From the Department of Cell Biology and Neuroscience
A1, Osaka University Graduate School of Medicine, Yamadaoka 2-2, Suita, Osaka 565-0871, Japan, § Molecular and
Experimental Medicine MEM275, The Scripps Research Institute, La
Jolla, California 92037, and the
Department of Molecular
Biodynamics, The Tokyo Metropolitan Institute of Medical Science
(RINSHOKEN), Bunkyo-ku Tokyo 113-8613, Japan
Received for publication, August 22, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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The amyloid A proteinase with In addition, recent analyses of the A The present study showed that the cytoplasmic tail of BACE binds
directly to phospholipid scramblase 1 (PLSCR1), another component of the plasma membrane lipid microdomain (22). The interaction required
a dileucine repeat in BACE (Leu499,500), which was required
for the endocytic transport of BACE (14). Coprecipitation experiments
also showed that BACE forms a protein complex with PLSCR1 in SH-SY5Y
cells, a neuroblastoma cell line. We also demonstrated that BACE and
PLSCR1 are fractionated in the DIGs at an endogenous expression level
in SH-SY5Y cells. These findings strongly suggest that PLSCR1 is a
binding partner of BACE in vivo and provide a functional
implication of PLSCR1 in the intracellular trafficking of BACE.
Cell Cultures and Transfection--
HeLa cells were
cultured in Cloning, Plasmids, and Sequence Analysis--
Wild-type BACE
cDNA was generated from human brain cDNA
(Clontech) and human placenta cDNA using PCR.
The DNA fragment obtained was cloned into pcDNA3 (Invitrogen) and
pcDNA-EGFP expression vectors to construct pHB1-full and pHB1-EGFP,
respectively. Epitope tagging of PLSCR1 with 3xHA was carried out with
subcloning of the PLSCR1 open reading frame to the pEF-BOS-3HA vector.
Site-directed mutagenesis of the BACE tail (amino acids 478-501) was
carried out by PCR using following primers containing the mutation
sites: 5'-gcgctcgagtcacttcgccgcggagat-3',
5'-ggcctcgagtcacttcagcagggcgatgtc-3', and
5'-ggcctcgagtcacttcagcaggtcgatgtc-3'. The cytoplasmic tails of BACE2
(amino acids 495-518), mouse CI-MPR (amino acids 2316-2482), and
human APP695 (amino acids 649-695) were cloned into pGBD-C1 using the
following primer pairs: 5'-gccgaattccggtgtcagcgtcgcccc-3' and
5'-ggcctcgagtcatttccagggatgtct-3', 5'-gccgaattccataagaaggagagaagg-3' and 5'-ggcctcgagttagatgtgtaagagggt-3', and
5'-gccgaattcaagaagaaacagtacaca-3' and 5'-ggcctcgaggttctgcatctgctcaa-3',
respectively. The deletion plasmids of pGAD10-PLSCR1-full, 290, 156, and 118 were constructed by subcloning of the amplified DNA fragments
into pGAD10. The PCR primers used for construction of pGAD10 plasmids
are as follows: 5'-ggcctcgagctaccacactcctgattt-3' (full-as),
5'-ggcctcgagtttcattttaacatcaaggtc-3' (290-as),
5'-ggcctcgagtggcccacagcaatttcg-3' (156-as),
5'-ggcctcgagttccagaagttcaatttg-3' (118-as).
Yeast Two-hybrid Screening--
The DNA fragment corresponding
to the BACE cytoplasmic region was inserted into the pGBD-C1 vector to
construct pGBD-HB1cyt. PJ69-4A cells (kindly provided from Dr. Philip
James, University of Wisconsin; Ref. 24), harboring pGBD-HB1cyt were
transformed with a HeLa cDNA library
(Clontech), and Ade+-positive clones
were obtained, and the library clones were subsequently subjected to
DNA sequencing analysis after the reintroduction into
PJ69-4A/pGBD-HB1cyt. One of the obtained clones, pKM2-8 was found to
harbor a full-length open reading frame of the PLSCR1 gene.
In Vitro Binding Assay--
DNA fragments encoding the
cytoplasmic domains of wild-type or mutant BACE tail were introduced
into the pGEX6P-1 vector (Amersham Biosciences), and full-length PLSCR1
open reading frame amplified from pKM2-8 by the PCR method was
subcloned into the pMAL-C2 vector (New England Biolabs) for the
bacterial expression of the GST and MBP chimeric proteins,
respectively. After purification with glutathione-Sepharose 4B
(Amersham Biosciences) or amylose resin (New England Biolabs) columns,
the beads containing GST fusion proteins and the eluted MBP-PLSCR1
protein were subjected to the following in vitro binding
assay. GST beads containing ~1 µg of each GST chimeric protein were
incubated with 5 µg of MBP-PLSCR1 in a binding buffer (25 mM phosphate buffer (pH 7.2), 0.5 mM
CaCl2, 150 mM NaCl, 0.5% Triton X-100,
10 mM Antibodies, Immunofluorescence Microscopy, and Immunoelectron
Microscopy--
To raise the antibodies against the BACE cytoplasmic
(BACE-cyt; Cys478 to Lys501) or lumenal
(BACE-lum; Asp48 to Thr80) region, bacterially
expressed GST fusion protein that contains each region was injected to
rabbits. Anti-human BACE monoclonal antibody (MAB5308) and
anti-APP antibody 22C11 were purchased from Chemicon. Anti-caveolin-1
polyclonal antibody and anti-flotilin-1 antibodies were purchased from
Santa Cruz Biotechnology, and BD Transduction Laboratories,
respectively. Anti-cation independent mannose 6-phosphate receptor
(CI-MPR) polyclonal antibody was used for a TGN marker as reported
previously (25), and anti-Lamp1 monoclonal antibody was provided from
the Developmental Studies Hybridoma Bank. Indirect immunofluorescence
microscopy was performed as described elsewhere (26) with a confocal
laser scanning microscope (LSM-510, Zeiss). For immunoelectron
microscopy, HeLa cells cotransfected with pHB1-EGFP and
pEF-BOS-3HA-PLSCR1 were fixed with 4% paraformaldehyde and 0.1%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) for 15 min on ice and further fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 1 h at 4 °C. The
cells were then scraped and subjected to immunoelectron microscopic
procedures using anti-GFP antibody (Invitrogen) and anti-hemagglutinin
antibody (3F10, Roche Molecular Biochemicals), as described elsewhere
(27).
Differential Solubilization--
HEK293 cells stably expressing
BACE were homogenized by passing through the 27-gauge needle for 25 strokes in phosphate-buffered saline containing 1× protease inhibitor
mixture and 1 mM phenylmethylsulfonyl fluoride and
centrifuged at 700 × g for 5 min to generate a
post-nuclear supernatant (PNS) fraction. The PNS fraction was separated
to six aliquots, and each aliquot was treated with buffer to a final concentration of 1 M NaCl, 0.2 M
Na2CO3 (pH 11.0), 2 M urea, or 1%
Triton X-100 on ice for 30 min. After incubation, each sample was
centrifuged at 100,000 × g for 1 h, and the
resultant pellet and supernatant fractions were subsequently subjected
to immunoblotting using anti-BACE-lum and anti-PLSCR1 (4D2) antibodies.
Cross-linking and Immunoprecipitation--
HeLa cells
transfected with pHB1-full or pHB1-AA were washed with ice-cold
phosphate-buffered saline twice, harvested, and resuspended in buffer X
(50 mM phosphate buffer (pH 7.5), 150 mM NaCl,
1 mM CaCl2, 5 mM MgCl2,
5% glycerol, and 1× protease inhibitor mixture (Roche Molecular
Biochemicals)). Then, a chemical cross-linker, dithiobis[succinimidyl
propionate] (DSP, Pierce), was added at the concentrations indicated
and incubated on ice for 30 min. After termination of the cross-linking
by the addition of 1/5 volume of 1 M Tris-HCl (pH
7.6), BACE, or PLSCR1 was immunoprecipitated with anti-BACE (MAB5308)
or anti-PLSCR1 (4D2) antibodies as described elsewhere (22, 28). The
precipitates were subsequently subjected to immunoblotting. For
coprecipitation of endogenous BACE and PLSCR1, ~1 × 108 of SH-SY5Y cells were homogenized by passing through
the 27-gauge needle for 30 strokes in phosphate-buffered saline (pH
7.4) containing 1× protease inhibitor mixture (Roche Molecular
Biochemicals) and 1 mM phenylmethylsulfonyl fluoride. After
centrifugation at 700 × g for 5 min to generate
a PNS, the PNS was further centrifuged at 100,000 × g
for 1 h to generate a total membrane fraction. The total membrane
fraction that contains ~10 mg of proteins was further subjected to
immunoprecipitation of BACE or PLSCR1, as described (22, 28).
Flotation of Detergent-insoluble Lipid Microdomains--
After
washing with phosphate-buffered saline twice, the cells were harvested
and suspended in buffer L (150 mM NaCl, 50 mM phosphate buffer (pH 7.2), 1% Lubrol WX (Serva Electrophoresis), 1×
protease inhibitor mixture) and incubated for 30 min on ice. The cell
lysates were then adjusted to a final concentration of 45% sucrose by
the addition of an equal volume of 90% sucrose and placed at the
bottom of an Ultracentrifuge tube and then overlaid with 35 and
5% sucrose solutions containing phosphate-buffered saline. The
discontinuous sucrose gradient was centrifuged at 160,000 × g for 20 h in an SW41 rotor (Beckman Instruments) and fractionated from the top of the gradient with Piston gradient fractionator (BioComp Instruments). An equal volume of each fraction was used for immunoblotting or immunoprecipitation. For preparation of
the lipid microdomain from SH-SY5Y cells, 3 × 108
cells were used, and endogenous BACE was immunoprecipitated from each
fraction with anti-BACE antibody (MAB5308) and detected by immunoblotting using MAB5308.
Phospholipid Scramblase 1 Interacts with the Cytoplasmic Domain of
BACE--
As shown previously, the short cytoplasmic tail of BACE is
essential for its endosomal distribution in cells (14). To better understand the molecular mechanism of BACE trafficking, BACE
tail-interacting proteins were searched with the yeast two-hybrid
method. From a HeLa cDNA library, we obtained a cDNA encoding
the phospholipid scramblase 1 (PLSCR1) gene. PLSCR1 was
first identified from the erythrocyte plasma membrane and shown to
mediate calcium dependent transbilayer movement of membrane
phospholipids in vitro (29-31). To gain information on the
binding region of PLSCR1, several deletion mutants were constructed and
assayed for interaction with BACE tail by the yeast two-hybrid system.
As depicted in Fig. 1A, a segment of PLSCR1 that interacts with BACE appeared to map to the
N-terminal half of PLSCR1. We also found that substitution of
the dileucine at the C terminus of the BACE tail to alanine drastically
reduced the molecular interaction in the two-hybrid system and the
in vitro binding assay using the recombinant proteins (Fig.
1, B-D). The Ser498 residue, which has been
shown to be phosphorylated in vivo and involved in the
regulation of the endosomal transport of BACE (15), was not essential
for the interaction with PLSCR1. Moreover, the cytoplasmic tails of
other type I integral membrane proteins such as APP695, CI-MPR, or
BACE2 were found to be incompetent to bind with the BACE tail (Fig. 1,
B-D). These results suggest that the molecular interaction
between BACE and PLSCR1 is specific and dependent on the C-terminal
dileucine residues of the BACE tail.
Direct Interaction between BACE and PLSCR1 in Vivo--
From its
primary structure, PLSCR1 is predicted to be an integral membrane
protein with a single hydrophobic stretch at the C terminus. Moreover,
recent studies revealed that PLSCR1 receives phosphorylation and
palmitoylation at the N-terminal region in vivo (32, 33),
suggesting that PLSCR1 is a type II membrane protein. As shown in Fig.
2, a differential
solubilization experiment showed that both BACE and PLSCR1 were
solubilized only with detergent treatment but not with other reagents
such as alkali, high salt, or urea. This result indicates that PLSCR1
is an integral membrane protein. We next examined the interaction of
BACE and PLSCR1 in vivo by means of
co-immunoprecipitation experiments. HeLa cells expressing
wild-type BACE (BACE-WT) were incubated in the absence or presence of
DSP, a membrane-permeable chemical cross-linker. After quenching, cells
were harvested, and BACE-WT was immunoprecipitated by a specific
antibody. As shown in Fig. 2, ~1.5% of cellular PLSCR1 was
coprecipitated with BACE in the absence of DSP, and precross-linking
increased the amount of coprecipitated PLSCR1 up to ~6.5% of total
PLSCR1. Next, we examined the molecular interaction between endogenous
PLSCR1 and expressed wild-type or dileucine motif mutant (AA-mutant;
BACE-AA) BACE. HeLa cells transfected with BACE-WT or BACE-AA were
lyzed, and endogenous PLSCR1 was immunoprecipitated with an anti-PLSCR1
antibody. Wild-type but not AA-mutant BACE was coprecipitated with
PLSCR1 (Fig. 2C).
To further confirm the in vivo interaction between BACE and
PLSCR1 at an endogenous expression level, total membrane fractions prepared from neuroblastoma SH-SY5Y cells were subjected to
immunoprecipitation analysis. As shown in Fig. 2D, PLSCR1
was coprecipitated with BACE in the absence of the cross-linker, and a
higher amount of PLSCR1 was coprecipitated in the presence of DSP.
These results strongly suggest that BACE forms a protein complex with
PLSCR1 in vivo, and the interaction depends on the dileucine
residues of the BACE tail.
BACE Is Colocalized with PLSCR1 in Vivo--
As reported
previously, ectopically expressed BACE is localized in the
post-Golgi organelles including the Golgi apparatus, TGN,
endosomes, and plasma membrane (14). Although PLSCR1 has been reported
to be localized mainly in the plasma membrane (34), the accurate
distribution remains uncertain. Thus, we examined the intracellular
localization of PLSCR1 and BACE precisely by immunofluorescent
microscopy. Endogenous PLSCR1 was localized mainly in the plasma
membrane, whereas positive signals were also detected in the
perinuclear Golgi area, which were also positive for CI-MPR
immunofluorescence in HeLa cells (Fig.
3A, a-c). In HeLa
cells transiently expressing BACE-EGFP, BACE and PLSCR1 were also
colocalized in the Golgi compartment and at the cell surface (Fig.
3A, g-i), suggesting colocalization at these
organelles. In addition, at the peripheral punctate structures, partial
colocalization of both proteins was observed (Fig. 3A,
g-l), suggesting the localization of PLSCR1 in
endosomal compartments. Essentially, similar staining patterns were
observed in other cultured cell lines, such as human embryonic kidney
(HEK293) and human neuroblastoma (SH-SY5Y) cell lines.2
Intracellular Trafficking of BACE and PLSCR1 via a
U18666A-sensitive Route--
To clarify the routes in which the two
proteins are trafficking, HeLa cells expressing BACE-EGFP were treated
with U18666A. U18666A is a class II amphiphile and known to cause the
selective accumulation of low density lipoprotein-derived cholesterol
in late endocytic compartments and blockage of the outward protein transport from the endosomal compartments (35, 36). As shown in Fig. 3,
U18666A treatment induced a drastic co-redistribution of BACE-EGFP and
PLSCR1 to the perinuclear large punctate structures (Fig.
3A, m-r). As has been shown previously, these
structures are positive for immunofluorescence of CI-MPR (35),
suggesting that the compartments are derived from late endosomes (Fig.
3A, d-f). For further analysis of the
structures, immunoelectron microscopic analysis was carried out with
the HeLa cells transiently cotransfected with BACE-EGFP and
hemagglutinin-tagged-PLSCR1 expression vectors. U18666A treatment
revealed that these proteins were colocalized in multivesicular bodies
(Fig. 3B), and the BACE-positive multivesicular bodies were
also co-labeled with a monoclonal antibody against a late
endosome-specific lipid, lisobisphosphatidic acid (LBPA, 36),2 suggesting that the compartments are derived from the
late endosomes (36, 37). Furthermore, the redistribution of PLSCR1 was
restored 4 h after washing out of U18666A.2 These
results suggest that both BACE and PLSCR1 are actively trafficking
through the overlapped pathway, including late endosomal compartments.
BACE Is Cofractionated with PLSCR1 in the Lipid
Microdomain--
Recent studies have suggested that APP and its
processing activities, namely presenilins, PLSCR1 Is a U18666A-dependent Redistribution of BACE and
PLSCR1--
As has been shown previously, BACE is endocytosed from
the plasma membrane in a dileucine residue-dependent manner
(14). Moreover, our photobleaching analysis in living cells showed that BACE-EGFP in the peripheral area can be transported back to the perinuclear Golgi area,2 suggesting that BACE is
dynamically trafficking between the Golgi area and plasma membrane,
presumably via endosomal compartments. The present study also
demonstrates that PLSCR1 is localized in intracellular organelles
besides the plasma membrane. Furthermore, U18666A treatment led to the
redistribution of PLSCR1 in perinuclear punctate structures, which were
positive for BACE-EGFP (Fig. 3) but only slightly positive for
BACE-AA-EGFP.2 This finding suggests that BACE is
transported to the late endosomal compartments and that the dileucine
residues are required for targeting of BACE to late endosomes from the
cell surface. Our immunoelectron microscopic observations supported the
notion that BACE and PLSCR1 are colocalized in the enlarged,
multivesicular endosomes in U18666A-treated cells. Although the
molecular mechanism of how U18666A perturbs the endosomal transport
remains unclear, the findings herein suggest that PLSCR1 moves in the
intracellular compartments via a pathway, which overlaps with that of
BACE.
PLSCR1 Is a Raft-resident Protein as Well as BACE in Neuronal Cell
Lineage--
Thus far, a series of studies focusing on the
relationship between A-Site amyloid precursor protein (APP)-cleaving
enzyme (BACE) is an integral membrane aspartic proteinase
responsible for
-site processing of APP, and its cytoplasmic region
composed of 24 amino acid residues has been shown to be involved in the endosomal localization of BACE. With the yeast two-hybrid screening, we
found that the cytoplasmic domain of phospholipid scramblase 1 (PLSCR1), a type II integral membrane protein, interacts with the
cytoplasmic region of BACE. In cultured cells, BACE and PLSCR1 were
colocalized in the Golgi area and in endosomal compartments, whereas
they were co-redistributed in late endosome-derived multivesicular bodies when treated with U18666A, suggesting that both proteins share a
common trafficking pathway in cells. Co-immunoprecipitation analysis
showed that both proteins form a protein complex at an endogenous
expression level in the human neuroblastoma SH-SY5Ycells, and
the dileucine residue of the BACE tail is also revealed to be essential
for the physical interaction with PLSCR1 in vitro and
in vivo. Moreover, both BACE and PLSCR1 were localized in a
low buoyant lipid microdomain in SH-SY5Y cells. The dileucine-defective BACE mutant was also fractionated into the lipid microdomain, but much
less stably than wild-type BACE. Taken together, our current study
suggests the functional involvement of PLSCR1 in the intracellular
distribution of BACE and/or recruitment of BACE into the
detergent-insoluble lipid raft.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-peptide
(A
),1 a principal
constituent of senile plaques, is a major hallmark of familial
Alzheimer's disease (AD). A
peptides are generated from a type I
membrane glycoprotein, amyloid precursor protein (APP) (1), by
proteolytic events that involve the participation of
- and
-secretases (2, 3). A number of studies on familial AD have shown
that mutations in genes of APP, presenilin-1 or -2, affect APP
processing and result in increases in the total levels of A
,
especially A
42. A
42 is known to form amyloid fibrils more readily
than A
40 (4, 5), and its overproduction may thus accelerate plaque
formation, leading to early onset AD (6) and sporadic AD as well. Thus, A
formation has been the subject of considerable interest as a key
event of AD.
-secretase activity has recently been cloned and
is referred to as BACE (beta-site of
APP-cleaving enzyme) (7, 8). The
cells develop
-site cleavage activity when BACE is ectopically
expressed, and the recently established BACE-deficient mouse shows
considerably diminished
-secretase activity, indicating that BACE
functions as a major
-secretase in vivo (9).
Intracellular sites for
-secretase activity have been investigated,
and at least three sites including endoplasmic
reticulum/intermediate compartments (10), Golgi/trans Golgi network
(TGN) (11), and endosomal compartments (12, 13) have been reported.
When BACE is ectopically expressed, it is mainly detected in the
intracellular compartments including Golgi apparatus, TGN, endosomes,
and the plasma membranes (14, 15). Recent studies also revealed that a
pathogenic Glu11-site cleavage of A
is increased with
the limited expression of BACE-furin chimeric protein in TGN (16), and
sialyltransferase, a TGN-resident protein, was also found to be another
substrate of BACE in vivo (17). These results suggest that
pathogenic cleavage with BACE could occur in TGN. Although the
cytoplasmic region of BACE has been shown to be essential for the
intracellular localization of BACE (14), the molecular mechanisms of
BACE trafficking between TGN, endosomes, and plasma membrane remain to
be elucidated.
formation have uncovered a
novel regulatory mechanism for APP processing. Caveolae in the plasma
membrane constitute a microdomain that has a unique lipid composition
with a high content of both cholesterol and glycosphingolipids. In the
brain, the caveolae-like microdomain has been referred to as a
detergent-insoluble glycolipid membrane complex (DIG) (18).
Brain-derived DIG, or so-called lipid raft, has been reported to
accumulate APP (19), presenilin-1 and -2, and A
peptides (20).
Moreover, BACE has also been shown to be localized in the lipid
microdomain in cultured cells in a cholesterol-dependent manner (21). Therefore, DIGs represent putative sites where amyloid
biogenesis or transport takes place, and cholesterol metabolism is
strongly assumed to be associated with the APP processing events.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-MEM (Invitrogen) containing 8% fetal bovine serum
supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin.
HEK293 (clone JCRB9068, provided from the Human Science Research
Resources Bank) and human neuroblastoma SH-SY5Y cells (23) were
maintained in Dulbecco's modified Eagle's medium supplemented with
4.5 g/liter glucose and 10% fetal bovine serum. For the transfection
of HeLa and HEK293 cells, the FuGene-6 reagent (Roche Molecular
Biochemicals) and LipofectAMINE Plus (Invitrogen) were used,
respectively, following the manufacturer's recommended protocols.
Wild-type and transfected HeLa cells were also cultured in the presence
or absence of 3 µg/ml U18666A (BIOMOL Research Laboratory).
-mercaptoethanol, 1× protease inhibitor mixture
(Roche Molecular Biochemicals), and 1 mM
phenylmethylsulfonyl fluoride) at 4 °C for 2 h. The beads were
then washed extensively with binding buffer, and the bead-bound
proteins were subjected to immunoblot analysis with anti-GST or
anti-MBP antibodies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Yeast two-hybrid assay of the
interaction between BACE tail and PLSCR1. In A,
the primary structure of PLSCR1 is illustrated. The proline-rich region
(Pro-rich) and Ca2+ binding domain
(EF-hand) are indicated by gray and black
boxes, respectively. PJ69-4A cells harboring pGBD-BACE-tail were
transformed with PLSCR1 deletion constructs and their interactions
assayed. TM, transmembrane region. B, interaction
between PLSCR1 and BACE tail mutants. PJ69-4A cell harboring
pGAD-C1-PLSCR1 was transformed with pGBD-C1-BACE-tail mutants (S498A,
S498D, or L499, 500A, indicated as
LL AA) or pGBD vectors harboring
cytoplasmic tails of BACE2, APP695, or CI-MPR and examined for growth
on SD (
Trp-Leu-Ade) (indicated as
TLA) and SD
(
Trp-Leu) (indicated as
TL) plates. C,
-galactosidase activity of the transformants shown in panel
B. D, in vitro binding of PLSCR1 with BACE
tails. Interaction of MBP-PLSCR1 with the GST-BACE-tail was examined as
described under "Experimental Procedures." The bound MBP-PLSCR1 was
detected with an anti-MBP antibody.
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Fig. 2.
A, differential solubilization of
PLSCR1. PNS from HEK293 cells transfected with pcDNA-HB-full was
treated with the reagents indicated on ice for 30 min and then
centrifuged at 100,000 × g for 1 h to generate
the precipitated (P) and supernatant (S)
fractions. Each fraction was subjected to immunoblotting of BACE and
PLSCR1 with anti-BACE-lum and anti-PLSCR1 (4D2) antibodies,
respectively. B, co-immunoprecipitation of BACE and PLSCR1.
HEK293 cells transiently expressing BACE were harvested and treated
with the indicated concentrations of DSP on ice for 30 min. After
quenching of DSP, cells were lysed, BACE was immunoprecipitated
(IP) with MAB5308, and the immunoprecipitates were subjected
to immunoblotting of BACE and PLSCR1 with anti-BACE-cyt and anti-PLSCR1
(4D2) antibodies, respectively. 5% of immunoprecipitated PLSCR1 from
the cell lysate was subjected to immunoblotting as a control.
WB, Western blot. C,
dileucine-dependent interaction of BACE and PLSCR1. HEK293
cells transiently expressing BACE-WT or BACE-AA were lysed, and
endogenous PLSCR1 was immunoprecipitated with anti-PLSCR1 monoclonal
antibody 4D2. The immunocomplex was subjected to immunoblotting of BACE
(MAB5308) and PLSCR1 (4D2). As shown in D, endogenous BACE
interacts with PLSCR1 in neuroblastoma cells. Total membrane fraction was prepared from SH-SY5Y cells, the membranes were
incubated in the absence or presence of 0.3 mM DSP, and
endogenous BACE was immunoprecipitated with anti-BACE tail antibody
(MAB5308) in the absence or presence of excess amount of the BACE tail
synthetic peptide (Pep). The immunocomplex was subsequently
subjected to immunoblotting of BACE (MAB5308) and PLSCR1 (4D2). 0.2%
of membrane fraction used for immunoprecipitation of BACE was subjected
to immunoblotting of PLSCR1 (4D2) as a control. Asterisks
indicate IgG.
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Fig. 3.
Co-localization of BACE and
PLSCR1. As shown in A, HeLa cells
(a-f) or HeLa cells transiently expressing BACE-EGFP
(g-r) were incubated for 20 h in the absence
(a-c and g-l) or presence (d-f and
m-r) of 3 µg/ml U18666A. After fixation and
permeabilization, the cells were labeled with anti-CI-MPR
(a and d and green image in
c and f) and anti-PLSCR1 (b,
e, h, k, n, and
q and red image in c, f,
i, l, o, and r).
Localization of BACE-EGFP is shown in g, j,
m, and p (green image in i,
l, o, and r). Merged images are shown
(c, f, i, l, o,
and r). The boxed areas indicated in i and o are shown
in higher magnification in j-l and p-r,
respectively. Scale bars, 10 µm. As shown in B,
HeLa cells expressing BACE-EGFP and 3HA-PLSCR1 were treated with
U18666A and examined by immunoelectron microscopy using anti-GFP (15 nm-gold) and anti-hemagglutinin (5 nm-gold: arrowheads)
antibodies. Scale bar, 200 nm.
-secretase activity, and
BACE accumulate in detergent-insoluble, cholesterol-enriched, membrane
microdomains called DIGs (21, 38, 39). PLSCR1 was also reported to be a
component of lipid raft in human oral epithelial carcinoma (22). We
next examined whether BACE and PLSCR1 could be fractionated in the
lipid microdomain at an endogenous expression level in a neuronal cell
line. SH-SY5Y human neuroblastoma cells were treated with a non-ionic
detergent Lubrol WX, and low buoyant, lipid-associated proteins were
separated by flotation in a discontinuous sucrose gradient. As shown in
Fig. 4, BACE and PLSCR1 were fractionated in the DIG fractions, which contain known DIG proteins such as flotilin-1 and APP. Lamp-1, a late endosomal integral membrane protein,
was not fractionated in DIGs, as reported previously (40). Next, to
examine the functional consequence of the dileucine residues of the
BACE tail in recruitment of BACE into the lipid microdomain, HEK293
cells stably overexpressing BACE-WT or BACE-AA were subjected to DIG
preparation. PLSCR1 was fractionated into the DIG fractions as well as
wild-type BACE (Fig. 4). Although BACE-AA was also fractionated in the
DIG fractions, the ratio of DIG-associated BACE-AA varies in each
experiment, whereas BACE-WT, PLSCR1, and caveolin-1 were reproducibly
fractionated in the DIG-fraction through five independent experiments.
These results suggest that the BACE-PLSCR1 molecular interaction is not
essential for the recruitment of BACE into DIGs, but it may be involved
in the stable association of BACE with the lipid microdomain.
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Fig. 4.
Co-fractionation of BACE and PLSCR1 in the
low buoyant lipid microdomain. A, SH-SY5Y neuroblastoma
cells were harvested and treated with 1% lubrol WX on ice for 30 min
and subjected to sucrose discontinuous density gradient centrifugation
as described under "Experimental Procedures." From each fraction,
BACE was immunoprecipitated with anti-BACE antibody (MAB5308) and
detected by immunoblotting using MAB5308. The asterisk
indicates IgG. For detection of other proteins, each fraction was
directly used for immunoblot analysis. As shown in B and
C, HEK293 cells transiently expressing BACE-WT
(B) or BACE-AA (C) were subjected to the DIG
preparation carried out as in panel A. Each fraction was
used for immunoblotting with anti-BACE, anti-PLSCR1, or anti-caveolin-1
(Cav-1) antibody. Fraction 4 contains the 5-35% interface
of sucrose gradient, and the detergent insoluble lipid microdomain
fractions are indicated as DIGs.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Secretase Binding Partner--
A series of recent
functional analyses revealed that the C-terminal cytoplasmic region is
required for the correct intracellular localization of BACE (14, 15).
Although the mutational analysis of the BACE tail uncovered that the
BACE tail contains several sites for post-translational modification
and signals for intracellular trafficking of BACE (14, 15, 41), the
molecular mechanisms responsible for the tail-dependent
cellular trafficking of BACE were still unknown. In the current study,
our yeast two-hybrid screening implicated PLSCR1 as a novel BACE
interacting molecule. PLSCR1 was first identified as a plasma membrane
protein that has phospholipid scrambling activity in vitro
(29, 30). PLSCR1 was also shown to physiologically and functionally
interact with epidermal growth factor receptors and other cell surface
growth factor receptors, as well as with intracellular kinases that are known to be activated by these receptors. Nevertheless, the biological function of PLSCR1 in growth factor-regulated proliferation and differentiation remains to be completely elucidated (22, 32, 42). We
showed here that BACE forms a protein complex with PLSCR1 in neuronal
cells under normal conditions, and the dileucine residues of the BACE
tail were revealed to be essential for the physical interaction with
PLSCR1 in vitro and in vivo. Generally,
cytoplasmic dileucine residues of integral membrane proteins can be
utilized as an endosomal retention signal or an internalization signal from the plasma membrane (43). In the case of BACE, indeed, the BACE-AA
mutant in which the dileucine residues are substituted with alanine
accumulated on the plasma membrane.2 A previous study also
revealed that the dileucine mutant exhibited defects in internalization
from the plasma membrane and is more readily recycled to the cell
surface from endosomal compartments (14). These results allow us to
imply that PLSCR1 is involved in the dileucine-dependent
transport of BACE.
formation and cellular cholesterol have
revealed that cholesterol is required for A
production (44).
Moreover, the correct intracellular distribution of cholesterol is also
important for A
metabolism (45), and in addition, the major
components required for APP processing, such as presenilin-1 and -2, APP,
-secretase activity, and BACE, have been fractionated into the cholesterol-enriched, low buoyant lipid microdomain called DIGs (20,
38, 39, 46, 47). Moreover, we recently reported that PLSCR1 is also a
component of lipid microdomain (22, 33). In this study, we showed that
both BACE and PLSCR1, as well as APP, are components of the lipid
microdomains in a neuronal cell line, SH-SY5Y cells, at an endogenous
expression level. Furthermore, interestingly, BACE-WT, but not BACE-AA
was reproducibly fractionated in the DIG fractions, suggesting that the
dileucine residues may be involved in efficient recruitment of BACE
into DIGs or in stable retention of BACE in the lipid microdomain.
Although the physiological function of PLSCR1 is still unclear, it was
shown that recombinant PLSCR1 protein solely has a
phospholipid-scrambling activity in vitro (30, 31). This
leads to an implication that PLSCR1 might alter the local composition
or topology of plasma membrane phospholipids so as to influence the
process of endocytosis of BACE and/or potentially other cell surface
components, such as the epidermal growth factor receptor (22).
Recently, a family of GGA adapter proteins has been shown to directly
interact with the BACE tail in a dileucine-dependent manner
(48, 49). Moreover, the molecular property of the GGA-BACE interaction
was found to be different from that of the PLSCR1-BACE interaction;
GGAs require both the acidic amino acid cluster
(495DD496) and dileucine in the BACE tail for
physical interaction (49), whereas PLSCR1 does not require the acidic
cluster.2 At present, the functional consequence of these
adapter molecules on BACE trafficking is also unclear. Further
functional analysis of the BACE interacting molecules will clarify the
complex molecular mechanisms responsible for the intracellular
trafficking of BACE and regulatory mechanisms of A
formation.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid for Scientific Research on Priority Areas, Advanced Brain Science Project, from the Ministry of Education, Culture, Sports and Science and Technology, Japan.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.
¶ Supported by grants HL36946 and HL63819 from The Heart, Lung, and Blood Institute, National Institutes of Health.
** To whom correspondence should be addressed. Tel.: 81-6-6879-3120; Fax: 81-6-6879-3129; E-mail: uchiyama@anat1.med.osaka-u.ac.jp.
Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M208611200
2 S. Kametaka, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid
-peptide;
AD, Alzheimer's disease;
APP, amyloid precursor protein;
BACE,
-site APP-cleaving enzyme;
PLSCR1, phospholipid scramblase 1;
WT, wild-type;
TGN, trans Golgi network;
DIG, detergent-insoluble
glycolipid membrane complex;
CI-MPR, cation independent
mannose-6-phosphate receptor;
GFP, green fluorsecent protein;
EGFP, enhanced GFP;
DSP, dithiobis[succinimidyl propionate];
PNS, post-nuclear supernatant;
lum, lumenal.
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REFERENCES |
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