From the Center for Molecular Biology Heidelberg,
University of Heidelberg, INF 282, D-69120 Heidelberg, Germany, the
¶ Department of Pathology, University of Melbourne, Parkville,
Victoria 3052, Australia, and the ** Massachusetts General
Hospital/Harvard Medical School,
Boston, Massachusetts 02114
Received for publication, October 1, 2002, and in revised form, November 6, 2002
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ABSTRACT |
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The amyloid precursor protein is cleaved within
its ectodomain by An early event in the pathogenesis of Alzheimer's disease is the
generation of the amyloid In addition to its broad substrate specificity, Because of the lack of direct experimental proof for the To resolve these controversies and to better understand the mechanism
of Our results show that the apparent TMD of C99 inserted into ER-derived
membranes is significantly shorter than previously assumed and
comprises only 12 amino acids instead of the 24 residues that are
predicted by computer algorithms. Importantly, we found that the
These data indicate that the exact positioning of the APP TMD with
respect to the membrane may determine the cleavage site and, thus, that
the cleavage may occur at the exact center of the lipid bilayer.
Plasmid Construction--
The single cysteine mutants of C99
were generated by PCR using the QuikChange site-directed mutagenesis
kit (Stratagene, Amsterdam), suitable oligonucleotides, and the plasmid
pBS/SPC99 as template (17). The sequence of all constructs was
confirmed by DNA sequencing.
In Vitro Transcription and Translation--
The experiments
described were performed with C99 mutants expressed from the SP-C99
encoding plasmids by in vitro transcription and translation
using the RiboMAX large scale RNA production system (Promega) for
transcription and the rabbit reticulocyte lysate system (Promega) for
translation. Alternatively, coupled transcription and translation
reactions were performed with the TnT T7 master mix system (Promega).
Reactions were incubated for 90 min at 30 °C with
[35S]methionine in the presence or absence of canine
microsomal membranes (Promega).
Immunoprecipitation--
5 µl of the in vitro
translation reaction before or after labeling with IASD were
diluted in 500 µl of solubilization buffer (50 mM Tris,
pH 7.5, 150 mM NaCl, 1% Nonidet P-40) and incubated for
2 h with 20 µl of protein A-Sepharose (100 mg/ml, Amersham Biosciences) and 5 µl of the polyclonal antibody 22/13, which was raised against the C-terminal 13 residues of C99. For the analysis
on isoelectric focusing (IEF) gels, 40 µl of the in vitro translation reaction were used and immunoprecipitated as described above but in a volume of 1 ml. Protein was recovered in 40 µl of
sample buffer for PAGE or in 30 µl of denaturing buffer for IEF gel
analysis (according to the IPGphor system instructions; Amersham Biosciences).
Membrane Sedimentation and Sodium Carbonate Extraction--
For
the IASD labeling of the C99 mutants with a cysteine placed at the
N-terminal lumenal side of the membrane, the microsomal vesicles were
converted into membrane sheets prior to IASD reaction (30). 30-50 µl
of the in vitro translation reaction were mixed with 750 µl of 100 mM sodium carbonate, pH 11.5, and incubated at
0 °C for 30 min. Membranes were recovered by ultracentrifugation in
the Beckman 45 TLi rotor at 50,000 rpm and 4 °C for 45 min. For the
subsequent IASD labeling reaction, the membrane pellets were rinsed
once with ice-cold distilled water and then dissolved in 5-10 µl of
0.5 M sodium phosphate.
Cysteine Modification by IASD and Gel-shift
Electrophoresis--
Each radiolabeled single cysteine mutant of C99
(6 µl of the in vitro translation reaction) was incubated
in 0.5 M sodium phosphate, pH 7.5, containing 10 mM dithiothreitol (6 µl) for 5 min at room temperature.
Then 100 mM IASD (MoBiTec, Göttingen, Germany) was
added to a final concentration of 20 mM IASD and 4 mM dithiothreitol (31). After incubation for 30 min
at room temperature, the remaining IASD was inactivated with 40 mM dithiothreitol. For each labeling experiment a negative
control, in which water was used instead of IASD, was run in parallel.
C99 was immunoprecipitated and analyzed on expanded Tris/Tricine gels
or on isoelectric focusing gels with an immobile pH gradient (pH 3-10,
13 cm, IPGphor isoelectric focusing system; Amersham Biosciences).
Radiolabeled proteins separated by electrophoresis were visualized
using phosphorimaging (BAS 1000), whereas those separated by
isoelectric focusing were subjected to autoradiography with the help of
the Kodak intensifying screen (BioMax TranScreen LE).
For IASD labeling after membrane disruption, the in vitro
transcription/translation reactions were incubated in an equal volume of 0.5 M sodium phosphate, pH 7.5, containing 10 mM dithiothreitol and 2% Nonidet P-40 for 10 min at room temperature.
Cysteine-scanning Mutagenesis of C99--
To biochemically
determine the TMD length of C99, we performed a single cysteine
mutagenesis scan of C99. Because C99 does not contain any cysteine
residue, we replaced individual residues within and just outside of the
TMD of C99 by a cysteine, such that each of the 23 C99 mutants
contained one cysteine (Fig. 1). In a
coupled in vitro transcription/translation experiment, these mutants were inserted into canine microsomes that derive from the
membrane of the endoplasmic reticulum. The C99-containing microsomes
were then incubated with the membrane-impermeable cysteine-labeling reagent IASD. Because of its hydrophilic character, IASD
cannot diffuse into the lipid bilayer and therefore only labels
cysteine residues that are located outside the membrane (29, 31). IASD labeling was subsequently analyzed by one- and
two-dimensional gel electrophoresis.
Membrane Insertion of SP-C99--
To ensure the correct insertion
of C99 into the microsomal membranes, an N-terminal signal peptide (SP)
was fused to the C99 constructs (SP-C99, Fig. 1). The signal peptide is
cleaved by signal peptidase upon membrane integration, thus converting
SP-C99 into C99 (32-34). The C99 proteins were immunoprecipitated from the solubilized membranes with antibody 22/13 that binds to the C
terminus of C99 and analyzed by polyacrylamide gel electrophoresis. A
single band with the expected apparent molecular mass of 12 kDa was
detected (Fig. 2). In a control
experiment without membranes, the signal peptide of SP-C99 was not
cleaved and the immunoprecipitated SP-C99 had an apparent molecular
mass of 11.5 kDa (Fig. 2). Furthermore, SP-C99 showed extensive
aggregation, as seen by the numerous high molecular mass bands (Fig.
2). Because of the presence of the signal peptide, SP-C99 contains 17 more amino acids than C99. Nevertheless SP-C99 showed a lower apparent
molecular mass than C99, which might be because of a more compact
secondary structure of SP-C99 induced by the presence of the
hydrophobic signal peptide. This observation is in agreement with
previous studies (32, 35). The difference in molecular mass between
SP-C99 and C99 was observed for C99 wt and all cysteine mutants (shown
in Fig. 2 for representative mutants). This verifies that all C99
proteins were completely inserted into the microsomal membranes and
that the signal peptide was cleaved. In addition to the 12-kDa band of
C99, a 6.5-kDa protein could be detected (Fig. 2) that is likely to be
the result of an internal transcription starting point at methionine
35. As a further control for the correct membrane integration of the
mutants, we verified that the C99 proteins translated in the presence
of microsomal membranes could be recovered by sodium carbonate
treatment and subsequent membrane sedimentation of the microsomes. This
method allows the recovery of integral membrane proteins, whereas
soluble proteins and peripheral membrane proteins do not sediment
together with the microsomes (30). C99 wt and the C99 mutants were
mainly detected in the integral membrane protein fraction (data not
shown), demonstrating efficient membrane integration of the C99
mutants.
Molecular Mass Shift after IASD Treatment--
To determine
whether the individual cysteine residues of the C99 mutants are located
within or outside of the microsomal membrane, we used IASD, a reagent
that specifically reacts with free sulfhydryl groups, i.e.
the single cysteine residues in the C99 mutants. IASD contains two
sulfonate groups (pKa ~-6.5), which are fully ionized under our reaction conditions (pH 7.5), preventing diffusion into the membrane (29, 31). Thus, cysteine residues of C99 that are
buried within the lipid bilayer are not labeled. Because the N terminus
of C99 is located in the lumen of the microsomal vesicles, those
residues located on the N-terminal (lumenal) side of the TMD are not
directly accessible to the membrane-impermeable IASD. Therefore, when
analyzing the mutants C99/24C-40C the membrane vesicles were converted
to membrane sheets by sodium carbonate treatment prior to IASD
treatment. To verify that this method did not change the positioning of
C99 within the membrane, we controlled that the sodium carbonate
treatment did not interfere with the C-terminal (cytosolic) borderline
of the IASD-inaccessible domain (data not shown) as we have identified
it in this study.
IASD modification adds a mass of 450 Da to C99, which may change the
electrophoretic mobility of C99, as has been reported previously for
other proteins (29, 31). After incubation with and without IASD, the
different C99 mutants were immunoprecipitated and the electrophoretic
mobility was compared. Under these conditions only a minor, not
significant, fraction of the negative control (C99 wt) showed
unspecific labeling (Fig. 3).
IASD treatment of the C99 mutants with a cysteine positioned N-terminal
of residue 33 and C-terminal of residue 49 led to a clear shift to a
higher apparent molecular mass (Fig. 3 and Table
I). This demonstrates that the individual
cysteine residue in these mutants (at positions 24-32 and 50-57) had
been labeled by IASD and thus was located outside the microsomal
membrane. In contrast, for cysteines at positions 33-49 and C99 wt no
gel shift was observed (Fig. 3 and data not shown). For the mutants with a cysteine between residue 33 and 40, an IASD-induced shift could
not even be detected in a control experiment in which the mutants were
IASD-treated in a non-membrane-embedded state (data not shown but
summarized in Table I). Therefore, we considered the possibility that
IASD modification at these residues may have occurred without an
alteration in the apparent electrophoretic mobility.
Analysis of IASD Modification by Isoelectric Focusing--
To
answer this question we used IEF. The two negative charges added to C99
upon IASD modification lower its isoelectric point (IEP) from an IEP of
6.09 to 5.52 (as calculated by the computer program DNAStar), which
should be detectable as a shift on isoelectric focusing gels with an
immobilized, linear pH gradient. To validate IEF as a method for
determining the IASD modification of C99, we first analyzed as positive
controls C99 mutants that had shown a shift in molecular mass
(C99-V24C, V50C, and M51C, Fig. 3) and as negative control C99 wt. C99
wt with or without IASD treatment showed a single band at the expected
calculated IEP of pH 6 (Fig. 4) and thus
was not modified by IASD. In contrast, IASD treatment of C99/V24C,
-V50C, and -M51C led to a clear shift from an IEP of 6 to 5.5, as
expected (Fig. 4). This shows that IASD labeling can be detected well
on IEF gels. In some experiments an additional band with an IEP of 5.8 was observed for unknown reasons. However, a protein with an IEP of 5.8 carries only one additional negative charge compared with C99, which
therefore cannot be because of IASD labeling. This is further confirmed
by the fact that this protein band with an IEP of 5.8 did not occur
consistently in identically performed experiments and even in the
absence of IASD (see Fig. 4, C99/L34C as an example). This verifies
that this IEP isomer of C99 is irrelevant for our analysis because it
does not interfere with the analysis of IASD modification. The identity of the protein bands for C99 was confirmed by two-dimensional gels on
which the proteins showed the expected molecular mass of 12 kDa
(without IASD modification) and 12.5 kDa (after IASD modification; data
not shown, but compare Fig. 2). The clear shift seen on IEF gels after
IASD treatment of C99 mutants validates IEF as a method for determining
whether IASD modification of C99 has occurred.
We next analyzed whether those C99 mutants that did not show a gel
shift in their molecular mass upon IASD treatment showed a shift in
their IEP. IASD incubation of the C99 mutants L34C, M35C, V36C, and
L49C (Fig. 4) resulted in a clear shift in the IEP from 6 to 5.5, indicating that IASD modification had occurred and that this
modification can be detected on IEF gels. These data suggest that the
corresponding cysteine residues at positions 34, 35, 36, and 49 were
located outside the microsomal membrane. In contrast, C99 mutants with
cysteines at positions 37, 38, 39, 40, 44, 45, 47, and 48 did not show
a shift in IEP on IEF gels (Fig. 4), suggesting that they are buried
within the membrane bilayer. Moreover, when the microsomal membranes
were disrupted by the addition of a detergent (1% Nonidet P-40, for 10 min), the mutants showed the expected shift upon IASD labeling,
revealing that they can be labeled with IASD when no longer shielded by the membrane. This finding confirms that IASD does not label
membrane-inserted protein portions, which is in good agreement with
previous work of Krishnasastry et al. (29) that showed that
a cysteine mutant of
In summary, the IASD labeling analysis of all membrane-inserted C99
mutants shows that only 12 of the 24 residues of the putative TMD of
C99 (residues at position 37 to 48) are not accessible to IASD
modification and thus constitute that part of C99 that is buried in
microsomal membranes and constitute the TMD of C99 in ER-derived
membranes (schematically shown in Fig.
5).
The In our analysis we found that IASD modification occurred for all
cysteine residues of C99 located N-terminal of residue 37 and
C-terminal of residue 48, but cysteine residues at positions 37-40,
44, 45, 47, and 48 were not modified with IASD. We conclude from this
that the 12 residues, 37 to 48, of C99 are shielded from IASD
modification by the microsomal membrane and thus represent the actual
domain of C99 or APP, respectively, that is embedded within the
ER-derived membrane. This domain is significantly shorter on both
sides than the 24 hydrophobic residues that are predicted by computer
algorithms (37).
IASD is highly charged and therefore cannot diffuse into or through the
lipid bilayer, but it might enter the region of the polar head groups
of the lipids on the surface of the membrane. Furthermore, the
thiol-reactive iodoacetyl group of IASD is a few Å apart from the
charged sulfonate groups. Although the sulfonate groups are supposed to
stay outside the membrane and confer membrane-impermeability to IASD,
the hydrophobic thiol-reactive group may slightly dip into the membrane
and react with a cysteine positioned just below the membrane boundary.
This suggests that the exact border of the membrane-embedded protein
portion may slightly differ, depending on the hydrophilicity, charge,
and structure of the molecule interacting with the protein. The 12 amino acids that are IASD-inaccessible are flanked on both sides by
hydrophobic residues and may permit a perpendicular movement of the TMD
with respect to the membrane, such that over a given time more than the
12 residues are in contact with the membrane. In such a scenario it
would be expected that a partial IASD labeling of the neighboring
residues occur. Because labeling with IASD is supposed to be a very
fast reaction, this effect may be visible only under less severe
labeling conditions. However, we never detected such partial labeling,
not even with reduced IASD concentrations and very short incubation
times (data not shown). Accordingly, our study clearly shows
that C99 inserted into an ER-derived membrane has a domain of 12 residues (37 to 48) that is never accessible to IASD-modification and
therefore represents the effective TMD of C99, which is expected to be
inaccessible to cytosolic proteases.
This result is in good agreement with theoretical considerations of the
expected number of residues needed to span the ER membrane as well as
with previous experiments analyzing the membrane integration of APP.
The thickness of cellular membranes, and thus the number of residues
needed to span the membrane, increases along the secretory pathway from
the ER toward the plasma membrane because of increasing cholesterol and
sphingolipids in the corresponding membranes (38). A comparison of
proteins typically localized at the Golgi or plasma membrane shows that
the minimum number of hydrophobic residues in plasma membrane TMDs is
20 residues whereas that in Golgi TMDs is 15 (39). Because the ER
membrane is thought to be even thinner than the Golgi membrane (38), less than 15 hydrophobic residues is expected to be sufficient to span
ER-like membranes, which agrees well with the 12 residues of C99 that
we have determined to be IASD-inaccessible. Because the bilayer
thickness increases along the secretory pathway, presumably all 24 hydrophobic residues of the predicted TMD of C99 are used to span
the plasma membrane (39, 40).
Our results are also in agreement with previous mutagenesis studies
that suggested that the TMD of APP might be shorter than predicted on
its C-terminal side (18, 19, 28). One of these studies showed that the
introduction of negatively charged aspartyl residues into the putative
TMD of APP interfered with membrane integration and We demonstrate in this study that in the relatively thin membrane of
the ER residues 37 to 48 of C99 are shielded by the membrane. This has
important implications for the mechanism of Recently, it has been found that presenilin 1 is required not only for
In summary, our study strongly supports the existence of a real
intramembrane cleavage for APP. Furthermore, it suggests a model of the
mechanism of -amyloid-converting enzyme (BACE) yielding
C99, which is further cleaved by
-secretase within its putative
transmembrane domain (TMD). Because it is difficult to envisage how a
protease may cleave within the membrane, alternative mechanisms have
been proposed for
-cleavage in which the TMD is shorter than
predicted or positioned such that the
-cleavage site is accessible
to cytosolic proteases. Here, we have biochemically determined
the length of the TMD of C99 in microsomal membranes. Using a single
cysteine mutagenesis scan of C99 combined with cysteine modification
with a membrane-impermeable labeling reagent, we identified which
residues are accessible to modification and thus located outside of the membrane. We find that in endoplasmic reticulum-derived
microsomes the TMD of C99 consists of 12 residues that span from
residues 37 to 48, which is N- and C-terminally shorter than predicted. Thus, the
-cleavage sites are positioned around the middle of the
lipid bilayer and are unlikely to be accessible to cytosolic proteases.
Moreover, the center of the TMD is positioned at the
-cleavage site
at residue 42. Our data are consistent with a model in which
-secretase is a membrane protein that cleaves at the center of the membrane.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
peptide
(A
),1 which is later
deposited in the amyloid plaque. The 4-kDa peptide A
is a product of
the complex proteolytic processing of the amyloid precursor protein
(APP) (for a review, see Ref. 1). Some features of the APP processing
are shared with the processing of the cell surface receptor Notch (for
a review, see Ref. 2). Both are type I membrane proteins that undergo a
first proteolytic cleavage within their ectodomain, leaving a
C-terminal fragment within the membrane. These C-terminal fragments may
then undergo proteolytic cleavage within their transmembrane domains
(TMD), a process termed intramembrane proteolysis (for a review, see
Ref. 3). Upon intramembrane proteolysis, the cytosolic portions of
these proteins may move to the nucleus and stimulate the transcription
of target genes (4-9). In the proteolytic pathway that leads to the
generation of A
, APP is first cleaved within its lumenal domain by
the recently identified aspartyl protease,
-amyloid-converting
enzyme (for a review, see Refs. 10 and 11). The resulting
99-residue-long C-terminal fragment of APP (C99) can then be cleaved
intramembranously by a protease activity called
-secretase, which
leads to the release and secretion of the A
peptide (for a review,
see Ref. 1). Despite its importance for the pathogenesis of
Alzheimer's disease,
-secretase has not yet been unequivocally
identified. However, the
-cleavage of C99 depends on the presence of
the membrane protein presenilin 1, which itself could be
-secretase or part of a larger
-secretase complex (for reviews, see Refs. 2 and
12).
-Cleavage occurs predominantly after residue 40 of C99 and to a
minor extent after residue 42, thus generating the 40- and 42-residues-long peptides A
40 and A
42
(13-16). Little is known about the mechanism of
-cleavage and the
factors that determine whether the cleavage takes place after residue
40 or 42 of C99. Intensive mutagenesis of amino acid residues within
the TMD of C99 has shown that the generation of A
is not
sequence-specific (17-23). In contrast, the specific position where
-cleavage takes place (i.e. cleavage after residue 40 or
residue 42) strongly depends on the length of the TMD of C99 (18,
21).
-cleavage is
remarkable because it occurs within the predicted TMD of C99. Recently,
an increasing number of proteins have been reported to be cleaved
intramembranously, but so far no protease has been proven to cleave
within the membrane. Nevertheless, the metallo-protease S2P, the
putative serine protease rhomboid, and presenilin 1 have their active
site residues within their predicted TMDs (24-27).
-cleavage
taking place within the lipid bilayer, alternative structures and
membrane boundaries for the TMD of C99 have been proposed. These models
suggest that the C-terminal half of the putative TMD and the
-cleavage site are not buried within the membrane but instead are
exposed to the cytosol and accessible for a soluble or
membrane-associated protease (18, 19, 28). However, the true mechanism
remains elusive because a thorough characterization of the length of
the TMD of C99 in biological membranes is still lacking.
-cleavage, we biochemically determined the length and the
position of the TMD of C99. In a cysteine mutagenesis scan, we created
several single cysteine mutants of C99 with a cysteine placed within or
adjacent to its predicted TMD. In vitro, the single cysteine
mutants of C99 were translated directly into microsomal membranes,
which derive from the endoplasmic reticulum. The C99-containing
microsomes were incubated with IASD (4-acetamido-4'-((iodoacetyl) amino) stilbene-2,2'-disulfonate), which specifically labels cysteine residues. Because IASD is membrane-impermeable (29), only those cysteine residues are labeled that are located outside the membrane.
-secretase cleavage site after residue 42 is positioned exactly at
the center of the newly defined TMD.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the SP-C99 single
cysteine mutants. SP-C99 consists of the 17-residue-long signal
peptide (SP) of APP followed by a two-amino acid spacer (Leu
and Glu) and the C-terminal 99 amino acids (C99) of APP.
Amino acids are shown in the one letter code. The residues of wild type
C99 that were replaced by a cysteine are indicated in black.
Every SP-C99 mutant contains only a single cysteine residue. The
hydrophilicity plot of SP-C99 (using the scale of Kyte and Doolittle
(47) over a window of 9 residues) indicates hydrophobicity by negative
numbers (kcal/mol) and shows that residues 29 to 52 are the predicted
TMD of SP-C99.
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Fig. 2.
Membrane integration and removal of the
signal peptide (SP) demonstrated by gel shift
electrophoresis. SP-C99 was in vitro-translated in the
presence (+) or absence ( ) of microsomal membranes
(membr.), metabolically labeled with
[35S]methionine, immunoprecipitated with antibody 22/13,
and separated by PAGE. C99 (after signal peptide cleavage (+)) has a
higher apparent molecular mass than SP-C99 (without cleavage of the
signal peptide (
)). Numbering refers to the residue of C99 that was
exchanged by a cysteine.
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Fig. 3.
IASD modification analysis of membrane-bound,
35S-labeled C99 single cysteine mutants. In
vitro translations in the presence of microsomal membranes of C99
wt and C99 single cysteine mutants were treated with IASD (+) or water
( ), immunoprecipitated, separated by high-resolving Tris-/Tricine
PAGE, and visualized using phosphorimaging (shown for a selection of
the C99 mutants). The appropriate section of the gel shows a shift to a
higher apparent molecular mass for IASD-treated samples of some C99
mutants, whereas for other mutants no shift could be detected
(summarized in Table I).
Results of the IASD labeling analysis of all C99 single cysteine
mutants and C99 wt with (middle column) and without membrane insertion
(left and right column) by molecular mass (MW) and by isoelectric point
(IEP)
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Fig. 4.
a, IASD modification of membrane-bound
C99 mutants demonstrated by a shift in the IEP on isoelectric focusing
gels with an immobilized, linear pH gradient. The in vitro
transcription/translation sample was IASD-treated, immunoprecipitated,
separated by its IEP, and visualized by autoradiography. Numbers
indicate the position of the individual cysteine mutation in the C99
sequence. After IASD treatment, (+)-labeled C99 mutants shifted from an
IEP of ~6 for untreated samples ( ) to an IEP of ~5.5 (marked by a
rectangle), as expected. The absence of such a shift in IEP
seen for C99 wt and several mutants indicates that IASD labeling did
not occur (highlighted in gray). b, control of
general accessibility of the cysteines to IASD after membrane
solubilization. After incubation of the in vitro
transcription/translations in the presence of a detergent, all mutants
show the IASD-induced shift in the IEP, confirming that the absence of
the shift in Fig. 4a for some mutants is because of the
position of their cysteines within the lipid bilayer.
-hemolysin could not be labeled with IASD when
embedded into membranes, although it was rapidly labeled in the absence
of membranes. Therefore, the absence of a shift in the IEP for a
membrane-inserted single cysteine mutant indicates that this cysteine
is located within the microsomal membrane. This experiment further
shows that IEF provides a method to overcome problems in the detection of IASD labeling that sometimes occur with analysis on conventional PAGE and that IEF can be used to analyze IASD labeling of all our C99
single cysteine mutants.
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Fig. 5.
Schematic representation of the IASD
modification pattern for membrane-bound C99 mutants. Amino acids
are shown in the one letter code. Residues exchanged to a cysteine in
one of the 19 single cysteine mutants are indicated by
color; those that were labeled by IASD are marked in
gray; those, that could not be labeled, in black.
Please note that the middle of the predicted transmembrane domain is
positioned at -cleavage site 40, whereas the middle of the TMD in
microsomal membranes is positioned at
-cleavage site 42.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-secretase cleavage of the C-terminal APP fragments is a
remarkable proteolytic mechanism as it occurs around the center of the
putative TMD of APP (for a review, see Ref. 2). Because direct evidence
is lacking that the
-cleavage takes place within the lipid bilayer,
alternative boundaries for the TMD of C99 have been proposed such that
a cytosolic or membrane-associated protease could cleave C99 (18, 19,
28). However, these studies only analyzed one half but not the total
length of the TMD. Thus it remained unclear whether the
-cleavage
occurs within the membrane. To analyze whether the
-cleavage sites
of APP are indeed located within the membrane or are exposed to
cytosolic cleavage, we biochemically determined the whole length of the
TMD of C99 in microsomal membranes. For this analysis, we carried out a
cysteine mutagenesis scan of C99, which is a widely employed technique
for topology studies of membrane proteins (for a review, see Ref. 36).
The cysteine mutants of C99 were inserted into membranes and labeled
with the membrane-impermeable, cysteine-specific reagent IASD.
-secretase
processing, whereas aspartyl residues introduced into the C-terminal
half of the putative TMD did not (19). The authors concluded that the
C-terminal membrane boundary is placed around residues 46 or 47. Given
that the introduction of a negatively charged residue near the membrane
may easily shift the position of C99 with respect to the membrane,
their finding is nearly identical to our biochemical analysis, which
identifies the TMD boundary to be after residue 48.
-cleavage. Because the
major
-cleavage sites after residues 40 and 42 of C99 are positioned
around the middle of the IASD-inaccessible domain, they are also
expected to be inaccessible to cytosolic or membrane-associated
proteases, which had been considered a possibility in previous studies
(18, 19, 28). This suggests that C99 undergoes intramembrane cleavage
by a membrane protein having its active site residues within its TMD. A
candidate for such a protease is the polytopic membrane protein,
presenilin 1, which is required for
-cleavage (for review, see Refs.
2 and 12). In this context it would be interesting to know whether the
aspartate residues in the putative active site of presenilin 1 are
indeed located in an appropriate position. Alternatively, presenilin 1 may be an essential cofactor for
-secretase activity, e.g. by activating
-secretase or providing a channel for
the addition of water during proteolysis. Moreover, this effective TMD
of 12 IASD-inaccessible residues is not located directly in the middle
of the stretch of 24 hydrophobic residues that were predicted as TMD of
APP by hydropathy plot (37). Instead, on the N-terminal side the TMD is
8 residues shorter, whereas on the C-terminal side it is only 4 residues shorter than previously predicted. This implies that the
center of the newly determined TMD is exactly between residues 42 and
43. In contrast, in the predicted 24-residue-long TMD of C99, which may
represent the TMD of C99 at the plasma membrane, the center is located
between residues 40 and 41. This observation is striking because the
center of these TMDs coincides with the C terminus of the major A
species found in the corresponding compartments: at the plasma membrane (and the trans Golgi network) mostly A
40 is
found, whereas in the ER A
42 is observed almost
exclusively (41). This finding is consistent with a model in which
-cleavage always takes place at the center of the actual
transmembraneous part of C99. In agreement with this model, we and
others have recently shown that in C99 mutants with an altered length
of the TMD the preferred
-cleavage site is determined by the
location of the
-cleavage site with respect to the hydrophobic
domain (18, 21).
-cleavage but also for an additional cleavage of C99 between
residues 49 and 50 close to the C terminus of the predicted TMD
(
-cleavage), leading to the release of the APP intracellular domain
(42-45). This C-terminal
-cleavage site is at a similar position
within the TMD to the so-called S3 cleavage site of the cell surface
receptor Notch, which also is presenilin 1-dependent (46).
The position of the
-cleavage site of C99 is located just outside
the IASD-inaccessible, membrane-embedded TMD in microsomal membranes.
Thus, it is topologically different from the
-cleavage site in the
middle of the TMD and could be envisaged to be accessible to cytosolic
or membrane-associated proteases. However, as the membrane thickness
increases along the secretory pathway, the
- and S3 cleavage sites
of C99 and Notch could be shielded by a thicker membrane typical for
later compartments of the secretory pathway and thus become
inaccessible to cytosolic cleavage. Nevertheless, a comparison of the
newly defined TMD of APP with the TMD predicted for Notch shows that
the APP intracellular domain and S3 cleavage sites are topologically
more related to each other than to the
-cleavage site in the middle
of the TMD (8). This would account for different or modified cleavage
mechanisms of
-cleavage, on the one hand, and APP intracellular
domain and S3 cleavage on the other.
-secretase that uses membrane thickness and thereby
lipid composition to explain the mechanism of
-cleavage site selection.
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ACKNOWLEDGEMENT |
---|
We thank Oezcan Talay for technical assistance.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Graduate College for Biotechnology (to K. B.), by a grant of the Bundesministerium für Bildung und Forschung and the DFG (to T. H. and K. B.), and by the European Union (QLRT-2002-172) (to T. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-6221-546848; Fax: 49-6221-545891; E-mail: bgrziwa@ix.urz.uni-heidelberg.de.
Both authors contributed equally to this work.
Supported by an Emmy Noether Fellowship of the DFG.
Published, JBC Papers in Press, November 25, 2002, DOI 10.1074/jbc.M210047200
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ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid
;
APP, amyloid precursor protein;
TMD, transmembrane domain;
IASD, 4-acetamido-4'-((iodoacetyl) amino) stilbene-2,2'-disulfonate;
ER, endoplasmic reticulum;
IEF, isoelectric focusing;
SP, signal peptide;
wt, wild type;
IEP, isoelectric point.
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