(Received for publication, November 10, 1995; and in revised form, February 8, 1996)
From the
The 4-kDa -amyloid peptide (A
), a principal component
of parenchymal amyloid deposits in Alzheimer's disease, is
derived from amyloid precursor proteins (APP). To identify potential
intracellular compartments involved in A
production, we expressed
human APP-695 (APPwt) and APP-695 harboring the Swedish double mutation
(APPswe) associated with familial early-onset Alzheimer's
disease, in mouse N2a cells. We demonstrate that cells expressing
APPswe secrete high levels of A
peptides and
-secretase-generated soluble APP derivatives
(APP
) relative to cells expressing APPwt. In addition,
we observed a concomitant diminution in the levels of
-secretase-generated soluble APP derivatives
(APP
). Our interpretation of these findings is that
-secretase cleavage occurs in an intracellular compartment and
disables those substrates which would normally be cleaved by
-secretase. As anticipated, the levels of APPswe are diminished
relative to the steady-state levels of surface-bound APPwt; moreover,
surface-bound APPswe and APPwt molecules are released from the plasma
membrane after cleavage by
-secretase, but not by
-secretase.
Finally, by examining the rate of appearance of specific APP
metabolites generated by
-secretase, we now unequivocally
demonstrate that
-secretase cleavage of APPswe occurs within the
Golgi apparatus, as early as the medial compartment.
Alzheimer's disease, a progressive neurodegenerative
disorder of the elderly, is characterized by the presence of
parenchymal deposits of A, (
)a 39- to 43-amino acid
peptide derived from
APP(1, 2, 3, 4) . APP are integral
membrane glycoproteins that mature through the secretory
pathway(5) . A fraction of newly synthesized APP appears on the
cell surface (6, 7, 8, 9) and some
of these molecules are cleaved by
-secretase (7, 8) within the A
sequence (10, 11, 12, 13) , resulting in the
release of the APP ectodomain (APP
). In a cell-type
specific manner, APP
is generated in the trans-Golgi network or other late compartments of the
constitutive secretory pathway (14, 15, 16, 17) . A fraction of APP
are directly sorted, or reinternalized from the cell surface, to
endosomal/lysosomal compartments(7) , where a complex set of
A
-containing membrane-bound fragments
accumulate(7, 18) . Finally, it is fully established
that A
(
4 kDa) and a truncated form of A
(
3 kDa)
are released constitutively in vitro and in
vivo(19, 20, 21, 22, 23) .
The biochemical mechanism(s) and cellular compartments involved in
A production have not been fully elucidated. Despite earlier
excitement created by the discovery of potential amyloidogenic
fragments generated in endosomal-lysosomal pathways, several lines of
evidence now suggest that lysosomal degradation of APP is unlikely to
contribute to the production of A
(reviewed in (24) ).
However, agents that interfere with pH gradients (i.e. ammonium chloride and chloroquine) inhibit the production of
A
(19, 21) , suggesting that A
may be
generated in acidic compartments (i.e. endosomes and/or late
Golgi). Indeed, biochemical studies by Koo and Squazzo (23) confirmed that A
production and release involves the
endocytosis of full-length APP from the cell surface and subsequent
recycling. In this model,
-secretase cleavage occurs within
endocytic compartments while
-secretase cleavage of the residual
100-amino acid membrane fragment occurs virtually simultaneously
with A
formation and release. However, the stoichiometry of A
contributed by reinternalized APP to A
generated in the
biosynthetic pathway is not known.
To identify potential
intracellular compartments involved in A production, we examined
the metabolism of APP with a double mutation at codons 670 and 671 (of
APP-770) described in two large Swedish pedigrees with familial
Alzheimer's disease. Cells expressing this mutant APP secrete
6-8-fold higher levels of A
relative to cells
expressing wild-type APP(25, 26) . In preceding
efforts, we examined the trafficking and metabolism of APPwt and APPswe
in polarized epithelial cells (MDCK)(27) . We demonstrated that
APPswe was cleaved at the
-secretase site and that the resulting
soluble derivative, termed APP
, was detectable in cell
lysates. These results lead to the suggestion that
``
-secretase'' cleavage occurs in an intracellular
compartment. In the present report, we extend the observations in MDCK
cells by characterizing the maturation of APPswe in transiently and
stably transfected N2a cells, a mouse peripheral neuroblastoma line. We
demonstrate that N2a cells expressing APP harboring the Swedish
mutations secrete high levels of A
peptides and APP
derivatives relative to cells expressing APPwt. Interestingly, we
observed a concomitant diminution in the levels of secreted
APP
. One scenario to explain this intriguing result is
that normal
-secretase substrates are disabled by the prior action
of
-secretase. This model predicts that
-secretase must
cleave APPswe in an intracellular compartment proximal to the cellular
site(s) of
-secretase activity. We now confirm that
-secretase cleavage occurs within the Golgi apparatus, as early as
the medial compartment.
Mouse N2a neuroblastoma cells were transfected using a
high-efficiency CaPO co-precipitation
procedure(30) . To generate cell lines that express human APPwt
and APPswe, N2a cells were transfected with pAPPwt or pAPPswe and
stable transfectants were selected in medium containing 0.4 mg/ml G418
(Life Technologies, Inc.) (the pCB6 vector contains a gene encoding
resistance to the neomycin analog, G418). Expression of c-Myc-modified
human APP was assayed by immunoblotting and immunoprecipitation with a
polyclonal antiserum Myc-I raised against the synthetic peptide
MEQKLISEEDLN(31) .
Cell-associated and soluble APP derivatives were immunoprecipitated
as described previously(11) . To immunoprecipitate full-length
APP and APP COOH-terminal fragments, we used 369, a polyclonal
antiserum raised against the entire cytoplasmic tail of
APP(32) , or Myc-I, a polyclonal serum raised against a
12-amino acid epitope in the c-Myc oncoprotein (see above). To
immunoprecipitate full-length and soluble APP derivatives, we used a
monoclonal antibody (mAb), P2-1, which recognizes an epitope in the
NH-terminal cysteine-rich region of human APP(33) .
6E10, a mAb that recognizes residues 1-17 of human A
(34) was used to immunoprecipitate soluble APP generated
following cleavage by
-secretase, while 4G8, a mAb that recognizes
residues 17-28 of A
(34) was used to
immunoprecipitate A
and related p3 peptides. Antibody 54 was used
to immunoprecipitate the soluble APP derivative generated following
cleavage of APPswe(35, 36) . Polyclonal anti-VSV serum (28) was used to immunoprecipitate VSVG protein. Quantification
of immunoprecipitated polypeptides was performed by phosphorimaging
using Molecular Dynamics software.
To assess the level of cell surface-bound APP, radiolabeled
cell lysates were subject to immunoprecipitation with 369 antiserum.
The immunoprecipitates were boiled in immunoprecipitation buffer
containing 1% SDS to dissociate APP-related polypeptides from the
antibody. The resulting soluble fraction was subsequently incubated
with streptavidin-agarose beads (Pierce) to recover biotinylated APP,
as described previously(27) . To examine the fraction of
soluble derivatives which were biotinylated, i.e. that were
released from the cell surface, equivalent aliquots of medium collected
from cells incubated at 37 °C following biotinylation was incubated
with either mAb 22C11, an antibody that recognizes a region between
residues 66 and 81 of APP (6, 38) or mAb 6E10 (see
above). Immunoprecipitated proteins were dissociated by boiling in
immunoprecipitation buffer containing 1% SDS. The resultant soluble
fraction was adjusted to 1 immunoprecipitation buffer and
subjected to a second round of binding to streptavidin-agarose beads.
Figure 1:
Metabolism
of human APP-695 in mouse N2a cells. A, analysis of APP
metabolism in transiently transfected cells. N2a cells transfected with
plasmid vector or plasmids encoding human APPwt (Wt) or APPswe (Swe) were labeled for 3 h with
[S]methionine. Full-length APP were
immunoprecipitated from cell lysates with antibody 369. Secreted
APP
molecules were immunoprecipitated from conditioned
medium (Supt) using either mAb P2-1 or 6E10. Note that 6E10
immunoprecipitated considerably lower amounts of APP
from
APPswe as compared with P2-1. A
and p3 were immunoprecipitated
from conditioned medium using mAb 4G8. B, characterization of
termini generated by
-secretase cleavage of APPswe. Aliqouts of
[
S]methionine-labeled conditioned medium from
cells transiently transfected with empty vector (lane 1),
APPwt cDNA (lane 2), or APPswe cDNA (lane 3) were
immunoprecipitated with antibody 54, specific for the carboxyl terminus
of APP
. APPwt and APPswe panels,
radiosequencing of A
molecules from conditioned medium of cells
expressing APPwt or APPswe, respectively. C, analysis of APP
metabolism in stable N2a lines. Stable N2a cell lines 695.12 (Wt) and Swe.1 (Swe), expressing human APPwt or
APPswe, respectively, were labeled for 3 min (lanes 1 and 2) or 3 h (lanes 3-10) with
[
S]methionine. Full-length human APP were
immunoprecipitated from cell lysates using Myc-I antiserum or mAb P2-1.
Secreted APP
were immunoprecipitated with either mAb P2-1
or 6E10. Note that the vast majority of APP
derived from
APPswe (lane 6) was not immunoprecipitated by 6E10 (lane
8). A
and p3 were immunoprecipitated from conditioned medium
using mAb 4G8. The
95-kDa APP species (marked by an arrowhead) observed in lysates of Swe.1 cells (lane
4) likely represents
APP
(27) .
Finally,
and to confirm that endoproteolytic cleavage by -secretase occurs
between Leu
and Asp
of APPswe, we
characterized the carboxyl terminus of the secreted soluble derivative
and the amino terminus of A
. To confirm the authenticity of the
-secretase-generated soluble derivative, we subjected parallel
aliquots of conditioned medium shown on Fig. 1A, lanes
4-6, to immunoprecipitation analysis with antibody 54,
specific for APP
(35, 36) . This
antibody fails to detect
-secretase-cleaved
APPswe(35, 36) , and hence is specific for an epitope
of the APPswe soluble derivative which is ``exposed'' upon
truncation of the full-length precursor by
-secretase. As
anticipated, antibody 54 recovered high levels of
95-kDa species
from medium of cells expressing APPswe (Fig. 1B, lane
3); this result is in sharp contrast to the isolation of
nonspecific polypeptides of
85 and
95 kDa from medium of
cells transfected with empty vector or wtAPP cDNA (Fig. 1B,
lanes 1 and 2, respectively). To analyze the amino
terminus of A
derived from cells expressing APPwt and APPswe, we
radiolabeled cells with [
H]phenylalanine and
immunoprecipitated A
and p3 from the conditioned medium; the
isolated 4-kDa band was subjected to radiosequencing. Predominant peaks
of radioactivity in cycles 4, 19, and 20 clearly show that >95% of
the 4-kDa peptides secreted by cells expressing either APPwt and APPswe
initiate at Asp
(Fig. 1B, panels APPwt and APPswe). Taken together with studies demonstrating that the
membrane-bound fragment generated by
-secretase initiates at
Asp
(39) , our demonstration that the carboxyl
terminus of the soluble derivative extends to Leu
fully
confirms our view that
-secretase cleaves APPswe between
Leu
and Asp
.
In order to further
characterize intracellular trafficking and processing of APPwt and
APPswe APP in N2a cells, we generated 20 and 18 independent stably
transfected cell lines which expressed variable levels of APPwt or
APPswe, respectively. We did not observe any toxicity-related
phenotypes in the N2a lines expressing either APPwt or APPswe.
Biosynthetic rates of c-Myc epitope-tagged transgene-derived
polypeptides in individual cell lines were assessed by short (3 min)
pulse-labeling with [S]methionine and
immunoprecipitation analysis with Myc-1 antibody. Two cell lines,
695.12 and Swe.1, that express essentially indistinguishable levels of
newly synthesized APPwt and APPswe, respectively (Fig. 1C, compare lanes 1 and 2,
respectively), were selected for further analysis. Immunoprecipitation
of APP-related species using APP NH
-terminal antibody,
P2-1, from lysates of 695.12 and Swe.1 cells labeled for 3 h, revealed
the presence of accumulated
105- and
120-kDa forms (Fig. 1C, lanes 3 and 4). Interestingly, we
also detected a species with an apparent molecular mass of
95 kDa
in lysates of cell line Swe.1 (Fig. 1C, lane 4),
similar to a species observed in lysates of MDCK that constitutively
express APPswe(27) . Analysis of soluble APP derivatives in
radiolabeled conditioned medium of lines 695.12 and Swe.1 (Fig. 1C, lanes 5-8) fully supported our findings
in transient assays. For example, relative to the level of soluble
derivatives in 695.12 cell medium (Fig. 1C, lane 5) the
total level of soluble derivatives in Swe.1 cell medium (Fig. 1C, lane 6) was clearly elevated; by
phosphorimaging analysis, the total level of secreted derivatives
generated by line Swe.1 was increased
3-fold relative to line
695.12, and the vast majority of these species exhibited accelerated
electrophoretic mobility. Moreover, the levels of APP
in Swe.1 cell medium was clearly diminished relative to the level
of APP
in 695.12 cell medium (Fig. 1C,
compare lanes 8 and 7, respectively); phosphorimaging
analysis showed
3-fold higher levels of APP
in
695.12 cell medium compared to Swe.1 cell medium. Hence, we argue that
the principal contributor to the elevated level of accumulated
derivatives secreted by Swe.1 cells are species which are generated
following cleavage at the
-secretase site. Finally, a 4-fold
elevation of accumulated
4-kDa A
was evident in Swe.1 cell
medium relative to 695.12 cell medium. However, and in contrast to our
findings in transiently transfected N2a cells, a corresponding
diminution in the levels of the p3 fragment was not observed. At
present, there is not a satisfactory explanation for this phenomenon.
Earlier studies have suggested that the penultimate precursor for p3 is
the membrane-retained fragment generated by
-secretase(24) . It is also conceivable that a fraction of
-secretase-generated, carboxyl-terminal
derivatives are substrates for cleavage by
-secretase and that
this truncated fragment is ultimately cleaved by
-secretase to
generate p3. Thus, we would propose that the trafficking and
proteolysis of the
-secretase-generated fragment is intrinsically
different in stable and transiently transfected N2a cells, and may
reflect differences in expression levels of transgene-encoded APPswe or
the corresponding
-secretase-generated carboxyl-terminal fragment.
Despite this inconsistency in p3 levels between stable and transiently
transfected N2a cells, these data do not detract from our principal
findings that the total levels of secreted derivatives generated by the
stable APPswe cell line are increased relative to cells expressing
APPwt and that over 80% of these species exhibited accelerated
electrophoretic mobility. Moreover, concomitant dimunition in
APP
levels in medium of the stable cell line
expressing APPswe strongly supports our view that potential
-secretase substrates are disabled by the prior action of
-secretase.
In the aggregate, we have demonstrated that in
transient and stably transfected N2a cells, the levels of soluble
derivatives and A in medium of cells expressing APPswe are
elevated relative to respective species secreted by cells expressing
APPwt. Moreover, the vast majority of soluble derivatives in medium of
cells expressing APPswe are APP
, molecules generated
following cleavage at the
-secretase site. The production of
elevated levels of APP
from cells expressing APPswe is
essentially identical to results obtained by expression of chimeric
human placental alkaline phosphatase-APP molecules harboring the
Swedish substitutions in H4 glioblastoma cells. These studies
demonstrated that human placental alkaline phosphatase-APPswe were
sensitive to endoproteolytic cleavage at the
-secretase site and
that high levels of chimeric human placental alkaline
phosphatase-APP
species were secreted into the
conditioned medium(40) . More importantly, we document that the
total level of APP
derivatives is diminished in medium
of cells expressing APPswe. This result can be accommodated by a model
in which a significant population of substrates which might normally be
subject to cleavage by
-secretase are disabled by the prior action
of
-secretase. Implicit in this model is that
-secretase must
cleave APPswe substrates in an intracellular compartment proximal to
the cellular site(s) of
-secretase activity. Thus, we argue that
APPswe is obligatorily cleaved by
-secretase in an intracellular
compartment wherein
-secretase is either inactive or absent.
Figure 2:
Kinetics of APP processing in N2a cells. A, analysis of APP carboxyl-terminal fragments generated by
proteolytic processing. Detergent lysates of transiently transfected
N2a cells (Fig. 1A) were analyzed by
immunoprecipitation with either Myc-I or 369 antiserum. B and C, kinetics of cleavage and secretion of APP molecules.
Parallel dishes of N2a cells transfected with plasmids encoding APPwt (Wt) or APPswe (Swe) were pulse-labeled for 10 min
with [S]methionine and chased for the times
indicated. At each time point, medium was collected and lysates were
prepared from cell monolayer. Carboxyl-terminal APP fragments were
immunoprecipitated from lysates with 369 antiserum. Secreted APP
molecules were immunoprecipitated from conditioned medium using
mAb P2-1. The
13.5-kDa fragment whose levels were elevated in
cells expressing APPswe is marked by an arrow. The
12-kDa
band (marked by an arrowhead) likely represents the
carboxyl-terminal fragment generated after
-secretase cleavage of
APP.
To evaluate the
kinetics of cleavage of APPwt and APPswe at the - or
-
secretase sites specifically, we performed pulse-chase analyses.
Parallel dishes of N2a cells were transiently transfected with cDNAs
expressing APPwt or APPswe. Cell monolayers were pulse-labeled with
[
S]methionine for 10 min, then chased at 37
°C for varying periods of time up to 45 min. At the end of each
chase period, medium was saved and detergent soluble lysates were
prepared from cell monolayers. Lysates or conditioned medium were
subjected to immunoprecipitation with Ab369 or P2-1 antibodies,
respectively. Fig. 2, B and C, depicts the
production of APP carboxyl-terminal fragments in cell lysates and the
appearance of APP
in the medium, respectively. Analysis of
the carboxyl-terminal fragments generated in cells expressing APPwt
revealed the nearly concomitant generation of both
12- and
13.5-kDa fragments at 20 min into the chase period, species which
are continually generated (or accumulate) over the next 25 min (Fig. 2B, left panel). We suggest that the
13.5-kDa fragment is the penultimate precursor of A
and that
the kinetics of appearance is consistent with previous studies which
demonstrated A
generation from plasma membrane-bound APP after
endosomal recycling(23) . In parallel, APP
first
appear in the conditioned medium of cells expressing APPwt APP at 30
min into the chase period (Fig. 2C, left panel). Taken
together with the kinetics of appearance of the
12-kDa
carboxyl-terminal fragment, we argue that the APP
species
are produced by endoproteolytic cleavage by
-secretase, an
activity which occurs late in the secretory pathway, and predominantly
on the plasma membrane (see below, Fig. 3). In striking contrast
to the results obtained in cells expressing APPwt, cells expressing
APPswe contain a prominent
13.5-kDa fragment which first appears
within 10 min into the chase period (Fig. 2B, right
panel). Notably, a
12-kDa carboxyl-terminal fragment is also
present, albeit at somewhat lower levels, in cells expressing APPswe,
and the kinetics of appearance of this species fragment is
indistinguishable to that observed in cells expressing APPwt.
Interestingly, APP
first appear in the conditioned medium
within 20 min of chase (Fig. 2B, right panel), a rate
considerably accelerated relative to the secretion of APP
species from cells expressing APPwt. Moreover, the soluble
derivatives in medium of cells expressing APPswe clearly exhibit
accelerated migration relative to APP
. Our
interpretation of this result is that these APP
species which are produced early in the secretory pathway are
liberated from specific intracellular sorting/retention signals
contained within the core protein and hence rapidly transit through the
secretory pathway prior to secretion. Thus, the early production of the
13.5-kDa carboxyl-terminal fragment and accelerated appearance of
secreted APP
supports our view that
-secretase
cleavage of APPswe is initiated early in the secretory pathway.
Interestingly, we have failed to recover APP carboxyl-terminal
fragment(s) that encompasses sequences between Ala
(in
APP-695) and the myc-epitope tag, a species which would be generated by
the action of
-secretase (data not shown). The reason for this
discrepancy is unclear but suggests that
-secretase-generated
56-59 amino acid fragment is subject to rapid degradation.
Figure 3:
Cell
surface biotinylation and release assay. Transiently transfected N2a
cells were labeled with [S]methionine and cell
surface biotinylated. One set of cells were incubated at 37 °C for
10 min, while the other set was kept at 4 °C. Conditioned medium
and cell lysates were analyzed as described under ``Materials and
Methods.'' Lanes 1 and 2 represent secreted
APP
molecules immunoprecipitated with mAb P2-1 from
conditioned medium collected during the 3-h
[
S]methionine labeling period. Lanes
3-6 represent cell-surface biotinylated APP molecules
immunoprecipitated with 369 antiserum from lysates of cells incubated
at 37 °C for 0 or 10 min. Lanes 7-10 represent mAb
22C11 or 6E10 immunoprecipitates of biotinylated APP
molecules secreted during 10 min incubation at 37
°C.
Figure 4:
Kinetics of -secretase cleavage of
APPswe molecules. A, kinetic analysis of carboxyl-terminal APP
fragments in Swe.24, an N2a line expressing APPswe. Parallel dishes of
Swe.24 cells transiently transfected with plasmid encoding VSVG protein
were pulse labeled for 5 min with [
S]methionine
and chased for the times indicated. Carboxyl-terminal fragments of APP
were immunoprecipitated with 369 antiserum from cell lysates prepared
at each time point. B, kinetics of acquisition of
endoglycosidase H resistance by APP molecules. Full-length APP
molecules were immunoprecipitated from cell lysates of selected time
points with Myc-I antiserum. One-half of the sample was incubated with
endoglycosidase H (Endo H). Arrowhead indicates the
endoglycosidase H-resistant APP species. C, kinetics of
maturation of VSVG. VSVG protein was immunoprecipitated from cell
lysates with rabbit anti-VSV serum. Arrowhead indicates the
mature
67-kDa species which is resistant to endoglycosidase H
digestion (data not shown; 41).
A, the principal component of parenchymal amyloid
deposits in Alzheimer's disease, is derived from integral
membrane glycoproteins, APP. Although A
is normally secreted by a
variety of cultured cells, the molecular mechanisms involved in A
production have not been fully clarified. The present report provides
several insights into the cellular compartments involved in
-secretase cleavage and the production of A
. We have assessed
the metabolism of a APP (APPswe) harboring a double mutation at codons
670 and 671 (of APP-770) in N2a cells. Using surface biotinylation and
release approaches, we demonstrate that relative to the steady-state
levels of surface-bound wild-type APP, the levels of APPswe are
diminished. Moreover, surface-bound APPswe and APPwt molecules are
released from the plasma membrane after cleavage by
-secretase,
but not by
-secretase. Finally, using kinetic approaches, we
provide compelling evidence that
-secretase cleavage of APPswe
occurs early in the secretory pathway and unequivocally demonstrate
that a population of APPswe molecules are cleaved at the
-secretase site within the Golgi apparatus, as early as the medial
compartment. Moreover, and consistent with the cell surface labeling
and release studies, cleavage of both the APPwt and APPswe substrates
occurs several minutes later, concomitant with the appearance of
soluble APP
derivatives in the conditioned medium. The
principal conclusions of this work are summarized in Fig. 5.
Figure 5:
Model depicting sites of - and
-secretase cleavage of APPwt and APPswe in N2a cells. The middle panel depicts selected cellular compartments of the
central vacuolar pathway involved in APP trafficking/processing. For
APPwt, the majority of secreted APP
are
generated by cleavage of full-length molecules by
-secretase on
the plasma membrane. Low levels of secreted A
presumably occurs
via endocytic recycling of plasma membrane-bound APP(23) . For
APPswe,
-secretase cleaves full-length membrane bound molecules in
the medial Golgi and compartments proximal to the plasma membrane.
Resulting soluble APP
traffic through the secretory
pathway and are released into the medium. As a consequence of early
cleavage by
-secretase, the levels of full-length APPswe that
arrive on the plasma membrane is considerably diminished. Finally, the
membrane-bound,
-secretase-generated 100-amino acid
carboxyl-terminal fragment is cleaved by
-secretase at, or near
the plasma membrane to release high levels of soluble A
. Asterisks indicate the position of the Swedish mutation in
APPswe; solid box, A
domain.
Our finding that -secretase cleavage of APP harboring the
Swedish mutations is initiated in the Golgi apparatus provides a
conceptual framework for developing a model to explain the increase in
A
secretion by APPswe cells. We propose that the production of the
membrane-bound
13.5-kDa A
-containing carboxyl-terminal
fragment early in the secretory pathway liberates the fragment from
potential sorting/retention signals in the APP lumenal domain. Support
for a role for APP lumenal sequences in intracellular trafficking has
recently emerged from studies of soluble APP molecules expressed in
MDCK cells (45, 46) . Thus, in sharp contrast to APP
or APP with a deletion of the cytoplasmic sequence, YENPTY, in which
only
20 or
60%, respectively, of newly synthesized molecules
reach the cell surface and are secreted(9) , we suggest that
the
13.5-kDa fragment is efficiently shunted through the secretory
pathway. These molecules would subsequently encounter
-secretase
near, or at, the plasma membrane immediately prior to A
release.
Addendum-While our manuscript was under review, we learned of studies reported by C. Haass and colleagues (47) which concluded that in human embryonic kidney 293 cells, APPswe is cleaved in Golgi-derived vesicles, most likely within secretory vesicles.