidence that fibroblasts derived from
patients with presenilin mutations produce more and longer forms of the
A4 amyloid peptide suggests that the mutations exert their effect on
cellular metabolism or trafficking of amyloid precursor protein (21).
The localization of PS1 in the early compartments of the biosynthetic
pathway makes it unlikely that the presenilin mutations directly
influence
- and/or
-secretase activity, since these operate in
the late-Golgi and transport vesicles, at the cell surface and in
endosomes (27, 41-43). The possibility that PS mutants influence the
balance between amyloidogenic and non-amyloidogenic processing of APP
in an indirect way by changing its intracellular trafficking should now
be further explored in polarized Madin-Darby canine kidney cells and
neurons (16, 44, 45).
Fig. 10.
Localization of antibody epitopes and
orientation of PS1 in the endoplasmic reticulum membrane. PS1 is
inserted in the endoplasmic reticulum membrane with both its
amino-terminal (NH2-) and hydrophilic loop domain at the
cytoplasmic side. The localization of the peptides used to raise the
different antibodies used to study the topology of PS1 are indicated by
bars above the sequence (see also "Materials and
Methods" and Note Added in Proof).
[View Larger Version of this Image (13K GIF file)]
*
This investigation was supported by grants from the Fonds
voor Geneeskundig Wetenschappelijk Onderzoek (FGWO-NFWO), the Human Frontiers of Science Program (HFSP), the Action Program for
Biotechnology of the Flemish Government (VLAB), the Flemish Institute
for Biotechnology (VIB), the Katholieke Universiteit Leuven, the
Alzheimer's Society of Ontario, and the Medical Research Council of
Canada. 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.
§
Onderzoeksleider of the National Fund for Scientific Research,
Belgium (NFWO). To whom correspondence should be addressed: Campus
Gasthuisberg, O&N 06, 3000 Leuven, Belgium. Fax: 32-16-345871; E-mail:
bart.destrooper{at}med.kuleuven.ac.be.
1
The abbreviations used are: PS2, presenilin 2;
PS1, presenilin 1; APP, amyloid precursor protein; mAb, monoclonal
antibody; PBS, phosphate-buffered saline; TBS, Tris-buffered saline;
FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine
isothiocyanate; PAGE, polyacrylamide gel electrophoresis; Pipes,
1,4-piperazinediethanesulfonic acid; NSP, neuroendocrine-specific
protein.
2
P. Fraser, unpublished results.
3
B. De Strooper and K. Craessaerts, unpublished
results.
4
D. Moechars, B. De Strooper, and F. Van Leuven,
unpublished results.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.