From the
The role of the Ras-related GTP-binding protein, Rab1B, in
intracellular trafficking of
The 4-kDa amyloid
Proteins belonging
to the Rab subgroup of the Ras superfamily of small GTP-binding
proteins have emerged as important regulatory components of the
exocytic and endocytic vesicular transport pathways in mammalian cells
(23, 24, 25, 26) . Individual members of
the Rab family are uniquely localized in specific organelles and
membrane compartments
(27, 28, 29, 30, 31, 32, 33) .
This suggests that different Rab proteins may control the
directionality and/or specificity of particular steps in intracellular
protein trafficking. Support for this concept has come from studies
conducted with permeabilized cells and cell-free systems that
reconstitute discrete steps in vesicular transport. Such assays have
implicated Rab1B in ER
The targeting mechanisms that determine the alternative fates of
Before proceeding with further analysis of
As in the case of wild-type
Previous studies by Balch and co-workers
(35, 53, 55) have established that Rab1A and
Rab1B mutants that are defective in GTP-binding can block ER
Although both
Rab1B
As mentioned
earlier, the initial endoproteolytic cleavage of
The experiments conducted with the
Swedish variant of
The differential decline in
A
Although this study focuses on well defined early steps in the
secretory processing of
-amyloid precursor protein (
APP)
was studied in cultured 293 cells.
APP is processed via one of two
alternative routes. In the major secretory pathway,
APP is cleaved
by
-secretase within the region comprising the
-amyloid
peptide (A
), resulting in release of a soluble
NH
-terminal exodomain (APP
) and a 3-kDa
peptide (p3) derived from the carboxyl-terminal tail. In the
alternative amyloidogenic pathway,
APP is cleaved by
-secretase, with the release of a truncated exodomain
(APP
) and an intact A
peptide. When
APP
was coexpressed with Rab1B(wt) or
dominant-negative Rab1B mutants (Rab1B
or
Rab1B
) there was a marked decrease in conversion of the
immature Endo-H sensitive form of
APP
(108 kDa) to
the mature O-glycosylated form of
APP
(130
kDa) in cells expressing the mutant forms of Rab1B. The block in
Golgi-dependent processing of
APP was accompanied by inhibition of
secretion of APP
(APP
). A similar decrease
in secretion of APP
(APP
+
APP
) was observed in cells that were coexpressing
Rab1B
with the ``Swedish'' variant of
APP
( i.e.
APP SW
), which undergoes increased
amyloidogenic processing. Coincident with the decline in APP
secretion, the cells coexpressing
APP SW
with Rab1B
showed a 90% decrease in A
secretion. The data indicate that Rab1B plays a key role in endoplasmic
reticulum
Golgi transport of
APP, and that
APP must
pass through a late Golgi compartment before entering either the
-secretase or the amyloidogenic
-secretase pathway. The
results also suggest that mutant versions of other Rab proteins that
function in different parts of the exocytic and endocytic pathways may
be useful in defining the specific routes of
APP transport
involved in the biogenesis of A
.
-peptide (A
)
(
)
is a major component of cerebrovascular amyloid deposits
associated with Alzheimer's disease
(1, 2, 3) . A
originates as a product of
proteolytic processing of
-amyloid precursor protein (
APP), a
ubiquitously expressed membrane glycoprotein that exists in three major
isoforms (
APP
,
APP
, and
APP
)
(2, 3, 4, 5, 6, 7) . The
nascent or ``immature'' forms of
APP undergo several
modifications upon translocation from the endoplasmic reticulum (ER) to
the Golgi apparatus. These include addition of O-linked
oligosaccharides, tyrosine sulfation, and trimming of N-linked
carbohydrates
(8, 9, 10, 11) .
APP
is processed via one of two alternative proteolytic pathways
(2, 3, 4) . In the major pathway,
APP is
cleaved within the A
sequence by an endoprotease activity termed
-secretase
(12, 13, 14, 15) ,
releasing a 102-115-kDa amino-terminal exodomain which is
secreted as a soluble protein, APP
. The
carboxyl-terminal remnant is then further degraded to yield a 3-kDa
fragment (p3) which is also secreted
(16, 17) . In the
alternative processing pathway,
APP is cleaved by an endoprotease
activity termed
-secretase in a manner that preserves the entire
A
sequence and releases a truncated exodomain, APP
(18) . A
is then generated after further proteolysis
of the carboxyl-terminal stump
(19, 20, 21, 22) .
Golgi transport
(28) , Rab5 in
early steps of endocytosis
(34) , Rab8 in basolateral transport
from the trans-Golgi network to the plasma membrane
(33) , and Rab9 in transport from late endosomes to the
trans-Golgi compartment
(32) . The roles of specific
Rab proteins have been further defined by studies in which
overexpression of mutant Rab proteins with defective guanine nucleotide
binding properties has been used to suppress the function of the
corresponding endogenous Rab proteins in intact cells. For example,
Tisdale et al.(35) showed that mutants of Rab1 or
Rab2 inhibited ER
Golgi transport of vesicular stomatitis virus
glycoprotein in HeLa cells. Similar studies with Rab5 indicate a role
for this protein in the endocytosis of transferrin
(36) and
horseradish peroxidase
(37) in baby hamster kidney cells.
APP remain to be defined, as do the exact subcellular sites of the
various proteolytic processing events involved in the genesis of
APP
/p3 or APP
/A
. We hypothesize
that many, if not all, of the steps in the intracellular trafficking of
APP are mediated by distinct members of the Rab family.
Consequently, functional perturbation of Rab proteins known to be
localized in specific subcellular compartments may help to define the
routes by which
APP is directed to the alternative secretory or
amyloidogenic processing pathways. As a first step in testing this
approach, we have examined the effects of dominant-negative mutations
in Rab1B on the posttranslational processing of
APP. Herein we
show that coexpression of GTP-binding-defective Rab1B mutants with
APP
in cultured human 293 cells inhibits the
Golgi-dependent maturation of
APP and decreases the secretion of
APP
. Mutations in Rab1B also inhibit secretion of
A
, suggesting that
APP is transported at least as far as the
medial Golgi compartment before entering the amyloidogenic processing
pathway.
Mutagenesis of Rab1B
Mutations were introduced
into the Rab1B sequence by means of overlap extension PCR
(38) using pGEM3Z- Rab1B(39) as the template.
The following 5` and 3` anchoring oligonucleotide primers were used in
all PCR reactions: 5`-GTTGTAAAAC-GACGGCCAGTG and
5`-TCTGCTAGTGGTGGCTGCTGTTAGATAGGATCCCGT. The following mutator
oligonucleotides and their complements were used to generate the
indicated point mutations; 5`-GTGGGCAAGAATTGCCTGCTT
(Rab1B) and 5`-CTGGTAGGCA-TCAAGAGTGAC
(Rab1B
). All PCR products were subcloned into pGEM3Z as
EcoRI/ BamHI inserts. Rab1B proteins that were tagged
by an amino-terminal myc epitope (EQKLISEEDL) were obtained by PCR
modification of the wild-type or mutant Rab1B cDNAs, utilizing
the following 5`- and 3`-oligonucleotide primers:
5`-GCCAGCGAATTCC-ATATGGAGCAGAAGCTGATCAGCGAGGAGGACCTGAACCCCGA-ATATGACTAC
and 5`-ACGGGATCCTATCTAACAGCAGCCACCACTAGCAGA. The resulting PCR products
were digested with EcoRI and XbaI and subcloned into
pCMV5neo
(40, 41) to generate pCMV Rab1B,
pCMV Rab1B, or pCMV Rab1B. The sequences of all
Rab1B constructs generated by PCR modification were verified
by the dideoxy chain termination technique using Sequenase 2.0 (U. S.
Biochemical Corp.).
Construction of
phCK751 and pohCK751sw were used for expression of
wild-type or Swedish forms of APP
Expression
Vectors
APP
, respectively. In
both vectors the 655-bp fragment from HincII to AvaII
of the cytomegalovirus immediate early gene promoter (CMV) drives
expression of the NruI to SpeI fragment encoding
human
APP
. The splice sequence positioned
immediately 3` to the CMV promoter and 5` to the
APP-encoding
region was derived from a hybrid sequence of the adenovirus major late
region first exon and intron and a synthetically generated IgG variable
region splice acceptor. The 162-bp PvuII to HindIII
fragment of the adenovirus major late region, containing 8 bp of the
first exon and 145 bp of the first intron, was fused with a
synthetically derived splice acceptor identical to the 99-bp
HindIII to PstI fragment of the IgG variable region
splice acceptor clone-6
(42) . A similar splice signal has been
shown to enhance expression when placed 5` to the coding sequences
(43) . Polyadenylation sequences are provided by the 884-bp
HpaI to EcoRI fragment of SV40 containing the early
polyadenylation signal. The BamHI site in this fragment was
destroyed by mutagenesis. The PvuII to EcoRI fragment
of pBR322 provides for phCK751 replication in Escherichia
coli, with an engineered NotI site replacing the
PvuII site. Herpes simplex type-1 viral replication and
packaging sequences located between the plasmid and CMV sequences were
derived from pON812 as described
(44) . These sequences are not
relevant to the experiments described in this report. The differences
between phCK751 and pohCK751sw are in the presence of a familial
Alzheimer's disease mutation and pUC-derived plasmid replication
sequences in the latter vector. The mutation encodes a dual amino acid
change (KM to NL) at positions 651 and 652 of
APP
(45) . In pohCK751sw, the NotI to PvuI
fragment of phCK751 (containing the plasmid origin of replication) was
replaced with the PvuII to PvuI fragment from pGEM3,
thus allowing more efficient replication in E. coli.
Transient Expression of Rab1B and
Transformed
human embryonic kidney cells (line 293), obtained from American Type
Culture Collection, were grown in Dulbecco's modified
Eagle's medium (DMEM) with 10% (v/v) fetal calf serum. Cells were
plated in 60-mm dishes at 1.8 APP
in Cultured Cells
10
cells/cm
on the day before transfection. A calcium phosphate precipitation
technique
(46) was used to transfect cultures with phCK751 or
pohCK751sw, alone or in combination with pCMV Rab1BWT,
pCMV Rab1B, or pCMV Rab1B. After a 3-h exposure to the
precipitated DNA, cells were subjected to glycerol shock (15% (v/v)
glycerol in PBS), then fed with fresh DMEM with 10% serum. Transfection
efficiencies and co-localization of transiently expressed proteins were
monitored by dual-antibody immunofluorescence. Briefly, the transfected
cells were fixed in 4% paraformaldehyde and permeabilized with 0.05%
Triton X-100 in PBS. After blocking with 5% powdered milk in PBS, cells
were incubated with an affinity-purified rabbit polyclonal antibody
(Anti-6) directed against residues 590-695 of human
APP
(10, 47) and the 9E10 monoclonal antibody against the
Rab1B myc epitope tag. In parallel control reactions one or both
primary antibodies were omitted. Bound primary antibodies were
visualized with rhodamine B-conjugated goat anti-mouse IgG and
fluorescence isothiocyanate-conjugated goat-anti rabbit IgG. A Nikon
Optiphot-2 microscope was used with filter combinations
EX546/DM580/BA590 and EX450-490/DM510/BA520 to distinguish red
and green fluorescence, respectively.
Immunoblot Assays for Expression of
Washed cell pellets derived from 60-mm dishes were
solubilized in sample buffer containing 8 M urea, 62.5
mM Tris-HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, and 5%
2-mercaptoethanol. For detection of APP
, APP
, and
Rab1B
APP, one-tenth of the total
cell protein was subjected to SDS-PAGE on a 6.25% polyacrylamide gel
(48) . For detection of Rab1B, one-fifth of the total protein
was run on a 12.5% polyacrylamide gel. Proteins were transferred to
Immobilon-P and the membranes were preincubated for 1 h in blotting
solution (PBS containing 5% powdered milk and 0.1% Tween 20).
Expression of Rab1B was monitored with monoclonal antibody 9E10
(Oncogene Science) directed against the c-Myc epitope tag on the Rab
protein. Initially, the expression of
APP
was
monitored with the Anti-6 polyclonal antibody, but in later studies
(Figs. 5 and 6) a monoclonal antibody (8E5) with specificity against
residues 444-592 of human
APP
(49) was used. The
latter reagent was provided by Dr. Dale Schenk (Athena Neurosciences).
All primary antibodies were used at a final concentration of 0.1
µg/ml, and incubations were carried out for 1 h. Secondary
antibodies were either horseradish peroxidase-conjugated goat
anti-rabbit IgG or horseradish peroxidase-conjugated goat anti-mouse
IgG (Bio-Rad), used at a 1:3000 dilution. Chemiluminescent detection
was performed using the ECL kit (Amersham). In some experiments (Figs.
5 and 6),
I-labeled goat anti-mouse IgG (0.45 µCi/ml;
Du Pont NEN) was used as the secondary antibody to facilitate
quantitation of the 8E5 monoclonal antibody bound to
APP
. After autoradiographic detection of bound
I-labeled IgG, radioactivity was quantitated by placing
sections of the blot in a
-counter. The same immunoblotting
procedure was used to quantitate the secreted exodomain derived from
APP in samples of culture medium. Since the 8E5 antibody does not
differentiate between APP
and APP
, the
term APP
is used to denote both possibilities.
Metabolic Labeling and Immunoprecipitation of
Cell cultures were pulse-labeled for 10 min
at 37 °C with 1 ml methionine-free DMEM containing 10% dialyzed
fetal calf serum and 100 µCi of
[APP
and APP
S]methionine/cysteine
(Tran
S-label, 1100-1200 Ci/mmol, ICN Inc.). The
cells were then washed twice with PBS and subjected to a 40-min
``chase'' in medium containing 10% fetal calf serum, 2
mM methionine, and 2 mM cysteine. Cells from parallel
cultures were harvested at the end of the pulse and chase periods and
collected by centrifugation. Cell pellets were lysed in 500 µl of
RIPA solution (100 mM Tris, pH 7.4, 2 mM EDTA, 0.1%
SDS, 0.5% sodium deoxycholate, 0.5% Nonidet P-40) and lysates were
cleared by centrifugation at 100,000
g for 20 min. The
supernatant solution was incubated for 2 h at 4 °C with 5 µl of
the 8E5 antibody, and immune complexes were collected by incubation for
1 h with protein A-Sepharose beads coupled with rabbit anti-mouse IgG.
The beads were washed four times with RIPA, one time with PBS, and
eluted by boiling for 10 min in 100 µl of SDS-PAGE sample buffer.
Proteins were subjected to SDS-PAGE followed by fluorography
(50) . The relative amounts of processed and unprocessed
APP
were determined from radioanalytic gel scans
with an AMBIS 4000 detector. Endo-H sensitivity was determined by
incubating the immunoprecipitated proteins overnight at room
temperature with 10 milliunits of endoglycosidase H (Boehringer
Mannheim) in 50 µl of 30 mM sodium citrate, pH 5.5, 0.75%
SDS, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl
fluoride, prior to elution from the protein A-Sepharose beads
(51) .
ELISA for A
The concentration of A was
determined in samples of 48-h conditioned medium from transfected
cultures by means of a sandwich-type ELISA, which employs a monoclonal
antibody directed against an epitope in the A
peptide that spans
the site cleaved by
-secretase and a second antibody directed
against the first 16 amino acids of A
(7) .
Generation of Rab1B Mutants
To begin to explore
the relationship between Rab-mediated transport pathways and APP
processing, two specific mutations that alter the GTP binding
properties of Rab proteins and other Ras-related proteins were
introduced into Rab1B. The first mutation entails substitution of Asn
for Ser at position 22 (Rab1B
). This Ser is analogous to
Ser-17 in H-Ras, which is known to coordinate Mg
and
the
phosphate of the guanine nucleotide
(52) . Previous
studies have indicated that this mutation greatly reduces the affinity
of Rab1 proteins for GTP without altering their affinity for GDP
(35, 53) . The second mutation entails substitution of
Ile for Asn at position 121 (Rab1B
). This Asn is
equivalent to Asn-116 in the conserved NK XD sequence element
in H-Ras, which is involved in binding the purine ring of GTP and GDP
(54) . Rab1A and Rab1B proteins bearing this mutation fail to
bind GTP and GDP in vitro(35, 55) . Both
Rab1B
and Rab1B
behave as
dominant-negative suppressors of endogenous Rab1 function when
expressed in cultured HeLa cells, as evidenced by their ability to
impair ER
Golgi transport of vesicular stomatitis virus
glycoprotein
(35) . To facilitate quantitation of expressed Rab
proteins by immunoblot assays, our wild-type and mutant Rab1B cDNAs were engineered to encode a Myc epitope tag at the
amino-terminal end of the protein. Addition of such epitope tags to
Ras-related proteins, including several Rab proteins, has not been
found to alter their function or subcellular localization
(53, 55, 56, 57, 58, 59) .
Coexpression of Rab1B with
To test the effects of mutant Rab1B proteins on the
intracellular transport and processing of APP in 293
Cells
APP, we developed a
co-transfection/transient expression assay utilizing cultured human 293
cells. The wild-type and mutant Rab1B constructs described
above were subcloned into the mammalian expression vector, pCMV5neo.
The coding sequence for human
APP
was inserted into
the vector, phCK751. When cells were transfected with DNA mixtures
containing phCK751 combined with pCMV Rab1B,
pCMV Rab1B, or pCMV Rab1B, expression of both the
APP
and Rab1B proteins was readily detected by
immunoblot analysis of cell lysates after 24 h (Fig. 1). In
cultures that were transfected with phCK751 alone, the Myc antibody
gave no discernable signal in the 20-30-kDa region of the blot,
which contains endogenous Rab proteins. Based on the results from a
separate immunoblot performed with a polyclonal antibody directed
against the carboxyl-terminal hypervariable region of Rab1B (not
shown), the overall expression of Myc-tagged Rab1B
in the
transfected cultures was estimated to be 44 times that of the
endogenous Rab1B. In accord with findings reported with other Rab
proteins
(60) , the expression of the Rab1B
and
Rab1B
mutants was reduced by 55-75% compared to
the wild-type protein (Fig. 1). However, these proteins were
still expressed at levels that were 10-19-fold above that of
endogenous Rab1B.
Figure 1:
Immunoblots demonstrating coexpression
of APP
and Rab1B proteins in human embryonic kidney
293 cell cultures. Cells were transfected with 2 µg of phCK751,
either alone or in combination with pCMV Rab1B (10 µg),
pCMV Rab1B (30 µg), or pCMV Rab1B (30 µg).
Cells were harvested 18 h after transfection and solubilized in SDS
sample buffer. One-tenth of the total cellular protein was subjected to
SDS-PAGE on a 6.25% polyacrylamide gel and immunoblotted with Anti-6
antibody against
APP ( upper panel). One-fifth of the
total protein was run on a 12.5% polyacrylamide gel and immunoblotted
with 9E10 antibody against the myc-epitope on Rab1B ( lower
panel). ECL was used for detection of bound IgG. Positions of
molecular weight markers are indicated at the right of each
panel.
APP
was strongly overexpressed
in 293 transfected with the phCK751 construct, regardless of whether or
not pCMV Rab1B was cotransfected in the same culture
(Fig. 1). Although 293 cells normally express some
APP
(10) , the endogenous
APP was not detectable with the brief
ECL exposures used to assay the transfected cells in this study. In the
cultures that were transfected with phCK751 alone or in combination
with pCMV Rab1B, the immunodetectable
APP
migrated on 12.5% SDS-polyacrylamide gels as a closely spaced
doublet of approximately 108 and 130 kDa, corresponding to the
unprocessed nascent polypeptide and the mature fully glycosylated
protein, respectively
(8, 10) . Although the nascent and
mature forms of
APP
were poorly resolved in these
initial studies of protein expression, it is noteworthy that in the
cultures that had been cotransfected with plasmids encoding mutant Rab
proteins ( i.e. Rab1B
and
Rab1B
), there appeared to be a diminution in the 130-kDa
band, with little or no change in the expression of the 108-kDa band.
This observation provided the first hint that overexpression of the
mutant Rab1B proteins might be affecting the posttranslational
processing of
APP.
APP processing in the cotransfected cells, an immunofluorescence
study was conducted to determine the extent to which cotransfection
resulted in the transient expression of both
APP
and
Rab1B in the same population of cells. Cultures that had been
transfected with phCK751 and pCMV Rab1B were analyzed by
dual-antibody immunofluorescence. The overall transfection efficiency
for the phCK751 + pCMV Rab1B combination was determined to
be approximately 30%. Among the positive cells, 89% stained with both
the anti-
APP and anti-myc antibodies (data not shown). The
staining with both antibodies exhibited a punctate distribution in
discrete regions surrounding the nucleus, similar to the pattern
recently described for Rab1B in NRK cells
(61) . This is
consistent with the expected localization of the majority of
APP
precursor and the Myc-tagged Rab1B in membranes of the ER and Golgi
network. Transfection efficiencies and coexpression percentages similar
to those just described were also seen in cultures that were
transfected with phCK751 combined with the mutant pCMV Rab1B (data not shown).
Posttranslational Processing of
Because of
the high degree of overlap between cell populations overexpressing
APP
Is Impaired in Cells Expressing Mutant Rab1B
APP
and Rab1B in the cotransfected cultures, it was
feasible to conduct a pulse-chase study to determine whether the
overexpression of wild-type or mutant Rab1B might have functional
consequences for the posttranslational processing of
APP.
Cell-associated radiolabeled
APP was immunoprecipitated from
parallel transfected 293 cell cultures, either immediately after a
10-min pulse with [
S]methionine, or after a
40-min chase to allow time for nascent
APP to undergo ER
Golgi transport and oligosaccharide maturation. As shown in
Fig. 2A, when the immunoprecipitated
APP was
resolved by SDS-PAGE immediately after the pulse, the nascent 108-kDa
form of
APP
was the only radiolabeled protein
detected in all of the cultures. After the 40-min chase, significant
accumulation of the mature 130-kDa form of
APP
could
be seen in cells that were overexpressing
APP
,
either alone or in combination with Rab1B
. In contrast,
the mature form of
APP
was almost undetectable in
cells that were coexpressing
APP
with
Rab1B
. Instead, a poorly resolved
APP
band with a mobility slightly slower than that of the 108-kDa
protein became visible after the 40-min chase (compare 0 versus 40 min lanes). The marked impairment of conversion of
APP
to the 130-kDa form in cells expressing
Rab1B
resembled the arrest of
APP processing seen
in cells treated with brefeldin A, an agent that is known to disrupt
Golgi architecture and interfere with ER
Golgi trafficking of
glycoproteins
(62, 63) (Fig. 2 A).
Cultures in which
APP
was coexpressed with
Rab1B
(compared to Rab
) showed a
noticeable, but less severe, decrease in accumulation of radiolabeled
130-kDa
APP after the 40-min chase (Fig. 2 A).
Figure 2:
Pulse-chase analysis indicates that
posttranslational processing of APP is impaired in cells
expressing mutant Rab1B. 293 cells were cotransfected with phCK751 and
the indicated pCMV Rab1B constructs as described in the legend
to Fig. 1. Eighteen hours after transfection, parallel cultures were
pulse-labeled for 10 min with [
S]methionine and
harvested immediately (0 min), or chased in medium with unlabeled
methionine for 40 min. In one pair of cultures that had been
transfected with phCK751, brefeldin A (2.5 µg/ml) was added to the
medium 1 h prior to the pulse, and was maintained throughout the
subsequent pulse-chase incubations. Cell pellets were solubilized and
APP was immunoprecipitated as described under ``Experimental
Procedures.'' Panel A shows the results of fluorography
(48 h exposure) of dried SDS gels containing the immunoprecipitated
APP from parallel cultures harvested before and after the 40-min
chase. Panel B shows the results of two experiments in which
the radioactivity in regions of the dried gel containing the processed
(130 kDa) and unprocessed (108-115 kDa) forms of
APP was
used to calculate the ratio of processed to unprocessed protein at the
end of the 40-min chase.
Because the levels of expression and labeling of nascent
APP
were somewhat different in each of the
transfected cultures, fluorographic analysis was extended by direct
quantitation of the radioactivity in gel segments containing the
130-kDa versus 108-115-kDa forms of
APP at the end
of the 40-min chase. When this ratio was used as an index of
APP
processing, it was clearly evident in two separate experiments that
overexpression of Rab1B
, and particularly
Rab1B
, resulted in substantial inhibition of the
posttranslational maturation of
APP
(Fig. 2 B).
Low Molecular Mass Forms of
Based on the localization of Rab1B in the ER,
Golgi membranes, and transitional vesicles between these organelles
(61) , it is reasonable to infer that the impaired maturation of
APP Are Sensitive to
Endoglycosidase H
APP
observed in the foregoing pulse-chase study was
a result of Rab1B
or Rab1B
interfering
with Rab1-mediated transport steps required for delivery of nascent
APP to the Golgi compartment. To further explore this possibility,
cell-associated radiolabeled
APP
was
immunoprecipitated from cotransfected cultures after a 1-h chase, and
the precipitated protein was digested with Endo-H. The latter enzyme
removes high-mannose N-linked oligosaccharides, which are
present on newly synthesized glycoproteins in the ER, but does not
cleave complex N-linked carbohydrates that have been trimmed
by
1,2-mannosidase II and subjected to further modification
( e.g. addition of sialic acid) in the medial and late Golgi
compartments
(64) . As expected, the mature 130-kDa form of
APP in cells expressing
APP
alone or in
combination with Rab1B
was insensitive to Endo-H
(Fig. 3). In contrast, the 108-kDa form of
APP isolated from
all of the cultures, including those overexpressing the Rab1B
and Rab1B
, exhibited a mobility shift equivalent
to approximately 2 kDa after treatment with Endo-H (Fig. 3). The
magnitude of this mobility shift was identical to that previously
reported when N-linked carbohydrate was removed from the
nascent forms of
APP by digestion with endoglycosidase F
(10) . In the cells expressing Rab1B
, the
intermediate
APP band (approximately 112 kDa), which was poorly
resolved in the earlier pulse-chase study (see Fig. 2 A),
was clearly separated from the 108-kDa form and also exhibited a
mobility shift when exposed to Endo-H (Fig. 3). These findings
support the conclusion that the lower molecular mass forms of
APP
, which accumulate in 293 cells expressing mutant
Rab1B proteins, are localized in a pre-Golgi or early Golgi
compartment.
Figure 3:
Low molecular mass forms of APP in
cultures expressing Rab1B mutants are sensitive to endoglycosidase H.
Eighteen hours after transfection, 293 cell cultures expressing
APP
alone, or with the indicated Rab1B proteins,
were pulse-labeled with [
S]methionine for 10 min
and chased for 40 min. At the end of the chase,
APP was
immunoprecipitated and incubated with (+) or without (-)
Endo-H as described under ``Experimental Procedures.'' All
samples were then subjected to SDS-PAGE and fluorography. The
fluorograms shown in the figure were exposed for 40
h.
Secretion of APP
Endoproteolytic cleavage of
Is Suppressed in Cells
Expressing Mutant Rab1B
APP
by an enzymatic activity termed
-secretase
appears to occur in a late compartment of the secretory pathway,
resulting in the release of the 120-kDa NH
-terminal
exodomain as a soluble protein, APP
(3, 4) . Likewise,
-secretase activity, which
results in the production of APP
and an amyloidogenic
carboxyl-terminal tail, is thought to occur in a post-ER compartment.
Therefore, impairment of protein trafficking between the ER and Golgi
membranes might reduce the access of mature
APP to the secretase
enzymes and diminish the output of APP
to the extracellular
medium. To test this possibility, cells that were coexpressing
APP
with Rab1B
or Rab1B
were pulse-labeled with [
S]methionine for
10 min to label nascent
APP. The intracellular
APP and the
extracellular APP
were then immunoprecipitated at the end
of a 1-h chase. Because most of the APP
generated from
APP
in 293 cells is the
form, the results of
this study reflect the activity of the
-secretase pathway. As
shown in Fig. 4, cells expressing Rab1B
effectively
processed
APP
to the mature 130-kDa form and
secreted radiolabeled APP
into the culture medium. Addition
of brefeldin A to a parallel culture expressing Rab1B
blocked the deposition of APP
in the medium,
confirming a previous report that trafficking of
APP through an
intact Golgi apparatus is essential for secretory processing
(16) . When
APP
was coexpressed with
Rab1B
instead of Rab1B
, intracellular
processing of
APP
was markedly inhibited and
accumulation of radiolabeled APP
in the medium was
undetectable after the 1-h chase (Fig. 4).
Figure 4:
Secretion
of APP is inhibited in cells coexpressing
APP
with Rab1B
. Eighteen hours after
transfection, 293 cell cultures that were coexpressing
APP
with either Rab1B
or Rab1B
were
pulse-labeled for 10 min with [
S]methionine.
Cells were then chased for 1 h in medium containing 10% serum and both
the cell monolayer and the conditioned medium were collected for
immunoprecipitation with the 8E5 monoclonal antibody, which recognizes
epitopes in
APP and its secreted exodomain, APP
. Where
indicated, brefeldin A (2.5 µg/ml) was added to cells 1-h prior to
the pulse, and maintained throughout the subsequent pulse and chase
incubations. All immunoprecipitated proteins were subjected to SDS-PAGE
and fluorography (72-h exposure). The radiolabeled
APP
immunoprecipitated from the cells before and after the 1-h chase is
shown in the lanes marked Cells. The radiolabeled APP
immunoprecipitated from the medium at the end of the chase is
shown in the lanes marked Medium.
To further evaluate
the effect of Rab1B expression on APP secretion,
conditioned medium was obtained from cultures that were coexpressing
APP
with either Rab1B
or
Rab1B
for 48 h. Aliquots of the medium were subjected
to immunoblot analysis, using the 8E5 monoclonal antibody and a
secondary goat anti-mouse
I-labeled IgG to quantitate the
relative amounts of accumulated APP
in each sample. Data
derived from two separate transfection studies indicated that
coexpression of Rab1B
with
APP
resulted in a marked reduction in the total APP
that
accumulated in the conditioned medium (Fig. 5 A). Similar
results were obtained when the values for extracellular APP
were normalized to the values for total intracellular
APP in
the cell monolayers, to compensate for variations in
APP
expression among the cultures (Fig. 5 B). In a separate
study we determined that the amount of APP
that accumulated
in conditioned medium from cultures transfected with pCMV Rab1B alone was only 15-20% of that measured in cultures where
pCMV Rab1B was cotransfected with phCK751. Thus, the small
amount of APP
in the medium from cultures coexpressing
APP
with Rab1B
primarily reflects
residual secretory processing of the expressed
APP
,
rather than a background level of APP
production from
endogenous
APP in the non-transfected cell population.
Figure 5:
Accumulation of APP is reduced
in medium from 293 cells coexpressing
APP
with
Rab1B
. Cells were cotransfected with phCK751 and either
pCMV Rab1B or pCMV Rab1B. Immediately after
transfection, cultures were fed with 2 ml of DMEM containing 10% fetal
calf serum and were maintained in the same medium for 48 h. Both the
conditioned medium and the cell monolayer were collected from each
culture. Aliquots of conditioned medium (30-70 µl) were
subjected to SDS-PAGE and immunoblot analysis, using
I-labeled goat anti-mouse IgG to detect the 8E5 antibody
bound to APP
. The bound
I-labeled IgG counts
were used to determine the relative amount of accumulated APP
in the 2 ml of conditioned medium. In parallel blots the total
intracellular
APP (both processed and unprocessed forms) was
determined by the same method. For each culture the results were
expressed as the total secreted APP
in the medium
( Panel A), and as a normalized value obtained by forming a
ratio between total secreted APP
and total intracellular
APP (cpm of bound
I-labeled IgG) ( Panel B).
The figure shows the results of two separate experiments, the first
indicated by stippled bars and the second indicted by
cross-hatched bars. Transfections were done in triplicate and
each bar indicates the mean (± S.D.) of the
determinations from three parallel cultures.
Production of A
Since the foregoing studies indicated that Rab1B
plays an essential role in early steps of trafficking of via Alternative Processing of
APP
Is Inhibited in Cells Expressing
Rab1B
APP along
the major
-secretory pathway, we next asked what effect
perturbation of Rab1B function might have on the processing of
APP
along the alternative
-secretase pathway responsible for the
biogenesis of A
. To facilitate detection of A
, these studies
were conducted with cells that were transfected with the plasmid
pohCK751sw, which encodes a variant form of
APP
found in a Swedish family exhibiting an autosomal dominant
pattern of Alzheimer's disease. Although cells expressing the
Swedish variant of
APP continue to generate APP
through the
-secretase pathway, they exhibit substantially
increased production of the
-secretase product,
APP
, and the amyloid peptide, A
(22, 65, 66) .
APP
, coexpression of Rab1B
with the
Swedish variant of
APP
( i.e.
APP SW
) resulted in a 70-80%
decrease in the accumulation of APP
in conditioned medium
from the transfected cultures (Fig. 6, A and
B). This was true regardless of whether the results were
expressed as total APP
(Fig. 6 A) or a ratio
of extracellular APP
to intracellular
APP
(Fig. 6 B). Although it is likely that these results
reflect a diminished production of both APP
and
APP
, this cannot be stated definitively, since the
antibody used in this study does not discriminate between the
and
forms of APP
. A more direct measure of the activity
of the amyloidogenic pathway in the transfected cells was obtained by
using an ELISA to quantitate the level of A
in samples of
conditioned medium from the same cultures used for the APP
assays. The results indicate that the decline in production of
APP
in cultures expressing Rab1B
was
accompanied by a parallel decrease in the secretion of A
into the
medium (Fig. 6, C and D). Thus, both the
amyloidogenic and secretory processing of
APP were clearly
affected by perturbation of Rab1B-mediated trafficking events. In fact,
the data suggest that interference with the function of Rab1B may have
an even greater impact on the events leading to A
release than it
is does on the secretion of APP
. This point is underscored
by a comparison of the A
/APP
ratios in samples of
medium derived from cultures that were coexpressing
APP SW
with either Rab1B
or
Rab1B
(Fig. 6 E). At present, the
mechanism underlying the apparent differential inhibition of A
versus APP
biogenesis in cells expressing
Rab1B
remains to be defined.
Figure 6:
Accumulation of both A and
APP
is reduced in medium from 293 cells coexpressing an
amyloidogenic variant of
APP with Rab1B
. Cells
were cotransfected with pohCK751sw and either pCMV Rab1B or
pCMV Rab1B. Immediately after transfection, cultures were fed
with 2 ml of DMEM containing 10% fetal calf serum and were maintained
in the same medium for 48 h. Both the conditioned medium and the cell
monolayer were collected from each culture. Aliquots of conditioned
medium and cell lysate were subjected to SDS-PAGE and immunoblot
analysis to determine the relative amounts of secreted APP
and intracellular
APP in the cultures. The concentration of
A
was determined by ELISA in aliquots of conditioned medium from
the same cultures. The results of the APP
assays were
expressed as total secreted APP
(based on cpm of bound
I-labeled IgG on the immunoblots) per 2 ml of conditioned
medium ( Panel A) and as a ratio of total secreted APP
(cpm) to total immunodetectable cellular
APP (cpm) for each
culture ( Panel B). The results of the A
assays were
expressed as the total A
(ng) accumulated in 2 ml of conditioned
medium ( Panel C) and as nanograms of extracellular A
per
unit of cellular
APP (1 unit = 10
cpm of bound
I-labeled IgG) ( Panel D). The ratio of total
A
(ng) to total immunodetectable units of APP
(1 unit
= 10
cpm bound
I-labeled IgG) in the
conditioned medium from each culture is depicted in Panel E.
Transfections were done in triplicate and each bar indicates
the mean (± S.D.) of the determinations from three parallel
cultures.
Golgi transport and processing of virus-encoded glycoprotein when
expressed in mammalian cells infected with vesicular stomatitis virus.
These dominant-negative effects have suggested that Rab1 proteins may
play a global role in ER
Golgi transport of a variety of other
integral membrane glycoproteins that undergo oligosaccharide maturation
and/or O-glycosylation as they traverse the Golgi compartment
en route to the cell surface. The present demonstration that
Rab1B
, and to a lesser extent Rab1B
, can
impair the conversion of Endo H-sensitive precursor forms of
APP
to the higher molecular weight O-glycosylated forms, provides
strong independent evidence to support this view.
and Rab1B
inhibited the conversion
of
APP
to the fully glycosylated 130-kDa form, the
effect of Rab1B
was much more pronounced than the
effect of Rab1B
(see Fig. 2). Recent studies
focusing on Rab1A, a functionally interchangeable isoform of Rab1B,
indicate that these two mutations may interfere with the physiological
function of Rab1 proteins through different mechanisms. The S25N
mutation in Rab1A reduces the affinity of the protein for GTP without
altering its affinity for GDP, thus favoring retention of the protein
in the GDP state
(53) . In contrast, the N124I substitution in
Rab1A (equivalent to N121I in Rab1B) decreases the affinity of Rab1A
for both GTP and GDP
(55) . Morphological analyses suggest that
the inhibition of vesicular stomatitis virus glycoprotein transport by
Rab1A
or Rab1B
occurs primarily at the
level of vesicle budding from the ER
(53) , whereas the block in
ER
Golgi transport produced by Rab1A
or
Rab1B
occurs as a result of interference with the
targeting or fusion of transitional vesicles with the
cis-Golgi
(35, 55) . Interestingly, both
mutants have been shown to cause dispersion of the Golgi apparatus when
they are microinjected into rat fibroblasts, possibly indicating a
disruption of the equilibrium between anterograde and retrograde
transport through this organelle
(67) . The latter observation
is consistent with the hypothesis that in addition to regulating ER
Golgi transport, Rab1B may play a role in regulating early steps
in intra-Golgi vesicle trafficking
(28) .
APP by
-secretase is believed to occur after the protein has traversed
the trans-Golgi compartment and undergone
O-glycosylation and tyrosine sulfation
(13, 20, 68, 69) . The studies described
in Figs. 4 and 5 confirm this model by showing that the decreased flux
of
APP into the Golgi compartment in cells coexpressing
APP
with Rab1B
is accompanied by a
substantial reduction in secretion of APP
. It is noteworthy
that the inhibition of secretion of APP
in cells expressing
Rab1B
was not complete; i.e. the cumulative
levels of APP
in samples of 48-h conditioned medium were
approximately 30% of those detected in cultures expressing
Rab1B
. There are several possible explanations for this
finding. The most obvious possibility is that the block in ER
Golgi transport mediated by Rab1B
is leaky, so that
over an extended period of time enough
APP might reach the
trans-Golgi network and plasma membrane to generate detectable
quantities of secreted APP
. An alternative possibility is
that a small portion of the relatively large intracellular pool of
immature
APP in transfected 293 cells is subject to
endoproteolytic cleavage by an unidentified enzyme in the ER,
generating an amino-terminal APP fragment that is recognized by the 8E5
antibody. If the resulting APP fragment could be translocated to the
cell surface without traversing the Golgi apparatus, one might expect
its extracellular accumulation to be insensitive to the block in ER
Golgi trafficking by Rab1B
. In this regard,
Gabuzda et al.(70) have recently described a protease
activity that degrades the immature form of
APP in COS cells,
yielding an 11.5-kDa peptide that contains the intact A
sequence.
It is not yet known whether this activity results in the release of an
intact amino-terminal exodomain.
APP
( i.e.
APP SW
) provide new information about
the pathway for generation of A
. When Rab1B
was
coexpressed with
APP SW
, the secretion of
APP
was suppressed to the same extent as in the experiments
with wild-type
APP
. Since the APP
derived from APP SW
is a mixture of
APP
and APP
, whereas the APP
derived from wild-type
APP
is predominantly
APP
(65) ,
(
)
these
findings suggest that transport of
APP from the ER to the Golgi
apparatus is required for entry of the protein into the
-secretase
pathway as well as the
-secretase pathway. This conclusion is
strengthened by the finding that the concentration of A
was
markedly reduced in conditioned medium from cells that were
coexpressing
APP SW
with
Rab1B
. The foregoing observations are entirely
consistent with previous studies in which both
APP maturation and
A
production were inhibited by brefeldin A
(16) . The
results also indicate that if immature
APP can undergo proteolysis
in the ER or early Golgi to form potentially amyloidogenic
carboxyl-terminal fragments, as suggested in a recent study
(70) , then Rab1B-mediated transport events may be required for
translocation of these fragments to a compartment where they can be
further processed to yield A
.
versus APP
in conditioned medium from cells
coexpressing
APP SW
with Rab1B
instead of Rab1B
(see Fig. 6 E) was
unexpected, and raises the intriguing possibility that interference
with Rab1B-mediated export of
APP from the ER has a more severe
impact on amyloidogenic processing of
APP than it does on the
-secretase processing of the protein. At present we can only
speculate about the types of mechanisms that might underlie this
phenomenon. One possibility is that the
-secretase has a higher
K
for mature
APP substrate than does
the
-secretase, so that even a modest decline in the pool of
mature
APP traversing the Golgi compartment has a substantial
impact on
-cleavage and A
production, while allowing some
-cleavage to continue. Alternatively, it is possible that the
concentration of mature
APP residing in a specific compartment
( e.g. the trans-Golgi network) must exceed a
particular threshold value before the steps involved in A
production (
-secretase cleavage and possible endosomal processing
of the carboxyl-terminal domain) can operate at optimal efficiency. The
latter model would be consistent with a recent study in which amino
acid substitutions that impair cleavage of
APP at the
-secretase site were shown to increase A
production, possibly
by increasing substrate availability for the
-secretase pathway
(71) . Future studies using antibodies that can discriminate
between the APP
and APP
secretory
products should help to distinguish between these potential mechanisms.
APP, it should be emphasized that the
transport routes which determine alternative amyloidogenic processing
of
APP remain poorly characterized. For example, while acidic
compartments such as the endosome or lysosome have been implicated in
the production of A
(16, 72, 73) , it is
uncertain whether entry of
APP and its carboxyl-terminal remnants
into this pathway occurs via internalization from the plasma membrane,
translocation from an intracellular compartment such as the
trans-Golgi network, or both. Thus, aside from documenting a
role for Rab1B in ER
Golgi transport of
APP, the results of
the present study clearly support the feasibility of using
dominant-negative mutations in additional members of the Rab protein
family to define and distinguish other lesser known segments of the
APP processing routes. This experimental approach provides a
powerful new tool for dissection of this critical pathway in the
pathogenesis of Alzheimer's disease.
, amyloid
-peptide;
APP,
-amyloid precursor protein; APP
, soluble
NH
-terminal exodomain derived from
APP; PAGE,
polyacrylamide gel electrophoresis; PCR, polymerase chain reaction;
Endo-H, endoglycosidase H; PBS, phosphate-buffered saline; DMEM,
Dulbecco's modified Eagle's medium; ER, endoplasmic
reticulum; bp; base pair(s); CMV, cytomegalovirus; ELISA, enzyme-linked
immunosorbent assay.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.