(Received for publication, July 6, 1995; and in revised form, September 8, 1995)
From the
Secretory processing and polarized sorting of horseradish
peroxidase fused to the amyloid precursor protein transmembrane domain
were compared with those of wild-type amyloid precursor protein in COS
and polarized Madin-Darby canine kidney (MDCK) cells. The cellular and
secreted forms of the chimeric protein were enzymatically active in
colorimetric and cytochemical assays after reconstitution with hemin
and Ca. The peroxidase enzyme was secreted by a
proteolytic process, similar to the parent amyloid precursor protein.
In polarized MDCK cells, amyloid precursor protein was secreted
exclusively in the basolateral compartment, while the peroxidase
chimeric protein was secreted in both compartments. The basolateral
sorting determinant for secretion must therefore be located in the
extracellular domain of amyloid precursor protein. On the other hand,
cell surface-associated peroxidase chimeric protein was similar to cell
surface-associated wild-type amyloid precursor protein, mainly
expressed at the basolateral side. The basolateral cell-surface
expression, in contrast to the basolateral secretion, is therefore
controlled by determinants in the cytoplasmic domain. Methylamine
inhibited and bafilomycin slightly increased the basolateral secretion
of both proteins, but both drugs strongly increased apical secretion.
The default secretory pathway of COS cells and the basolateral (but not
the apical) secretory pathway of MDCK cells are therefore comparably
sensitive to methylamine and not to bafilomycin.
Amyloid precursor protein (APP) ()is the precursor of
the
A4-amyloid peptide, a 4-kDa protein that precipitates in the
amyloid plaques in the brains of Alzheimer's patients. The
metabolism of the protein is complex and occurs along several possible
pathways, all involving proteolytic cleavages of the precursor protein
(reviewed in (1) ). The normal secretory processing of APP
takes place in a late compartment of the default secretory pathway (2, 3, 4) by an
-secretase cleavage in
the
A4-amyloid peptide sequence
itself(5, 6, 7, 8) . This cleavage
step yields the soluble extracellular domain of APP (APP
)
and precludes further amyloid formation. APP that escapes from this
activity is transported to the cell surface and
endocytosed(9) . Alternative proteolytic cleavages, possibly in
the endosomes, release the
A4-amyloid peptide and/or the
p3-peptide(1) . These fragments are secreted with APP
by all cell types studied until now. Interestingly, amyloid
lesions in the brains of Alzheimer's patients are always
associated with polarized cell types, e.g. neurons and
endothelial cells. This raises the question whether APP and its
proteolytic products are transported and secreted in a polarized way, i.e. in neurons to the axons or dendrites and in epithelial
cells to the apical or basolateral side. It was recently demonstrated
that in primary cultures of hippocampal neurons, APP is transported
first to the axons and later to the
dendrites(10, 11) . In polarized epithelial
Madin-Darby canine kidney (MDCK) cells, APP and its various proteolytic
products (APP
,
A4, and p3) are strictly sorted to the
basolateral side(12, 13, 14, 15) .
It is therefore clear that APP is transported in a polarized way in
polarized cells. While the study of APP sorting in hippocampal neurons
is obviously more relevant to Alzheimer's disease, the two
functionally distinct domains of axons and dendrites cannot be
independently studied or manipulated in vitro, which hampers
the biochemical analysis of this model. In contrast, MDCK cells provide
a powerful in vitro system to study polarized membrane
trafficking of APP. They form a tight monolayer on tissue culture
filters in vitro, which results in a complete physical
separation of an upper (apical) and lower (basolateral) compartment.
This allows us to label and analyze APP separately on the apical versus basolateral surface of the cells and in
addition to follow the progressive accumulation of secreted APP
and
A4- and p3-peptides in the two
corresponding compartments. Moreover, since APP is endogenously
expressed by these cells, as kidney cells do in vivo, the
study of this system is physiologically relevant.
Mutational
analysis of the tyrosines in the cytoplasmic domain of APP has
demonstrated one tyrosine residue (Tyr-653) to be critical for the
basolateral surface sorting of the protein(13) . Remarkably,
the ectodomain of APP continued to be secreted into the basolateral
compartment independently of the sorting of the integral membrane form (13, 15) . This suggested two possibilities: either
-secretase is only present in the basolateral pathway, or an
independent basolateral sorting signal in the ectodomain of APP is
responsible for its basolateral delivery. Both alternatives are not
necessarily exclusive: the possible relationship between
-secretase processing of APP and sorting remains to be further
explored. Given that the amino acid sequence specificity of
-secretase at the cleavage site in APP is
low(3, 8, 16, 17) , the question is
raised how other integral membrane proteins in the default secretory
pathway escape this enzymatic activity. Specific targeting of APP by
undefined sorting signals to a ``secretase compartment'' (3) or specific binding of the secretase to a region of APP
outside the
A4-amyloid peptide sequence, lending specificity to
the cleavage process, are two possibilities that remain to be further
explored.
The potential role of the cytoplasmic sorting determinants
for polarized secretion and -secretase processing of APP was
further studied here with a reporter molecule containing the
cytoplasmic domain of APP and the minimal structural information needed
for
-secretase processing. Horseradish peroxidase (HRP) was chosen
as a reporter protein because its enzymatic activity permits
considerable signal amplification, and the end product can be
visualized using either light or electron microscopy(18) . We
demonstrate that a membrane-bound chimeric protein of horseradish
peroxidase fused to the transmembrane and cytoplasmic sequences of
wild-type APP (HRP/
A4/TM/CD) is enzymatically active both in the
culture medium and in fixed cells in situ. We show that the
chimeric protein is processed by
-secretase, similar to wild-type
APP. In polarized MDCK cells, however, the secretion of
HRP/
A4/TM/CD, in contrast to the secretion of APP, is randomized,
providing direct evidence that the basolateral sorting determinant for
polarized secretion of APP resides in its extracellular domain.
HRP/
A4/TM/CD that escaped
-secretase activity, on the other
hand, is targeted to the basolateral surface, indicating that the
cytoplasmic basolateral sorting determinants act independently of the
extracellular domain of APP. Finally, we investigated the effect of
alkalizing agents on
-secretase processing and sorting of
HRP/
A4/TM/CD, demonstrating that the default secretory pathway in
unpolarized COS cells and the basolateral pathway in MDCK cells (but
not the apical pathway) are similarly sensitive to methylamine and not
to bafilomycin as far as processing of APP and HRP/
A4/TM/CD is
concerned.
The extracellular domain of APP was replaced by horseradish
peroxidase in a recombinant DNA construct (see Fig. 1). A
cassette containing HindIII and PstI sites was cloned
into the unique AspI site of the cDNA coding for the
A4-amyloid region and the transmembrane and cytoplasmic domains of
murine APP695 (APP695(M)) (19) in the eukaryotic expression
vector pSG5 (Stratagene). The HindIII/PstI fragment
of BBG10 horseradish peroxidase (British Biotechnology Products Ltd.,
Abingdon, United Kingdom) engineered to mammalian codon usage (20) was cloned into the cassette. The resulting cDNA codes for
a HRP/
A4/TM/CD fusion protein (see Fig. 1). The two small
linker sequences needed to fuse the APP and HRP proteins are also
displayed (see Fig. 1). In HRP/
A4/TM/A666*, part of the
cytoplasmic domain including the GY.NPTY sequence was deleted by
introducing a stop codon at Ala-666 in APP695(M)(3) .
Figure 1:
Deletion mutants of mouse APP. The
domain structure of APP is schematically indicated. SP, signal
peptide; A4,
A4-amyloid peptide; TM,
transmembrane domain; CD, cytoplasmic domain. In the
peroxidase constructs, the complete cDNA coding for the extracellular
domain of APP was replaced by the cDNA of horseradish peroxidase
(mammalian codon usage). The short linker sequences inserted after the
APP signal peptide and between the HRP and the
A4-amyloid peptide
sequence are displayed in one-letter code for amino
acids.
The mouse APP695(M)Y653* mutant, in which the complete cytoplasmic domain is deleted (see Fig. 1), was generated by site-directed mutagenesis in the pSG5 plasmid(21) , mutating the codon for Tyr-653 toward a TAG stop codon. APP695(M)A626*, which terminates at the transmembrane domain, was made by introducing a cassette in the BbeI restriction site of APP695(M), changing codon 626 to a TAG stop codon. All constructs were analyzed by restriction analysis and by sequencing of the mutated sites(15) . The stop codons were functionally active since carboxyl-terminal antibodies did not react with the mutated proteins when expressed in COS-1 cells.
Transfection of pSG5 plasmids in COS-1 cells with DEAE-dextran has
been described(3) . For cytochemistry, COS-1 cells
(150,000/slide) were seeded into chamber glass slides (Lab-Tek, Nunc)
and transfected in situ the next day. After 2 days in culture,
slides were washed with phosphate-buffered saline, fixed and
permeabilized for 10 min in acetone, incubated for 60 min with
Tris-buffered saline containing 1 mM Ca and
5 µM hemin (Sigma), and stained for 15 min with 0.5 mg/ml
diaminobenzidine in 50 mM Tris-HCl (pH 7.4) and 0.01% (v/v)
H
O
. Secreted apoperoxidase in cell culture
medium was reconstituted with hemin (5 µM) in
Tris-buffered saline and 1 mM Ca
(30 min,
room temperature), and peroxidase activity was assessed
spectrophotometrically at 450 nm by measuring the rate of conversion of
1,2-phenylenediamine. A standard curve was obtained by making dilutions
from a 250 µM stock solution of apoperoxidase C (Sigma) in
Tris-buffered saline containing 1 mM Ca
and
5 µM hemin.
Transfection and culture of COS cells and
polarized MDCK cells and immune precipitation of APP have been
described in detail before(3, 15, 22) . After
surface biotinylation of MDCK cells expressing
HRP/A4/TM/CD(23) , rabbit anti-HRP polyclonal antibodies
(Dako Corp.) were used to immunoprecipitate HRP/
A4/TM/CD.
Biotinylated HRP/
A4/TM/CD was isolated on streptavidin-agarose
beads (Pierce), solubilized by boiling in 1% SDS and 1%
-mercaptoethanol, and electrophoresed on 4-20%
polyacrylamide gels (Novex). Autoradiograms were taken and analyzed by
densitometric scanning as previously described(3) .
The cDNA sequence coding for the extracellular domain of APP
was replaced by cDNA coding for horseradish peroxidase (mammalian codon
usage). In the resulting protein, the HRP enzyme is fused to the
A4-amyloid region and the transmembrane and cytoplasmic domains of
APP (Fig. 1). The fusion protein (HRP/
A4/TM/CD) was
expressed in COS-1 cells. In fixed and permeabilized cells, the
enzymatic activity of the chimeric protein was detected by
diaminobenzidine staining. Untransfected COS cells or cells transfected
with wild-type APP did not stain, demonstrating the specificity of the
staining reaction. In different experiments, 15-30% of the cells
stained positively, which approximates the expected transfection
efficiency of this procedure (Fig. 2). The results demonstrate
that HRP/
A4/TM/CD is functionally active and can be detected in situ. Staining was mainly concentrated in the Golgi region,
in accordance with previously published studies using
immunocytochemical detection of wild-type APP(24) .
Figure 2:
Expression of HRP/A4/TM/CD in COS-1
cells in situ. COS-1 cells were transfected with pSG5
containing the cDNA for HRP/
A4/TM/CD. Cells were fixed,
reconstituted with hemin and calcium ions as detailed under
``Materials and Methods,'' and stained with diaminobenzidine
and hydrogen peroxide. Note that staining is most pronounced in the
Golgi compartment, as has previously been demonstrated for wild-type
APP (24) .
Secreted HRP activity in the culture medium increased exponentially with time. These kinetics were identical to those previously observed for the secretion of wild-type APP(3) . Deletion of the cytoplasmic domain by introducing a stop codon at Ala-666 (numbering as in APP695) doubled the rate of secretion very similar to truncated wild-type APP (3) . The doubling is not caused by differences in transfection or translation efficiency as assessed in parallel pulse-chase experiments, which demonstrated that both constructs yield equal amounts of APP precursor under the conditions used.
Methylamine
strongly inhibited the secretion of HRP in a concentration-dependent
way ( Fig. 3and Table 1), while phorbol esters stimulated
secretion of both ``wild-type'' and ``truncated''
HRP/A4/TM/CD
proteins(3, 22, 23, 24, 25, 26, 27, 28, 29) .
Other known inhibitors of the secretory pathway such as monensin and
brefeldin A also strongly inhibited HRP/
A4/TM/CD secretion (Table 1). Secreted HRP/
A4/TM/CD reacted with antibody R47 (6) , which is directed against amino acids 1-16 of the
A4-amyloid peptide (data not shown). No reaction with antibodies
against the cytoplasmic domain of APP (15) or with monoclonal
antibody 4G8 against amino acids 17-24 of the
A4-amyloid
peptide (30) could be demonstrated, providing further support
to the conclusion that HRP release reflects authentic
-secretase
processing.
Figure 3:
Secretion of HRP/A4/TM/CD. COS-1
cells were transfected with pSG5 containing the cDNA either for
HRP/
A4/TM/CD (labeled WT) or with the cytoplasmic domain
deleted (mutant HRP/
A4/TM/A666*; labeled TRUNC) in normal
medium and in the presence of 30 mM methylamine (MA).
The cell culture medium was sampled at regular time intervals, and
peroxidase activity was measured with a colorimetric assay. All points
are the means ± S.E. of three separate transfection
experiments.
The importance of the integral membrane domain for
-secretase processing of APP was further confirmed by
investigating the proteolytic processing of deletion mutants of APP (Fig. 1). Deletion of the entire cytoplasmic domain of APP
(APP695(M)Y653*) does not interfere with its normal secretion (Fig. 4A). Deletion of the cytoplasmic and
transmembrane domains of APP (APP695(M)A626*) yields a soluble protein (Fig. 4B). However, Western blotting of the secreted
products of the different constructs with monoclonal antibody 4G8 (30) revealed a strong signal only for APP695(M)A626* (Fig. 4B), demonstrating that this soluble APP mutant
is not proteolytically processed.
Figure 4:
Secretion of deletion mutants of APP. A, COS-1 cells were transfected with wild-type APP695(M) (WT), APP695(M)A666*, APP695(M)Y653*, and APP695(M)A626* (see Fig. 1). Cells were metabolically labeled for 4.5 h, and APP was
immunoprecipitated with antibody B2/3 against APP(3) . B, shown is an immunoblot of the cellular (C) and
secreted (S) forms of APP695(M)A626* with monoclonal antibody
4G8. This antibody recognizes an epitope in the A4-amyloid peptide
sequence carboxyl-terminal to the
-secretase cleavage
site.
HRP/A4/TM/CD was stably
expressed in MDCK cells using the Rous sarcoma virus
promoter(15) , and its polarized distribution was investigated
using selective cell-surface biotinylation (Fig. 5A).
More than 90% of the surface-expressed HRP/
A4/TM/CD was located at
the basolateral side of the MDCK cells, which is similar to wild-type
APP(13) . In contrast, 41% of the total pool of secreted
HRP/
A4/TM/CD was present in the apical compartment and 59% in the
basolateral compartment, indicating that HRP/
A4/TM/CD is secreted
essentially randomized (Fig. 5B). Wild-type
(endogenous) APP was detected only in the basolateral
compartment(12, 15) .
Figure 5:
Surface expression and secretion of
HRP/A4/TM/CD from MDCK cells. A, the apical (bar
A) and basolateral (bar B) cell-surface expression of
HRP/
A4/TM/CD in transfected MDCK cells was measured using a
biotinylation assay (see ``Materials and Methods'') and is
expressed as the relative amount of apical and basolateral detected
HRP/
A4/TM/CD relative to the total (apical + basolateral)
amount detected on the cell surface. Result are the means of two
independent experiments. B, the apically (bar A) and
basolaterally (bar B) secreted HRP activities were measured
and are expressed relative to the total (apical + basolateral)
secreted HRP. The means ± S.E. of four independent experiments
are displayed. C, the apical (bar A) and basolateral (bar B) secretion of peroxidase activity from transfected MDCK
cells preincubated for 30 min without or with 30 mM
methylamine (MA) or 100 nM bafilomycin (Baf)
was measured. The total amount of peroxidase activity in the apical and
basolateral compartments is displayed as the mean ± S.E. of four
independent experiments. SUP,
supernatant.
We compared the effects of
bafilomycin, a specific vacuolar proton pump inhibitor, and
methylamine, a drug that accumulates in and alkalizes acid cell
organelles, on the secretory processing and sorting of
HRP/A4/TM/CD in COS and MDCK cells. In COS cells,
HRP/
A4/TM/CD secretion was somewhat increased by 100 nM
bafilomycin, while secretion was strongly inhibited by 30 mM methylamine (Table 1). In MDCK cells, the same effect was
observed, but only for the basolateral pathway: HRP/
A4/TM/CD
secretion was somewhat increased by bafilomycin, but was strongly
inhibited by methylamine (Fig. 5C). Remarkably, a
4-5-fold increase in apical secretion was observed with both
drugs (Fig. 5C).
This work, combined with previously
observations(12, 13, 14, 15) ,
demonstrates unequivocally that two separate sorting mechanisms are
responsible for the basolateral targeting of APP in polarized MDCK
cells. The properties of these two mechanisms are summarized in Table 2. One mechanism is responsible for the basolateral secretion of soluble APP and interacts with
unknown determinant(s) that must be located in the extracellular domain
and are thus deleted in the HRP/
A4/TM/CD chimeric protein, as
demonstrated by the randomized secretion in the apical and basolateral
compartments. More important, since the chimeric protein used in this
study contained the entire
A4-amyloid peptide sequence, it follows
that the
A4-amyloid region is not involved in the basolateral
targeting of APP
. The second mechanism is responsible for
the polarized basolateral cell-surface expression of APP. This
mechanism must act on the cytoplasmic domain of APP and directs
HRP/
A4/TM/CD that escaped
-secretase cleavage to the
basolateral surface (Table 2). The results complement perfectly
previous observations that demonstrated that deletions or mutations of
the cytoplasmic domain of APP randomized its cell-surface distribution,
but did not affect its polarized secretion(13, 15) .
Although the sorting information in both domains works synergistically
when intact APP is concerned, the mechanisms do act independently from
each other ( (13) and (15) and this work). While no
precedent exists for a protein with a similar sorting behavior, the
conservation of the sequences in the cytoplasmic domain of APP (13, 15) strongly suggests that the mechanism
responsible for its basolateral cell-surface expression is identical to
that operative on the low density lipoprotein receptor(31) .
Since membrane-bound and soluble HRP/
A4/TM/CD proteins are sorted
differently, the present data further demonstrate that
-secretase
is active in MDCK cells before the last sorting station, the
basolateral endosome, is reached (3, 13, 15) .
We have previously shown
that pH and primary amines have profound differential effects on the
-secretase processing and targeting of APP, implying that
metabolic disturbances can change the fate of APP (3, 15) . Primary amines accumulate in acid
compartments of the cell, resulting in their alkalization, and
interfere with normal functions such as degradation in the lysosomes or
receptor-ligand dissociation in the endosomes. In addition, methylamine
inhibits the processing of proteins in the late compartments of the
default secretory pathway in certain cell
types(32, 33) . The randomized secretion of APP from
MDCK cells after treatment with alkalizing drugs (15) is most
likely caused by the disruption of its interaction with the proposed
basolateral sorting mechanism for soluble APP
. A similar pH
sensitivity of the polarized secretion of laminin and basal membrane
proteoglycan has been demonstrated(34) , as summarized in Table 2. Methylamine, but not other alkalizing drugs, inhibited
the secretion of APP in unpolarized COS cells(3, 35) .
In polarized MDCK cells, methylamine also inhibited APP secretion, but
only in the basolateral compartment, and not in the apical
compartment(15) . Since bafilomycin and concanamycin, both
specific inhibitors of the vacuolar proton ATPase(36) , did not
inhibit, but augmented secretion of the peroxidase chimeric protein
from COS cells, the inhibition by methylamine of both HRP/
A4/TM/CD
and APP secretion in COS cells cannot be caused by alkalization of the
acidic compartment that is sensitive to bafilomycin. Alternatively,
methylamine could block or divert the transport of HRP/
A4/TM/CD
and APP and thereby prevent the normal processing in the
-secretase compartment(3) . HRP/
A4/TM/CD secretion
from COS cells is inhibited by methylamine to the same extent as by
brefeldin A and monensin, drugs that are known to inhibit transport
along the default secretory pathway. Surprisingly, in MDCK cells,
methylamine blocks only the basolateral secretion of APP and
HRP/
A4/TM/CD. Clearly, the methylamine-sensitive step is operative
in the default secretory pathway of COS cells and in the basolateral
(but not in the apical) pathway of MDCK cells. This adds more evidence
to the emerging concept that different mechanisms are responsible for
the apical and basolateral transport of proteins in MDCK
cells(37, 38) . Finally, the increase in apical
secretion induced by methylamine and bafilomycin probably reflects the
redistribution of APP from a degradative pathway toward the apical
secretory pathway, which remains to be investigated.
Only recently
was soluble HRP expressed in an active form in eukaryotic
cells(19) . We demonstrate here that HRP is enzymatically
active when expressed as a chimeric integral membrane protein, which
opens the possibility of using HRP as a marker in a variety of protein
transport assays. Compared with alkaline
phosphatase(39, 40) , horseradish peroxidase offers
several advantages as a reporter molecule(19) . We anticipate
using the HRP/A4/TM/CD fusion protein to analyze the intracellular
pathways as well as to screen for candidate
-secretases in a
cellular environment(41) . This is possible because we
demonstrate here that the extracellular domain of APP, although
implicated in the normal transport and targeting of APP, is not needed
for normal
-secretase processing. This, together with the results
obtained with the APP deletion mutants, demonstrated that the essential
information for
-secretase processing is completely contained in
the
A4-amyloid and transmembrane sequences, which constitute
<8% of APP (and HRP/
A4/TM/CD). The molecular explanation of the
paradox that the nonspecific
-secretase is located in the default
secretory pathway, but does not cleave all integral membrane proteins
it encounters, must therefore be found in its interaction with and,
hence, the structure of the integral membrane domain of
APP(8) . Another integral membrane protein, transforming growth
factor
, is processed by a similar secretase mechanism,
characterized by low sequence specificity and activated by protein
kinase C(42) . The cytoplasmic domain of transforming growth
factor
, however, is absolutely needed for proteolytic cleavage,
and replacement of the terminal valine in this domain prevents
processing(43) . In the case of APP and HRP/
A4/TM/CD, the
cytoplasmic domain is not needed at all, indicating that at least two
different ``adaptor'' mechanisms are operating to permit
transforming growth factor
and APP to interact with the putative
nonspecific
-secretase proteolytic complex. This also implies that
many other proteins could be processed by the same mechanism provided a
suitable adaptor protein is present.