Received for publication, August 10, 2000, and in revised form, March 5, 2001
-Secretase is a membrane-associated
endoprotease that catalyzes the final step in the processing of
Alzheimer's
-amyloid precursor protein (APP), resulting in the
release of amyloid
-peptide (A
). The molecular identity of
-secretase remains in question, although recent studies have
implicated the presenilins, which are membrane-spanning proteins
localized predominantly in the endoplasmic reticulum (ER). Based on
these observations, we have tested the hypothesis that
-secretase
cleavage of the membrane-anchored C-terminal stump of APP
(i.e. C99) occurs in the ER compartment. When recombinant
C99 was expressed in 293 cells, it was localized mainly in the Golgi
apparatus and gave rise to abundant amounts of A
. Co-expression of
C99 with mutant forms of presenilin-1 (PS1) found in familial
Alzheimer's disease resulted in a characteristic elevation of the
A
42/A
40 ratio, indicating that the
N-terminal exodomain of APP is not required for mutant PS1 to influence
the site of
-secretase cleavage. Biogenesis of both
A
40 and A
42 was almost completely
eliminated when C99 was prevented from leaving the ER by addition of a
di-lysine retention motif (KKQN) or by co-expression with a
dominant-negative mutant of the Rab1B GTPase. These findings indicate
that the ER is not a major intracellular site for
-secretase
cleavage of C99. Thus, by inference, PS1 localized in this compartment
does not appear to be active as
-secretase. The results suggest that
presenilins may acquire the characteristics of
-secretase after
leaving the ER, possibly by assembling with other proteins in
peripheral membranes.
 |
INTRODUCTION |
Amyloid
-peptide
(A
)1 is the major
molecular component of the cerebral amyloid plaques associated with
Alzheimer's disease. The cellular pathways involved in the biogenesis
of A
have been the subject of intense investigation since the
discovery that A
originates from intracellular endoproteolytic
processing of a type I membrane-spanning glycoprotein termed amyloid
precursor protein (APP) (1-4). Extensive studies have established that APP can be processed via two alternative routes, one of which yields
the 4-kDa A
, whereas the other yields a truncated non-amyloidogenic peptide (p3) (5, 6). In most cells the non-amyloidogenic pathway
predominates. The first step involves the cleavage of APP
within the A
domain by a protease termed
-secretase
(7-9). After release of the N-terminal exodomain, the residual
83-amino acid membrane-spanning C-terminal fragment is further
processed by another protease termed
-secretase to remove the
cytoplasmic tail and generate p3 (6, 10). Because the latter cleavage occurs within the predicted membrane spanning region of APP (11-13),
-secretase is generally thought to be an intramembrane protease. In
the alternative amyloidogenic pathway, APP is initially cleaved proximal to the A
sequence by
-secretase, leaving a 99-amino acid
C-terminal fragment (C99) that contains the intact A
sequence and
the cytoplasmic tail (1, 14, 15). Thus, when
-secretase cuts the
latter substrate, A
is released. Cells can generate distinct species
of A
that differ in chain length (e.g. A
40 and A
42). It remains unclear whether the different forms
of A
arise through the action of separate
-secretases (11, 16, 17) or instead reflect the ability of a single enzyme to cleave C99 at
more than one site (18). Although A
40 is produced in greater abundance than A
42, the longer peptide has
particular significance for Alzheimer's disease pathology, since it
readily forms insoluble aggregates and accumulates in neuritic plaques (19-21).
A number of reports have provided information about the subcellular
compartments where APP is cleaved by the
- and
-secretases. Metalloproteases that function as
-secretases (22, 23) appear to
operate on APP at or near the cell surface (9, 24, 25). On the other
hand,
-secretase cleavage occurs predominantly in intracellular
membrane compartments such as the Golgi apparatus (26-28) and
endosomes (29-31). The latter findings have been verified in studies
of newly identified aspartic proteases that function as
-secretase
(32-35). The identity of
-secretase remained elusive until a recent
series of studies implicated the serpentine membrane-spanning proteins,
presenilin-1 (PS1) and presenilin-2 (PS2), as catalytic components of
this enzyme. Mutations in the presenilins have been linked to familial
forms of Alzheimer's disease that are characterized by elevations of
the A
42/A
40 ratio (36-39). It now
appears that presenilins may be unique aspartyl proteases that can
function as
-secretase (40-43) or play an essential role in
regulating
-secretase activity (44, 45). The subcellular location of the
-secretase cleavage step has not been established definitively, but several studies (46-48) have suggested that it occurs in the ER,
where most of the presenilin is localized (49-51). If this is true, it
would imply that the C83 and C99 fragments generated by
-secretase
and
-secretase in the plasma membrane and Golgi/endosomal compartments, respectively, must be transported back to the ER for the
final cleavage by
-secretase. However, the evidence supporting
-secretase processing of APP in the ER is not definitive. For example, in neurons A
42 is found in the ER, whereas
A
40 is localized in the trans-Golgi network
(52), but it is difficult to determine if the steady-state distribution
of the A
peptides reflects the primary site where they are
generated. Other evidence pointing to the ER as a site of
-secretase
activity centers around the observation that intracellular production
of total A
(46) or A
42 (47) can continue unchecked
when ER
Golgi transport is blocked by brefeldin A. However, the
interpretation of these observations is complicated by the fact that
brefeldin A causes mixing of various membrane compartments,
i.e. the Golgi apparatus merges with the ER and the
trans-Golgi network fuses with endosomes (53, 54). A final
line of evidence supporting the existence of
-secretase activity in
the ER comes from a report by Soriano et al. (48), who found
that secretion of A
40 and A
42 was not
markedly reduced when APP was retained in the ER by fusing the
extracellular and transmembrane domains with the cytoplasmic region of
the T cell antigen receptor CD3
chain. However, from this study it
is not entirely clear whether the susceptibility of the chimeric
APP/CD3
substrate to the ER protease accurately represents the
behavior of the native APP or C99. Indeed, there is considerable
evidence that the cytoplasmic domain of APP, which was removed in the
aforementioned study, contains important sorting determinants (55) and
sequence elements required for interaction with proteins like X11 (56) and nicastrin (57), which may influence APP processing and A
secretion.
In the present study we examined directly the contribution of the ER
compartment to
-secretase processing of the membrane-anchored C99
stump of APP in transfected cells, using two different strategies. The
first strategy involved the addition of minimal tetrapeptide extensions
to the C terminus of C99 to either retain the protein in the ER (KKQN)
or allow its trafficking to the Golgi and other distal compartments
(QLQN). The second strategy entailed the co-expression of C99 with
wild-type or dominant-negative mutant versions of the Rab1B GTPase,
which controls vesicular transport of proteins from the ER to the Golgi
compartment. The results show that when C99 was allowed to leave the
ER, it was a good substrate for
-secretase and responded to
co-expression of PS1 FAD variants with typical elevations of the
A
42/A
40 ratio. In contrast, cellular
production of both A
40 and A
42 was almost
completely eliminated when C99 was retained in the ER, where most of
the expressed PS1 was localized. These findings lead us to conclude
that the ER is not a major intracellular site for
-secretase
activity and, by inference, that the large pool of PS1 that is
typically localized in this compartment is not catalytically active as
-secretase. The results also indicate that the interactions that
occur between PS1 and APP to influence the site of
-secretase
cleavage (i.e. the production of long versus
short forms of A
) do not require the N-terminal exodomain of APP.
 |
EXPERIMENTAL PROCEDURES |
Expression Vectors--
The pohCk751sw mammalian
expression vector has been described previously (58). It encodes the
Swedish variant of APP751 (SwAPP) which contains a dual
amino acid change (K651N,M652L) that increases the
susceptibility of the protein to processing by
-secretase (59-61).
The cDNA encoding SwAPP was modified by standard PCR techniques
using Pfu polymerase (Stratagene) and appropriate primers so
that the last four amino acids of the protein were changed from QMQN to
KKQN. By using the pohCK751sw template, PCR was also used
to generate cDNA constructs encoding the APP-derived C99 fragment,
with the addition of a Myc epitope (EQKLISEEDL) followed by either QLQN
or KKQN at the C terminus. In generating the C99 DNA
constructs, the oligonucleotide primers were designed to add a
KpnI restriction site and an ATG start codon to the 5' end
of the cDNA and a BamHI site to the 3' end. The PCR
products were digested with KpnI and BamHI and
ligated into the pCMV5 mammalian expression vector (62), resulting in
plasmids designated pCMV5-C99(QLQN) and
pCMV5-C99(KKQN).
To generate Sindbis virus expression vectors, the cDNA inserts
encoding C99(QLQN) and C99(KKQN) were excised from the pCMV5 vectors
with KpnI and BamHI and cloned into the pKF3
enforcement vector (Takara/PanVera, Madison, WI) to pick up
XbaI and SmaI sites at the 5' and 3' ends,
respectively. The latter enzymes were then used to excise the C99
inserts and clone them between the XbaI and StuI
sites of the pSinRep5 vector (Invitrogen). The final constructs were
designated pSinRep5-C99(QLQN) and
pSinRep5-C99(KKQN). All constructs
were determined to be correct by automated DNA sequencing, using an ABI
377 system.
The mammalian expression vectors, pCMV5Rab1B(wt)
and pCMV5Rab1B(N121I), which encode Myc
epitope-tagged versions of Rab1B, have been described previously (58,
63). The vectors, pohCkPS1, pohCkPS1(M146L), and pohCkPS1(L286V),
which encode wt and FAD variant forms of presenilin-1, have also been
described in an earlier report (64).
Transfection of Cultured 293 Cells--
Human embryonal kidney
cells (line 293) were obtained from American Type Culture Collection
(Manassas, VA) and were maintained at 37 °C in a 5% CO2
atmosphere in Dulbecco's modified Eagle's medium (DMEM) containing
10% (v/v) fetal bovine serum (FBS). Tet-offTM 293 cells were obtained
form CLONTECH (Palo Alto, CA) and maintained in
DMEM with 10% tetracycline-free FBS and 100 µg/ml geneticin (G418).
Cells were plated in 60-mm dishes at 5 × 105
cells/dish on the day before transfection.
In studies of SwAPP or C99 expression and processing, cells were
transfected with pohCk751sw, pohCk751sw(KK),
pCMV5-C99(QLQN) or
pCMV5-C99(KKQN), using LipofectAMINE
Plus reagent according to the protocol recommended by the manufacturer
(Life Technologies, Inc.). The same approach was used in studies
involving co-transfection of cells with
pCMV5-C99(QLQN) and either
pCMV5Rab1B(wt) or
pCMV5Rab1B(N121I). For some studies,
the Myc-tagged Rab1B(wt) and
Rab1B(N121I) cDNA constructs were subcloned
into pTRE (CLONTECH) to permit suppression of gene
expression in 293 Tet-off cells by addition of doxycycline.
In studies aimed at assessing the effects of PS1 on C99 processing,
parallel cultures were co-transfected with
pCMV5-C99(QLQN) combined with pohCkPS1,
pohCkPS1(M146L), or pohCkPS1(L286V), using Superfect transfection
reagent (Qiagen, Valencia, CA) according to the protocol recommended by
the manufacturer.
Protein Expression in Cultured Neurons--
Cultures of NT2N
neurons were derived from Ntera2 teratocarcinoma cells (65) by
treatment with retinoic acid and mitotic inhibitors as described
previously (66). The Sindbis virus system (67, 68) was used to express
C99(QLQN) or C99(KKQN) in NT2N neurons. In brief,
pSinRep5-C99(QLQN) and
pSinRep5-C99(KKQN) were linearized with
XhoI, and RNA was generated by in vitro
transcription. The RNA was introduced into baby hamster kidney cells by
electroporation, along with dh26S helper virus RNA. The culture medium
enriched in recombinant virus was harvested 24 h after the
co-transfection, and the virus was used to infect NT2N cells. For each
60-mm culture, 150 µl of virus-conditioned medium was mixed with 300 µl of minimal essential medium with 1% FBS. Cells were incubated for
1 h at 37 °C, and then 4 ml of fresh minimal essential medium + 1% FBS was added. Cells and medium were harvested for assay of C99 and A
after 15 h.
Metabolic Labeling and Immunoprecipitation of APP--
Beginning
24 h after transfection, 293 cells were pulse-labeled for 15 min
at 37 °C with 1 ml of methionine-free DMEM containing 100 µCi of
[35S]methionine/cysteine (Trans-label, 1100-1200
Ci/mmol, ICN Inc.). The cells were then washed twice with PBS and
subjected to a 45-min chase in DMEM containing 10% FBS, 2 mM methionine, and 2 mM cysteine. Cells were
harvested from parallel cultures at the end of the pulse and chase
periods, and APP was immunoprecipitated from cell lysates as described
previously (58). Immature and mature forms of APP were separated by
SDS-PAGE and visualized by fluorography by using established procedures
(69).
Immunoblot Analysis--
For immunoblot analysis of APP or PS1,
cells were lysed in PBS containing 1% (v/v) Triton X-100. Aliquots of
cell lysate or conditioned medium were mixed with an equal volume of
2× sample buffer containing 8 M urea, 4% SDS w/v, 10%
2-mercaptoethanol v/v, 20% glycerol v/v, and 125 mM
Tris-HCl, pH 6.8. Proteins were subjected to SDS-PAGE, using 10%
polyacrylamide gels for analysis of PS1 and 6.5% polyacrylamide gels
for analysis of APP. For analysis of C99 and Rab1B, samples were
prepared as described above, except that SDS-PAGE was performed on
pre-cast Tricine 10-20% polyacrylamide gradient gels (NOVEX, San
Diego, CA). Proteins were transferred to Immobilon-P (Millipore Corp,
Bedford, MA), and the membranes were preincubated for 1 h in
blotting solution (PBS containing 5% w/v powdered milk and 0.05% v/v
Tween 20). Immunoblotting was carried out as described previously (27,
58). Intracellular APP and total secreted forms of APP
(s-APPtotal) were detected with monoclonal antibody 8E5
(70). The soluble exodomain fragments released from SwAPP as a result
of
-secretase cleavage (s-APP
) or
-secretase cleavage
(s-APP
) were detected with antibodies 2H3 (27) and SW192 (31),
respectively. PS1 was detected with an antibody against residues 1-79
from the N terminus of the protein (71), purchased from
Zymed Laboratories Inc. Laboratories (South San
Francisco, CA). C99 was detected with the following monoclonal antibodies: 9E10, which recognizes the Myc epitope (Calbiochem), 6E10,
which recognizes residues 1-12 of the A
sequence (Senetek, Napa,
CA), or 13G8, which recognizes the last 20 residues at the C terminus
of APP (gift from P. Seubert and D. Schenk, Elan Pharmaceuticals, South
San Francisco, CA). Rab1B was detected with an affinity-purified polyclonal antibody from Zymed Laboratories Inc..
Standard chemiluminescent detection was carried out with the ECL kit
(Amersham Pharmacia Biotech), using horseradish peroxidase-conjugated
goat anti-rabbit IgG or goat anti-mouse IgG (Bio-Rad) at 1:3000 v/v dilution.
For high sensitivity immunoblot analysis of total A
, an aliquot of
conditioned medium or cell lysate was diluted 3:1 with 4× concentrated
SDS sample buffer and loaded on a Tricine 10-20% polyacrylamide
gradient gel. Proteins were transferred to Immobilon-P at 4 °C at
400 mA for 3 h, with a transfer buffer consisting of 12 mM Tris, 96 mM glycine, 20% methanol, pH 8.3. The membranes were then incubated with the 6E10 antibody followed
sequentially by biotinylated goat anti-mouse IgG and streptavidin
conjugated to horseradish peroxidase(Bio-Rad). Bound HRP was detected
with SuperSignal reagent (Pierce).
ELISAs for A
--
Conditioned medium (2 ml total) was
collected from each culture 24 h after transfection. The
concentrations of A
peptides were determined by sandwich-type ELISAs
as described (27), with the following modifications. Polystyrene
96-well assay plates (Costar) were coated with the antibody specific
for A
40, 2G3, at 10 µg/ml, or with the antibody
specific for A
42, 21F12, at 5 µg/ml. Samples were
incubated on the plates overnight at 4 °C. The plates were washed
with Tris-buffered saline containing 0.05% Tween 20 (TTBS) and then
incubated with biotinylated reporter antibody. The 3D6 monoclonal
antibody (specific for residues 1-5 of A
) was used as the reporter
in the assays of cells transfected with full-length APP (Fig. 3).
Monoclonal antibody 266 (specific for residues 13-28 of A
) was used
as the reporter in the studies where C99 was co-expressed with PS1
(Fig. 11). The 6H9 monoclonal antibody (see below) was used as the
reporter in the assays of medium from cells expressing C99 with or
without an ER-retention signal (Fig. 7). The immunogen for the 6H9
monoclonal antibody was prepared by coupling a peptide encoding
A
-(17-28) plus a C-terminal extension of Gly-Gly-Cys to
sheep anti-mouse IgG using
-maleimidocaproic acid
N-hydroxysuccinimide. 100 µg of the immunogen was injected
into mice in Freund's adjuvant, and hybridomas were generated from the
highest titer mouse using standard methods. The reporter antibodies
were added at a final concentration of 0.25 µg/ml in specimen diluent
(17 mM NaPO4, pH 7.4, 7.7 mM sodium azide, 100 mM NaCl, 0.05% Triton X-405, 6 mg/ml bovine
serum albumin). The plates were incubated at room temperature for
1 h and washed with TTBS. Streptavidin-alkaline phosphatase (Roche
Molecular Biochemicals, 1000 units/ml) was added at a dilution of
1:1000 in specimen diluent, and the plates were incubated for 1 h
at room temperature. The plates were then washed in TTBS, and exposed to fluorescent substrate as described (72). Synthetic A
-(1-40) or
-(1-42) peptides were used as standards. The signals were read in a
CytoFluor 2350 (Millipore Corp., Bedford, MA) at 360/460 nm.
Immunofluorescence Microscopy--
Cells were plated on
laminin-coated coverslips in 60-mm dishes and transfected with the
indicated plasmids as described earlier. After 24 h, the cells
were fixed for 15 min in 3% (w/v) paraformaldehyde in PBS and
permeabilized with 0.1% (v/v) Triton X-100 in PBS for 2 min. Cells
were blocked with 0.1% (w/v) bovine serum albumin in PBS for 30 min
and subjected to immunofluorescence staining as described previously
(58). Detection of expressed C99(QLQN) or C99(KKQN) was accomplished
with the 9E10 mouse monoclonal anti-Myc antibody followed by Texas
Red-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR).
Where indicated, cells expressing C99 were also incubated with rabbit
polyclonal antibodies against either calreticulin (Affinity BioReagents
Inc, Golden, CO) or Rab6 (Santa Cruz Biotechnology, Santa Cruz, CA),
followed by FITC-conjugated goat anti-rabbit IgG (Sigma). In one case,
cells expressing C99 were incubated with a rabbit polyclonal anti-Myc
antibody (Upstate Biotechnology, Inc., Lake Placid, NY) combined with a
mouse monoclonal antibody H4B4 against the lysosomal membrane protein
LAMP-2 (73), obtained from the Developmental Studies Hybridoma Bank,
University of Iowa. To visualize PS1, cells were incubated with an
affinity-purified rabbit IgG (Zymed Laboratories Inc.,
South San Francisco, CA) that recognizes the full-length protein and
the N-terminal fragment (71). The secondary antibody was Texas
Red-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR).
Where C99 was co-expressed with PS1, C99 was detected with the 9E10
monoclonal antibody against the Myc epitope, followed by
FITC-conjugated goat anti-mouse IgG (Sigma). Photomicrographs were
taken with a Nikon Eclipse 800 fluorescence microscope equipped with a
digital camera. Images were merged and pseudo-colored through the use
of ImagePro software (Phase 3 Imaging Systems, Glen Mills, PA).
Proteolytic Cleavage of SREBP--
For studies of the
intracellular endoproteolytic processing of sterol regulatory
element-binding protein type 2 (SREBP2), the mammalian expression
vector pTK-HSV-BP2 was obtained from the American Type Culture Collection.
The latter encodes an N-terminal HSV epitope-tagged version of SREBP2
under the control of the thymidine kinase promoter (74). Parallel 10-cm
dishes of 293 cells were co-transfected with 2 µg of pTK-HSV-BP2
combined with either 2 µg of pCMV5Rab1B(wt) or
pCMV5Rab1B(N121I). Immediately after
transfection, cultures were fed with DMEM containing 10% delipidated
FBS and 25 µM lovastatin to promote sterol depletion and
SREBP cleavage. Cells were collected 20 h after transfection, with
addition of 25 µg/ml
N-acetyl-leucinal-luecinal-norleucinal to the medium
3.5 h prior to harvest. Cells from four dishes were combined and
fractionated into membrane components and nuclear extracts as described
by Hua et al. (75). Aliquots of protein from the membrane
fraction (100 µg) and nuclear extract (185 µg) were subjected to
SDS-PAGE and immunoblot analysis using a monoclonal antibody against
the HSV epitope (Novagen Inc., Madison, WI) to detect the SREBP2
full-length protein and the N-terminal fragment. Immunoblots were
scanned and quantified with a Kodak 440CF Image Station.
 |
RESULTS |
Addition of a Di-lysine Motif to SwAPP Prevents
Golgi-dependent Maturation of the Protein--
Numerous
studies have established that type I transmembrane proteins containing
C-terminal KKXX motifs are retrieved from early Golgi and
intermediate compartments and retained in the ER (76-78). To determine
whether the addition of such a motif to the cytoplasmic tail of APP
would prevent the protein from being transported beyond the
cis-Golgi compartment, plasmids encoding SwAPP with the
normal C-terminal ending (QMQN) or a modified sequence, KKQN, were
transfected into 293 cells, and the transiently expressed proteins were
monitored by pulse-chase analysis of the
[35S]methionine-labeled protein. Radiolabeled APP was
immunoprecipitated from parallel transfected 293 cell cultures, either
immediately after a 15-min pulse with [35S]methionine or
after a 45-min chase to allow time for nascent APP to undergo ER
Golgi transport and oligosaccharide maturation. Consistent with
previous observations, SwAPP migrates as two major bands with the
slower form (~130 kDa) representing the mature protein that has
undergone O-glycosylation in the medial-late Golgi
compartment and the faster form (~108 kDa) representing the immature
form of the protein that is localized predominantly in the ER (58, 79,
80). As shown in Fig. 1A, the
nascent 108-kDa form of SwAPP was the predominant radiolabeled protein detected in the cultures immediately after the pulse. Conversion of
immature SwAPP to the mature form was readily detected after the 45-min
chase in the cells overexpressing SwAPP with the normal C-terminal
sequence but not in the cells expressing the protein with the di-lysine
ER retention motif. As determined previously for SwAPP (58), the
108-kDa form of SwAPP with the KKQN motif was sensitive to digestion
with endoglycosidase H (not shown), indicating that it contains
N-linked high mannose oligosaccharides typical of immature
glycoproteins that have not yet been trimmed by
1,2-mannosidase II
in the Golgi compartment (81). The ability of the KKQN motif to retain
SwAPP in the ER was confirmed by SDS-PAGE and immunoblot analysis to
monitor steady-state levels of mature versus immature
protein (Fig. 1B). When the C terminus of SwAPP was changed
to KKQN, the amount of the mature form was substantially reduced.

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Fig. 1.
Addition of a di-lysine ER retention motif
prevents Golgi-dependent maturation of SwAPP in 293 cells. A, cells were transiently transfected with
vectors encoding SwAPP or the same protein in which the last amino
acids were altered to form a KKQN motif. Parallel cultures that were
not transfected (lanes marked None) were used to determine
the background levels of endogenous APP expressed in the 293 cells.
Pulse-chase analysis of APP processing was performed 24 h later.
Cells were harvested immediately after pulse-labeling with
[35S]methionine (0 min) or after the chase
with unlabeled methionine (45 min) as indicated. APP was
immunoprecipitated and subjected to SDS-PAGE and
fluorography. The positions of the mature O-glycosylated
form of APP (m) and the immature protein (i) are
indicated by the arrows. B, immunoblot analyses
were performed on aliquots of the cell lysates from the cultures
harvested at the end of the chase period to determine the steady-state
levels of immature and mature intracellular APP, using the 8E5
monoclonal antibody, which recognizes all forms of APP.
|
|
Addition of a Di-lysine Motif to SwAPP Inhibits the Secretion of
Soluble Exodomain Fragments and A
Peptides--
As mentioned
earlier, cleavage of APP by
-secretase occurs at the cell surface,
whereas alternative cleavage by
-secretase appears to take place in
late Golgi or endosomal compartments. Hence, one would predict that
retention of SwAPP in the ER would prevent the substrate from coming
into contact with both
- and
-secretase. To test this prediction,
we compared the relative amounts of the soluble exodomain fragments
(s-APP) released into the medium from cells expressing SwAPP with or
without the C-terminal di-lysine motif (Fig.
2). The results show that there was an
85-90% reduction in the amount of s-APP
and s-APP
released from
the cells expressing SwAPP with the ER retention signal. Essentially identical results were obtained when the immunoblot values for secreted
exodomain fragments were normalized to the values for total SwAPP
expressed in the transfected cells (not shown).

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Fig. 2.
Addition of the ER-retention signal to SwAPP
causes a decrease in secretion of soluble exodomain fragments (s-APP)
released by -secretase or
-secretase. HEK293 cells were transfected with
vectors encoding SwAPP with the normal C-terminal ending
(SwAPP) or the di-lysine ER retention motif
(SwAPP-KKQN). Parallel cultures that were not transfected
(None) were used to assess background levels of s-APP in the
medium. The medium was removed from all cultures 24 h after
transfection. Immunoblot assays were performed on equal aliquots of
medium using the following: A, monoclonal antibody 8E5,
which detects both s-APP and s-APP (s-APPTotal);
B, polyclonal antibody SW192, which detects only s-APP
derived from the Swedish variant of APP; or C, monoclonal
antibody 2H3, which detects s-APP . The results (mean ± S.E.)
from separate determinations on three cultures are shown in the
bar graphs next to the representative blots. Intracellular
levels of expressed SwAPP and SwAPP(KKQN) were very similar, so that
essentially identical results were obtained when the extracellular
s-APP values were normalized to the intracellular levels of SwAPP (not
shown).
|
|
Because the preceding findings indicated that SwAPP(KKQN) was not
efficiently cleaved by
-secretase, we postulated that the amount of
A
produced by cells expressing this construct would be significantly
diminished. This hypothesis was tested by measuring the concentrations
of A
released into the culture medium using ELISAs specific for long
and short forms of the peptide. As shown in Fig.
3, addition of the KKQN motif to SwAPP
caused a 75-80% decrease in the amount of A
40 released
into the medium. The amount of A
42 was similarly
reduced, so that percentage of A
42 relative to
A
40 (~5%) was not significantly altered.

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Fig. 3.
Addition of ER retention signal to SwAPP
causes a reduction of both A 40 and
A 42. HEK293 cells were
transfected with vectors encoding SwAPP with the normal C-terminal
ending (SwAPP) or the di-lysine ER retention motif (SwAPP-KKQN).
Aliquots of conditioned medium were subjected to ELISA to determine the
concentrations of A 40 and A 42. Each value
is a mean (±S.E.) from assays performed on two parallel
cultures.
|
|
Retention of the APP C99 Domain in the ER Decreases the Production
of A
--
The foregoing studies indicate that the biogenesis of
A
from full-length SwAPP is greatly diminished when the latter is
retained in the ER. The decline in A
seen in these studies is
probably due to the reduced contact of the SwAPP substrate with active
-secretase, based on the parallel reduction in the output of s-APP
(Fig. 2B). The small amount of residual A
produced in the cells overexpressing SwAPP(KKQN) could be related to
incomplete retention of the protein in the ER, possibly due to
saturation of coatomer binding sites (82). Alternatively, it could
reflect the presence of
-secretase activity in the ER, possibly
allowing a small pool of SwAPP to be transported to the
-secretase
compartment after removal of the C-terminal tail containing the KKQN
motif. In light of these uncertainties, we felt it was important to
obtain a more direct assessment of the activity of
-secretase in the ER. Toward this end, we generated a C99(KKQN) construct that
corresponds to the C-terminal portion of APP that remains after
-secretase cleavage. To facilitate detection, we added a short Myc
epitope tag to the C terminus, followed by the ER retention motif, KKQN (Fig. 4A). To verify that the
C-terminal additions did not in themselves affect
-secretase
cleavage, an identical construct was generated with QLQN instead of
KKQN following the Myc tag, i.e. C99(QLQN). Immunoblot
analyses using different antibodies to detect epitopes on the
N-terminal or C-terminal ends of the molecule indicated that both
C99(QLQN) and C99(KKQN) were transiently expressed in an intact form at
similar levels in 293 cells (Fig. 4B). However, the cells
expressing the construct with the di-lysine ER retention motif showed a
striking decrease in the amount of A
deposited into the culture
medium (Fig. 4C). To verify that the decrease in
extracellular A
reflects a decrease in the biogenesis of the peptide
rather than a block in its secretion, we also measured intracellular
A
in cells expressing the two C99 constructs (Fig. 4C).
Consistent with the absence of A
in the medium, intracellular A
was nearly undetectable in cells expressing C99(KKQN).

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Fig. 4.
Addition of an ER retention signal prevents
A production from the C99 C-terminal fragment
of APP. A, general description of the C99 protein
constructs showing relative positions of the A sequence,
-secretase cleavage sites, Myc epitope, and terminal tetrapeptide
extensions. The domains recognized by the 6E10 and 13G8 antibodies are
also indicated. B, immunoblots showing comparative
expression levels of C99(QLQN) and C99(KKQN) in equal aliquots of cell
lysate from 293 cells harvested 24 h after transfection. The
antibodies used are indicated below each panel. Standard ECL
was used for detection of bound IgG. C, immunoblots with the
6E10 antibody show that total A was reduced in the medium and cell
lysate from cells expressing C99(KKQN), compared with cells expressing
C99(QLQN). The samples consisted of 2% of the total cell lysate and
0.05% of the 48-h conditioned medium. The standard was 4 ng of
A 40 (Quality Controlled Biochemicals, Inc., Hopkinton,
MA). In the panel on the left side, equal aliquots of cell
lysate were blotted with the 6E10 antibody to verify that expression
levels of C99(KKQN) and C99(QLQN) were comparable. Bound IgGs were
detected using the high sensitivity method described under
"Experimental Procedures." The A signals cannot be compared
directly with the C99 signals, because of variations in the exposure
time (1 s for C99 and 2 min for A ). The results are representative
of two separate experiments. D and E, NT2N
neurons were infected with Sindbis virus encoding C99(QLQN), C99(KKQN),
or virus without insert (none), as indicated. The cells were
then labeled for 7 h with [35S]methionine (100 µCi/ml), and A was immunoprecipitated from 0.5 ml of culture
medium (E). To verify that similar levels of C99 expression
were obtained in the cells, equal aliquots of cell lysate were
subjected to immunoblot assay, using the 13G8 antibody (D).
Similar results were obtained in two separate experiments.
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To confirm that these observations were pertinent to human neurons as
well as 293 cells, the C99(QLQN) and C99(KKQN) constructs were
subcloned into Sindbis virus vectors and expressed in stable differentiated NT2N neurons. As shown in Fig. 4, D and
E, radiolabeled A
was clearly detected above background
levels in medium from NT2N cultures expressing C99(QLQN) but not in
cultures expressing similar amounts of C99(KKQN).
Subcellular Localization of C99--
When 293 cell lysates were
subjected to differential centrifugation, the expressed C99(QLQN) and
C99(KKQN) polypeptides were found predominantly in membrane-enriched
particulate fractions (not shown). However, because C99 lacks the
glycosylation sites of the APP exodomain, it was not possible to verify
retention of C99(KKQN) in the ER by monitoring
Golgi-dependent post-translational modifications of the
protein. As an alternative, we compared the subcellular distribution of
C99(QLQN) and C99(KKQN) with the distribution of an ER marker protein,
calreticulin (83), by immunofluorescence analysis. As shown in Fig.
5A, C99(QLQN) was concentrated
in a discrete region adjacent to the nucleus, with almost no overlap with calreticulin. In contrast, C99(KKQN) was localized in a
perinuclear ring and a diffuse reticular network throughout the
cytoplasm. The staining pattern of C99(KKQN) was nearly identical to
that of the calreticulin marker (Fig. 5A). In the absence of
the ER retention signal, most of the overexpressed C99(QLQN) was
co-localized with Rab6 GTPase, which is known to function within the
medial and trans-Golgi compartments (84, 85) (Fig. 5B).
Consistent with its retention in the ER, C99(KKQN) showed little or no
co-localization with Rab6 (Fig. 5B). It should be noted that
in the merged image of C99(QLQN) with Rab6 (Fig. 5B), a
small portion of the C99 did not overlap with the Golgi marker. This
non-Golgi pool of C99 may be localized in the endocytic compartment,
based on partial overlap of C99(QLQN) with LAMP-2, a marker for late
endosomes and lysosomes (73) (Fig. 5C).

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Fig. 5.
Immunofluorescence localization of C99
proteins with or without an ER retention motif. Cells were fixed
24 h after being transfected with expression vectors encoding
either C99(QLQN) or C99(KKQN) (see Fig. 4). Localization of C99 was
determined in A and B with a mouse monoclonal
antibody against the Myc epitope and in C with a rabbit
polyclonal antibody against the Myc epitope. Texas Red-conjugated goat
anti-mouse or anti-rabbit IgGs were used to detect the anti-Myc primary
antibodies (red). Cells were also incubated with
affinity-purified rabbit polyclonal antibodies against calreticulin
(A) or Rab6 (B), or with a mouse monoclonal
antibody against LAMP-2 (C), (followed by appropriate
FITC-conjugated goat anti-rabbit or anti-mouse IgGs) to highlight the
ER, Golgi, and endosome/lysosome compartments, respectively
(green).
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Subcellular Localization of C99 in Relation to PS1--
Because
PS1 has been proposed as a possible
-secretase or a critical
interacting protein controlling
-secretase activity, we decided to
compare the subcellular distribution of C99(QLQN) and C99(KKQN) with
the distribution of PS1. Previous studies have established that
endogenous PS1 is localized predominantly in the perinuclear region and
the ER, with very little protein localized in post-Golgi compartments
(50, 86). The staining around the nuclear envelope has been attributed
to the full-length protein, whereas the protein in the ER appears to
arise from the N-and C-terminal fragments (50). In Fig.
6A we examined the
distribution of overexpressed PS1, using a previously characterized
antibody that recognizes the full-length protein and the N-terminal
fragment. We observed both a perinuclear ring and a predominant ER
staining pattern. The latter was verified in separate studies where
there was good coincidence between PS1 and the ER markers, protein
disulfide isomerase, and calnexin (not shown). Identical results were
obtained when the localization studies were repeated with an antibody
against the C-terminal loop domain of PS1 (not shown), consistent with the notion that the N-terminal and C-terminal polypeptides remain together after endoproteolytic cleavage of PS1 (51, 87). As expected,
based on its demonstrated retention in the ER (Fig. 5), C99(KKQN) was
localized almost entirely in compartments that contained PS1 (Fig.
6A). In contrast, the staining pattern for C99(QLQN), which
was localized mainly in the Golgi and endosomal compartments, showed
much less overlap with PS1 (Fig. 6B). Thus, the sharp
decline in A
production that we observed when C99 was retained in
the ER (Fig. 4) occurred despite an increased
co-localization of C99(KKQN) with PS1.

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Fig. 6.
Intracellular localization of C99
polypeptides in relation to PS1. HEK293 cells were co-transfected
with vectors encoding PS1 and either C99(KKQN) (A) or
C99(QLQN) (B). The distribution of PS1 was determined with a
rabbit polyclonal antibody against the N-terminal portion of PS1,
followed by Texas Red-conjugated goat anti-rabbit IgG. C99 was
visualized with the 9E10 anti-Myc monoclonal antibody, followed by
FITC-conjugated goat anti-mouse IgG. Staining of endogenous PS1 in the
non-transfected cells was below the threshold of detection at the
exposure times used for the photographs.
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Retention of C99 in the ER Affects the Biogenesis of Both
A
40 and A
42--
Because the immunoblot
methods used in the preceding studies did not distinguish between long
and short forms of A
, it was conceivable that retention of C99 in
the ER selectively prevented the production of the more abundant
A
40, while having little or no effect on the formation
of smaller amounts of A
42. To examine this possibility,
the comparative studies of C99(QLQN) and C99(KKQN) were repeated in 293 cells, using specific and highly sensitive ELISAs to quantify
A
40 and A
42 (Fig.
7). The concentration of A
40 fell by ~98% in the cultures expressing C99 with
the ER retention motif, in accord with the major loss of A
signal in
the earlier immunoblot studies. In addition, these studies clearly
revealed that the concentration of the less abundant A
42
underwent a parallel 96% decline in the same cultures. It should be
noted that the reporter antibody used in these assays (6H9) is specific
for the region of A
distal to the
-secretase cleavage site. Thus,
the results indicate that retention of C99 in the ER caused a decline in all potential
-secretase products, including any
N-terminal "ragged" forms of A
that could arise from
-secretase cleavage at alternative sites (88), as well as any p3
peptides formed from
-secretase cleavage of C83 fragments remaining
after
-secretase cleavage of endogenous APP in the transfected
cells.

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Fig. 7.
Addition of ER retention signal to C99 causes
a similar reduction of both A 40
and A 42. HEK293 cells were
transfected with vectors encoding C99(QLQN) or C99(KKQN), and equal
aliquots of conditioned medium were subjected to ELISA to determine the
concentrations of A 40 and A 42. Each value
is a mean (± S.E.) from assays performed on three parallel
cultures.
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Co-expression of C99 with a Dominant-negative Rab1B GTPase Inhibits
A
Production--
Although the preceding studies strongly suggested
that reduced A
formation in cells expressing C99(KKQN) was due to
retention of the
-secretase substrate in the ER, we could not rule
out the alternative possibility that the introduction of the two lysine residues at the C terminus of the polypeptide, and its consequent association with the COP-I coatomer complex, might somehow interfere with the recognition of C99 by
-secretase. To address this issue, we
used a different approach to retain C99 in the ER. This entailed co-expressing C99 with either wild-type Rab1B or a dominant-negative Rab1B mutant, i.e. Rab1B(N121I). Previous studies have
established that the Rab1 GTPase functions as a molecular switch in the
ER
Golgi transport pathway (89, 90). Introduction of the amino acid
substitution, N121I, into Rab1B drastically reduces its affinity for
GTP and renders the protein a dominant suppressor of protein trafficking between the ER and Golgi compartments (58, 91).
Immunofluorescence analysis confirmed that Rab1B(N121I) had the
predicted effect on C99 localization (Fig.
8). That is when C99(QLQN) was
co-expressed with Myc-tagged Rab1B(N121I), it assumed a diffuse
reticular staining pattern similar to that previously observed for
C99(KKQN) (see Fig. 5). On the other hand, when C99 (QLQN) was
co-expressed with wild-type Rab1B, it accumulated mainly in the
juxtanuclear Golgi region (Fig. 8).

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Fig. 8.
Effects of Rab1B(N121I) on the intracellular
localization of C99(QLQN). Cells were fixed 24 h after being
co-transfected with expression vectors encoding C99(QLQN) and either
Rab1B(wt) or Rab1B(N121I), as indicated. The localization of C99 was
determined with the 13G8 mouse monoclonal antibody, followed by Texas
Red-conjugated goat anti-mouse IgG (red). The ER was
highlighted with an affinity-purified rabbit polyclonal antibody
against calreticulin, followed by FITC-conjugated goat anti-rabbit IgG
(green). At the exposure times used for the photographs,
staining of endogenous APP with the 13G8 antibody in the
non-transfected cells was below the threshold of detection.
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We next examined the effect of the dominant-negative Rab1B(N121I) on
A
production in 293 cells. Because Rab mutants that fail to bind
guanine nucleotides are unstable, they do not accumulate to the same
extent as their wild-type counterparts when transiently expressed in
cultured cells. This is evident in Fig.
9A, where the upper band,
representing the Myc-tagged Rab1B(N121I) was expressed approximately
2-fold over endogenous Rab1B, compared with an approximate 10-fold
overexpression for Myc-Rab1B(wt). However, it is important to note that
the transfection efficiency in this study is ~15-20%. Thus, the 2:1
ratio of Myc-Rab1B(N121I) versus endogenous Rab1B in the
total cell lysate reflects a 10:1 ratio in the subpopulation of
transfected cells. Our previous studies have clearly established that
this level of Rab1B(N121I) overexpression completely blocks ER
Golgi trafficking of the low density lipoprotein receptor (63) and APP
(58). As shown in Fig. 9C, co-expression of C99(QLQN) with
the Rab1B(N121I) caused a 90% reduction in extracellular A
,
compared with cells expressing the same C99 construct with Rab1B(wt).
As determined previously for cells expressing C99 with the di-lysine
retention motif, the decline in extracellular A
caused by
co-expression of C99(QLQN) with Rab1B(N121I) was matched by a similar
reduction of A
in the cell lysates (not shown), indicating that it
was not due to intracellular sequestration of the peptide. Furthermore,
the reduction in A
seen in the cells expressing the
dominant-negative Rab1B mutant could not be explained by an inhibitory
effect of Rab1B(N121I) on overall expression of the C99(QLQN)
substrate, which showed similar steady-state levels in cells expressing
either the wt or N121I Rab1B constructs (Fig. 9B). In this
regard, it might at first seem puzzling that intracellular levels of
the C99 substrate did not increase noticeably in conjunction with the
block in A
production caused by the Rab1B mutant. However, this was
not entirely unexpected since, under conditions of continuous C99
overexpression, the fractional conversion of C99 to A
is actually
much lower than is suggested by direct comparison of the blots shown in
Fig. 9, B and C. The A
blots shown in Fig.
9C were subjected to a special high sensitivity detection
method involving biotinylated IgG and streptavidin-horseradish peroxidase. Under the standard ECL conditions and exposure times used
for C99 (Fig. 9B), A
was almost undetectable. It is also important to mention that other proteases besides
-secretase probably contribute to the turnover of C99. For example, recent studies
(92, 93) have documented the existence of calpain-like proteases that
can degrade C99 in the ER. It is possible that these enzymes may assume
a greater role in C99 degradation when the protein is prevented from
exiting the ER in cells expressing Rab1B(N121I).

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Fig. 9.
Retention of C99(QLQN) in the ER by
co-expression with a dominant-negative Rab1B mutant inhibits
A production but does not block the cleavage
of PS1 or SREBP2. HEK293 cells were co-transfected with vectors
encoding C99(QLQN) and either Rab1B(wt) or Rab1B(N121I). A
and B, expression of the recombinant proteins was verified
by SDS-PAGE and immunoblot analysis of equal aliquots of cell lysate
using polyclonal antibody against Rab1B (A) or 6E10 antibody
to detect C99 (B). A, the recombinant Rab1B is
distinguished from the endogenous protein by its slightly slower
mobility, which is due to the presence of an N-terminal Myc epitope tag
(arrows). C, equal aliquots of medium from the
same cultures were subjected to electrophoresis and immunoblot analysis
to detect A as described under "Experimental Procedures." The
blots shown in the illustration are representative of determinations
performed on three parallel cultures. The A values in the culture
expressing Rab1B(N121I) were reduced to 8.5 ± 1.9 (S.E.) percent
of the values in the cultures expressing Rab1B(wt). D shows
an immunoblot performed with an antibody against the N terminus of PS1
in 293 cells where PS1 was co-expressed with either Rab1B(wt) or
Rab1B(N121I), as indicated. The full-length PS1 (FL) and
N-terminal fragments (NTF) are indicated to the
right of the panel. Material above 50 kDa represents PS1
aggregates typically seen on SDS gels. The lane on the right
contains lysate from 293 cells transfected only with the Rab1B(N121I)
construct. Expression levels of the wild-type and N121I Rab1B
constructs (not shown) were similar to those in A. E shows immunoblots of membrane and nuclear fractions from
sterol-deprived 293 cells co-expressing HSV-tagged SREBP2 with either
Rab1B(wt) or Rab1B(N121I). Relative expression levels of the wild-type
and N121I Rab1B constructs (not shown) were similar to those in
A. Full-length SREBP2 and the N-terminal fragment
(NTF) detected by the anti-HSV antibody are indicated by the
arrows. Other bands detected by the anti-HSV antibody are
nonspecific, as indicated by their presence in non-transfected 293 cells (control). Scanning of the blots indicated that ratio
of nuclear NTF to full-length SREBP in the cells expressing
Rab1B(N121I) was ~62% of the ratio determined in the cells
expressing Rab1B(wt). Similar results were obtained in two separate
experiments.
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To verify that the reduced
-secretase cleavage of C99 in the
preceding studies was related specifically to the inability of cells
expressing Rab1B(N121I) to transport the substrate protein from the ER
to the Golgi compartment, we carried out several follow-up studies to
rule out a global perturbation of membrane-associated proteases or
general disruption of cell function.
First, we asked whether or not the dominant-negative Rab1B mutant would
similarly impair the endoproteolytic cleavage of presenilin-1 (PS1). In
contrast to the striking reduction of A
production (Fig.
9C), we observed no difference in the production of PS1 N-terminal fragments when PS1 was co-expressed with either Rab1B(wt) or
Rab1B(N121I) in 293 cells (Fig. 9D).
Second, we examined the effect of the dominant-negative Rab1B(N121I) on
the proteolytic cleavage of a membrane protein unrelated to APP,
i.e. SREBP2. The latter is localized in the ER, where sterol
depletion triggers the release of the N-terminal transcriptional regulatory domain through the action of two sequential site-specific endoproteases, the second of which cleaves within a transmembrane region (94). As shown in Fig. 9E, co-expression of SREBP2
with Rab1B(N121I) did not have a major effect on the release of the N-terminal domain, which is typically found in the nuclear compartment (75). Since the cleavage of SREBP2 depends on the sequential action of
two endoproteases, as well as an interaction with a sterol-sensing
activating protein (95), these results strongly suggest that the block
of ER
Golgi transport by the Rab1B mutant does not cause a global
disruption of protein function in the ER.
Finally, to explore further the possibility of general cell damage, we
performed an experiment to determine if the suppression of A
production by the dominant-negative Rab1B(N121I) was reversible. The
cDNAs encoding Rab1B wt and N121I were subcloned into the pTRE
vector, where gene expression is controlled by the Tet operator. Each
of these constructs was then transfected together with
pCMV-C99(QLQN) into a 293 cell line (Tet-OffTM)
where the stably expressed tetracycline-controlled transactivator
complex (tTA) strongly represses transcription in the presence of
doxycycline. When the transfected cells were maintained in the absence
of doxycycline for 24 h, Rab1B(wt) and Rab1B(N121I) were
transiently expressed at levels comparable to those previously seen
with unregulated cytomegalovirus vectors (Fig.
10, A and B).
Also, as expected, the dominant-negative mutant completely blocked A
production during this period (Fig. 10C). When expression of
the Rab1B constructs was subsequently suppressed by addition of
doxycycline to the medium, Myc-Rab1B(N121I) declined to an undetectable
level within 24 h (Fig. 10B), whereas the more stable
Myc-Rab1B(wt) declined more slowly (Fig. 10A). Of particular importance, Fig. 10C shows that, in parallel with the
disappearance of Rab1B(N121I), the production of A
was restored to
levels comparable to those detected in cultures expressing
Myc-Rab1B(wt) or no exogenous Rab (control with doxycycline always
present).

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Fig. 10.
Effects of the dominant-negative
Rab1B(N121I) on A production are
reversible. Parallel cultures HEK293 Tet-offTM cells were
co-transfected with pCMV5-C99(QLQN) and either
pTRE-Rab1B(wt) (A) or
pTRE-Rab1B(N121I) (B). All
cultures were grown without doxycycline ( dox) for the
initial 24 h after transfection to allow expression of the Rab1B
proteins. At this point one set of cultures was harvested for
immunoblot analysis to determine the expression levels of Myc-Rab1B
relative to endogenous Rab1B (upper panel) and the
expression of C99(QLQN) (lower panel), as described under
"Experimental Procedures." The remaining cultures were changed to
medium containing 1 µg/ml doxycycline (+dox) and harvested
for immunoblot analysis at the indicated times (times are total hours
after transfection). C, equal aliquots of medium
(conditioned for 24-h) were removed from the cultures analyzed in
A and B and subjected to immunoblot analysis.
A was detected as described under "Experimental Procedures," and
the results were expressed as percent of the A values determined for
matched control cultures where doxycycline was added prior to
transfection to suppress the expression of the Rab1B constructs
throughout the entire 72-h post-transfection period.
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Effects of PS1 Mutations on A
Production from C99--
Several
studies have shown that co-expression of APP with mutant forms of PS1
can cause an increase in cellular production of
A
42 versus A
40 (38, 39, 64,
96). This implies that interactions between APP and presenilins play a
critical role in determining the exact site where
-secretase cuts
the polypeptide chain at the C-terminal end of the A
sequence.
Although it is generally assumed that APP is cleaved by
-secretase
after the N-terminal exodomain is removed by either
-secretase or
-secretase, it remains unclear whether or not the exodomain of APP
is involved in the initial protein interactions with
-secretase/PS1.
By using our C99 construct, we have been able to explore this issue by co-expressing C99 with wild-type or mutant forms of PS1 in 293 cells
and then determining the concentrations of A
40 and
A
42 in the conditioned culture medium. Immunoblot
analysis of PS1 in the transfected cells indicated that the wild-type
and mutant forms of the protein were very similar with respect to
overall expression and the extent of endoproteolytic cleavage (Fig.
11A). Quantification of long
and short forms of A
in these cells revealed a striking 90%
increase in the ratio of A
42 to A
40 when
C99 was co-expressed with either PS1(M146L) or PS1(L286V), compared with cells co-expressing C99 with PS1wt (Fig. 11B). This
increase was comparable to that previously observed when the same PS1
mutants were co-expressed with full-length SwAPP (64). Based on this finding, it appears that the exodomain of APP is not required for the
elevation of the A
42/A
40 ratio by the PS1
mutants.

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Fig. 11.
Mutant forms of PS1 increase the production
of A 42 versus
A 40 from C99. HEK 293 cells were co-transfected with the vector encoding C99(QLQN) combined
with vectors encoding either PS1(wt), PS1(M146L), or PS1(L286V) as
indicated. Cells and medium were collected 24 h after
transfection. A, immunoblot assays were performed on
aliquots of the cell lysates to confirm that comparable levels of C99
and PS1 expression were obtained in all of the cultures. The major PS1
bands at ~23 and 45 kDa represent the N-terminal fragment and the
full-length protein, respectively. Endogenous PS1 was not detectable at
the exposure times used for these blots (not shown). The blots shown in
the illustration are representative of determinations performed on
three parallel cultures. B, equal aliquots of medium from
the same cultures were subjected to ELISA to quantify
A 42 and A 40, as described under
"Experimental Procedures." Each bar shows the mean ± S.E. of separate determinations from three parallel cultures.
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DISCUSSION |
-Secretase catalyzes the terminal step in the proteolytic
processing of APP, leading to the release of A
. The subcellular localization of
-secretase remains uncertain, although a few reports
have implied that this activity may exist in early compartments of the
secretory pathway (46-48). The first part of this study was designed
to test the hypothesis that one or more
-secretase activities reside
in the ER. We began by adding a di-lysine ER retention motif to SwAPP.
Retention of this protein in the ER was clearly demonstrated by 1) the
impairment of Golgi-dependent post-translational
modifications and 2) reduction of exodomain products normally released
after SwAPP is cut by
-secretase or
-secretase in medial or late
compartments of the secretory pathway. These results confirm previous
reports (26, 28, 50, 97) indicating that most of the
-secretase
activity resides in subcellular compartments distal to the ER.
Moreover, they suggest that the recently described maturation and
cleavage of the
-secretase pro-peptide after it leaves the ER (98)
may be required for activation of the enzyme.
In the cells expressing SwAPP(KKQN), the amounts of both
A
40 and A
42 released into the medium were
markedly reduced. Because the full-length SwAPP(KKQN) construct was
unable to progress beyond the first step in the amyloidogenic pathway
(i.e. the translocation of APP to sites containing active
-secretase), its utility for assessing
-secretase localization
was limited. To circumvent this problem and obtain a direct measure of
-secretase activity, we generated a C99 construct, C99(QLQN) which
represents the C-terminal stump of APP that remains after
-secretase
cleavage, with a C-terminal Myc epitope and a tetrapeptide extension
similar to the normal APP C-terminal sequence, QMQN. Our initial
studies showed that this construct was a good physiological substrate
for
-secretase, giving rise to abundant amounts of
A
40 and A
42 that could be easily detected
with several different antibodies directed against epitopes at the N
terminus and C terminus of the peptide. When the di-lysine motif, KKQN,
was added to C99, retention of the protein in the ER was indicated by a
marked change in its immunofluorescence localization from a predominant
Golgi-like pattern to one that closely matched the ER marker, calreticulin.
Measurements of A
in the medium from cells expressing
C99(KKQN) revealed a near-complete block in deposition of both
A
40 and A
42 compared with cells
expressing C99(QLQN). If the decline in extracellular A
was due to
block in peptide secretion, we would have expected to see an increase
in the intracellular A
pool accompanying the decreased peptide
output. Instead, we observed a corresponding decrease in intracellular
A
, strongly suggesting that the ER retention signal was preventing
biogenesis of the peptide. To confirm that the reduced A
production
was specifically related to retention of C99 in the ER, rather than to
the di-lysine motif interfering with
-secretase substrate
interactions, we retained C99 in the ER without the di-lysine signal by
co-expressing it with a dominant-negative Rab1B mutant that blocks ER
Golgi trafficking (58, 63). These studies showed that C99(QLQN), which normally gave rise to substantial amounts of A
when expressed in 293 cells, was unable to generate A
when its transport out of the
ER was blocked. The latter effect was readily reversed when expression
of the Rab1B mutant was suppressed, and the functions of other ER
endoproteases operating on PS1 and SREBP2 were not disrupted. Taken
together, these observations lead us to conclude that the ER is not a
major site for
-secretase processing of the APP C99 fragment in the
well characterized 293 cell model. Our results contrast with those of
Soriano et al. (48), who found no reduction of
A
40 or A
42 secretion in Chinese hamster ovary cells expressing an APP/CD3
chimera that was retained in the
ER. At present we cannot offer a simple explanation for these conflicting results. One possibility is that major cell type-specific differences exist in the distribution of
-secretase. However, the
marked reduction of A
that we observed when C99(KKQN) was expressed
in NT2N neurons indicates that these cells are similar to 293 cells
insofar as most of the
-secretase activity appears to exist outside
of the ER.
It has been proposed that A
40 and A
42 are
generated by different
-secretases (16, 17), with the enzyme
responsible for producing the long form of A
residing in the ER and
the enzyme producing A
40 localized mainly in the
endosomes or other peripheral compartments (52). Although the present
study does not directly rule out the existence of multiple
-secretases that cut C99 at different sites, the parallel reductions
in the amounts of A
40 and A
42 produced by
293 cells expressing C99(KKQN) argue against the idea that a different
-secretase responsible for generating A
42
versus A
40 is selectively compartmentalized in the
ER. It remains to be determined precisely where, beyond the ER, the
final step in A
biogenesis actually occurs and how the peptide is
released from the cell. Under normal circumstances it is difficult to
detect substantial amounts of C99 or C83 in cultured cells, suggesting that these intermediate fragments are rapidly cleaved by
-secretase. However, in cells transfected with the C99 plasmids, overexpression of
the substrate appears to saturate the endogenous
-secretase processing pathway, with consequent accumulation of a large C99 pool
that can be readily detected by immunoblot analysis. The presence of
this material in the Golgi apparatus, and to a lesser extent in the endosomes, could indicate that these are normal sites of
-secretase processing. This would be consistent with numerous
studies pointing to the involvement of acidic compartments in A
biogenesis (6, 30, 31, 99).
The apparent absence of substantial
-secretase activity in the ER
has important implications for understanding the relationship between
presenilins and APP processing. Mutations in PS1 (100) and PS2 (101)
have been linked to early-onset familial forms of Alzheimer's disease,
with the majority of the known mutations occurring in PS1 (102, 103).
Several of the amino acid substitutions in the presenilins have been
shown to cause alterations in the amyloidogenic processing of APP, such
that cells produce an increased amount of A
42 relative
to A
40 (36-39, 104). The molecular mechanism underlying
this effect remains controversial. Presenilins are serpentine
polypeptides with multiple hydrophobic membrane-spanning segments and a
large cytoplasmic loop (105, 106), and there is some evidence for a
direct physical interaction between PS1 and APP, based on
co-immunoprecipitation studies (107). Both endogenous and overexpressed
presenilins are localized predominantly in the perinuclear region and
the ER (49-51, 108, 109). Nascent presenilins undergo endoproteolytic
cleavage in vivo to produce stable N-terminal and C-terminal
derivatives (87, 110, 111). This "presenilinase" activity is
completely abolished by substitutions of two conserved aspartate
residues that lie in the transmembrane domains flanking the cleavage
site (40, 41). Interestingly, the same studies show that the aspartate
substitutions in presenilin also reduce
-secretase activity in cell
lysates, raising the prospect that presenilins are autoactivated
aspartyl proteases that function as
-secretases. Further support for
this hypothesis comes from recent studies in which photoactivated
-secretase inhibitors have been shown to bind directly to the
cleaved forms of PS1 and PS2 (42, 43). If PS1 and
-secretase are
truly the same molecule, then one might expect to observe an increase in A
production in cells where the C99 substrate is retained in the
ER where most of the PS1 resides, compared with cells where C99 is
exported to other compartments. In fact, our studies show the exact
opposite, with A
production being almost completely abolished in
cells where C99 was retained in the ER by inclusion of the di-lysine
motif or disruption of ER
Golgi trafficking with a Rab1B mutant.
One interpretation of these findings is that most of the intracellular
PS1 residing in the ER is catalytically inactive as
-secretase,
whereas a small pool of PS1 is converted to a functional
-secretase
in one or more peripheral cellular compartments. This conversion could
involve a conformational change in the PS1 sub-domains or an unmasking
of cleavage sites in the APP substrate, perhaps related to the assembly
of these proteins into a large multimeric complex such as that
described by Li et al. (112) and/or interaction with
accessory proteins such as nicastrin (57). A similar model has been
invoked to explain why PS1 (
-secretase) cleaves the C-terminal
domain of Notch at the plasma membrane, even though PS1 apparently can
bind to Notch in the ER and Golgi compartments (113). In the latter
case, activation of the proteolytic cleavage may be related to a
ligand-induced conformational change in the Notch substrate that
changes the nature of the PS1/Notch interaction.
Regardless of whether the presenilins function as
-secretases
by themselves or as part of a larger protein complex, the present studies clearly show that mutant versions of PS1 can affect the biogenesis of A
when expressed with only the C99 stump of APP. Specifically, the increase in A
42/A
40
seen when C99 was co-expressed with PS1 L286V or M146I was similar in
magnitude to that previously reported when the same mutants were
expressed with full-length SwAPP (64). This demonstrates that the
exodomain of APP is not required for the relevant PS1 interactions.
When combined with earlier observations indicating that the association
of PS1 with APP can occur in the absence of the APP cytoplasmic tail
(107), our results suggest that the critical domain for interaction
with PS1 may be confined to a narrow region of APP that lies between the
-secretase cleavage site and the end of the transmembrane domain. This conclusion is consistent with the mutagenesis studies of
Lichtenthaler et al. (114), which indicate that the cleavage specificity of
-secretase (resulting in A
40 or
A
42) is determined largely by the eight amino acid
residues immediately downstream of the cleavage site within
the transmembrane region of C99. Thus, it is likely that modified C99
constructs will be particularly useful in future studies aimed at
defining the specific structural domains involved in the physical
interactions between PS1 and the transmembrane region of APP.
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