(Received for publication, October 19, 1995)
From the
The Ras-related GTP-binding protein, Rab6, is localized in late
Golgi compartments where it mediates intra-Golgi vesicular trafficking.
Herein we report that coexpression of Alzheimer's -amyloid
precursor protein (
APP
) with a dominant-negative
Rab6 mutant (Rab6
) in human embryonal kidney 293 cells
causes an increase in secretion of the soluble amino-terminal exodomain
(s-APP
) derived from non-amyloidogenic processing of
-APP
by
-secretase. The effect was specific to
Rab6
, since the corresponding mutation in Rab8 (i.e. Rab8
), which has been implicated in
protein transport to the plasma membrane, caused a modest reduction in
s-APP
secretion. While Rab6
stimulated secretion
of APP
, the accumulation of amyloid
peptide (A
) in the
medium was either moderately reduced or unaffected. Similar
differential effects of Rab6
on secretion of s-APP
versus A
were observed in cell cultures that were
overproducing A
after transfection with a plasmid encoding the
Swedish variant of
APP
. Moreover, assays of medium
from the latter cultures revealed a marked increase in secretion of
s-APP
relative to s-APP
(the immediate product derived from
cleavage of
APP by
-secretase). The results indicate that
vesicular transport events controlled by Rab6 occur at or near a
critical juncture in the trans-Golgi network where
APP is
sorted into either the constitutive
-secretase pathway or the
amyloidogenic
-secretase pathway.
The 4-kDa amyloid -peptide (A
) (
)has been
implicated in the pathogenesis of Alzheimer's
disease(1, 2) . A
is formed as the result of
intracellular proteolytic processing of
-amyloid precursor
proteins (
APP
,
APP
, and
APP
). The biogenesis of A
begins when
APP
is cleaved by an enzymatic activity termed
-secretase, releasing a
soluble NH
-terminal exodomain (s-APP
), which is
secreted from the cell, and leaving a membrane-anchored COOH-terminal
fragment containing the intact A
sequence(3, 4, 5, 6) . A
is
released when the latter fragment is further trimmed by another
proteolytic activity currently referred to as
-secretase(7, 8) . While amyloidogenic processing
of
APP is known to occur at low levels ubiquitously, cells that
produce
APP direct the substantial proportion of the precursor
protein to the constitutive secretory pathway, where it is processed
via a non-amyloidogenic mechanism. The latter entails initial cleavage
of
APP within the A
domain by an enzymatic activity
termed
-secretase, releasing a soluble exodomain (s-APP
)
containing part of the A
sequence(3, 9, 10, 11) . Because the
residual COOH-terminal stump lacks an intact A
domain, it cannot
give rise to A
when cleaved by
-secretase.
The mechanisms
by which cells control the flux of APP into the amyloidogenic versus non-amyloidogenic pathways are poorly understood. It
has been particularly difficult to determine the precise subcellular
sites of the various processing events because the relevant proteases
have not yet been isolated. However, there is sufficient evidence to
suggest that
-secretase acts on the mature form of
APP at the
cell surface or in a late compartment of the default secretory pathway,
after it has undergone tyrosine
sulfation(10, 12, 13, 14, 15) .
Less is known about the subcellular sites of the amyloidogenic
processing of
APP by
-secretase and
-secretase. A number
of studies have indicated that events occurring in acidic compartments (e.g. endosomes or vesicles of the trans-Golgi
network) are involved in the biogenesis of
A
(16, 17, 18, 19) . In
particular, a recent study suggests that
-secretase cleavage of
APP containing the Swedish mutation occurs within transitional
vesicles between the trans-Golgi compartment and the cell
surface(20) .
To elucidate the steps involved in
intracellular trafficking and processing of APP, we have adopted a
novel strategy, which is based on mutagenesis and expression of
GTP-binding proteins encoded by the rab gene family. There are
currently more than 30 distinct Rab proteins, which are localized in
discrete organelles and vesicles, where they play key roles in protein
trafficking between specific donor and acceptor compartments along the
exocytic or endocytic routes (21, 22, 23) .
Like other Ras-related proteins, Rab proteins are active when they are
in the GTP-bound state. Hence, mutant Rab proteins with reduced
affinity for GTP exert a dominant negative effect over their endogenous
counterparts when overexpressed in mammalian
cells(24, 25, 26) . We recently showed that
when
APP
was coexpressed with a dominant-negative
Rab1B mutant (i.e. Rab1B
) in HEK 293 cells,
the maturation of
APP
and the secretion of both
s-APP and A
were impaired(27) , consistent with the
established role of Rab1B in ER
Golgi
transport(24, 28, 29) .
In the present
study we focused on Rab6, which is known to reside in trans-Golgi cisternae, the trans-Golgi network, and
post-Golgi secretory vesicles(30, 31, 32) ,
and has been implicated in both intra-Golgi transport (33) and
the budding of exocytic vesicles from the trans-Golgi
network(34) . We found that when APP
was
coexpressed with a GTP-binding-defective Rab6 mutant
(Rab6
), secretion of s-APP
was stimulated, while
deposition of A
and s-APP
into the medium was either modestly
reduced or unaffected. These findings suggest that transport steps
mediated by Rab6 occur at a branch point where amyloidogenic and
non-amyloidogenic pathways for
APP processing diverge.
We recently showed that the inhibitory effects of a Rab1B
mutation (N121I) on early steps in the posttranslational maturation of
APP
can be readily detected by transiently
coexpressing both proteins in HEK 293 cells(27) . In the
present study, we used the same approach to examine the possible role
of a Golgi-localized Rab protein, Rab6, in the secretory and
amyloidogenic processing of
APP. By analogy to other members of
the Ras superfamily(46, 47) , incorporation of the
N126I substitution into Rab6 is predicted to drastically reduce the
affinity of the protein for GTP. Accordingly, Myc-Rab6
immunoprecipitated from 293 cells with anti-Myc antibody failed
to bind detectable [
P]GTP when compared to
Myc-Rab6
in a standard blot-overlay assay (not shown). To
determine the effects of this Rab6 mutation on secretion of s-APP and
A
in 293 cells, Myc-Rab6
and Myc-Rab6
were each coexpressed with
APP
for 48 h and
the relative amounts of s-APP and A
were measured in samples of
conditioned medium (Fig. 1). To exclude the possibility that
differences in extracellular s-APP or A
might reflect variations
in
APP
expression in the cell monolayers, the values
for s-APP and A
were normalized to the total intracellular
APP
in each culture. Compared to cultures that were
not transfected with Rab6 plasmid, the cultures that were coexpressing
Rab6
with
APP
showed a modest reduction
in secreted s-APP. The opposite effect was observed in cultures
expressing Rab6
, where levels of s-APP were markedly
elevated (Fig. 1A). In contrast to the level of s-APP,
the extracellular concentration of A
did not increase in the
cultures that were expressing Rab6
(Fig. 1B). In fact, the A
levels were lower
in these cultures than in the cultures expressing Rab6
or
no exogenous Rab6. The inverse effects of Rab6
on s-APP
and A
in this study are underscored by the 5-fold decline in the
direct ratio of extracellular A
to extracellular s-APP in the
cultures expressing Rab6
versus Rab6
(Fig. 1C).
Figure 1:
Rab6 has differential
effects on secretion of s-APP versus A
. Cells were
cotransfected with phCK751 alone or in combination with plasmids
encoding Myc-tagged Rab6
or Rab6
, as
indicated. All cultures were incubated without changing the medium for
48 h. Expression levels of the Myc-Rab6
or
Myc-Rab6
ranged from 3- to 5-fold over the endogenous
Rab6, as determined by immunoblot analysis with an affinity-purified
antibody against Rab6 (not shown). Expression levels of
APP
were approximately 40-fold over the endogenous
APP in 293 cells, so that measurements of products derived from
APP (i.e. s-APP) reflect almost exclusively the
processing of the overexpressed
APP
in the
subpopulation of cells expressing the Rab6 constructs. Upon harvesting
the cultures, levels of s-APP (counts/min) and A
(picograms) were
determined in aliquots of the conditioned medium by Western blot assay
using
IgG or ELISA, respectively (see ``Experimental
Procedures''). The values for total s-APP (panel A) and
A
(panel B) in 2 ml of medium were normalized to the
total intracellular
APP
in the cell monolayer. In panel C, the values for total extracellular A
were
expressed as a ratio to total extracellular s-APP. The results shown
are means (± S.E.) of separate determinations performed on three
parallel cultures.
To verify the specificity of the
results observed with the Rab6 mutant, the equivalent mutation (N121I)
was introduced into Rab8. The latter protein is localized to the cell
periphery (48, 49) and is normally expressed in a wide
variety of cells, including 293 cells. ()Rab8 has been
implicated as a mediator of protein transport from the trans-Golgi network to the basolateral (50) or
dendritic (51) plasma membrane in polarized epithelial and
neuronal cells, respectively. However, it is unclear whether Rab8 plays
a similar role in non-polarized cells. When
APP
was
coexpressed with Rab8
, the relative amount of secreted
s-APP was modestly reduced in comparison to parallel cultures
expressing Rab8
(Fig. 2A). The levels of
A
were also slightly reduced in the cultures expressing
Rab8
(Fig. 2B), and there was no change
in the direct ratio of A
to s-APP in the culture medium (Fig. 2C). These findings contrast with the striking
decrease in the ratio of A
/s-APP (mostly due to increased
secretion of s-APP) observed in the cultures expressing Rab6
(Fig. 1C).
Figure 2:
Rab8 affects s-APP
secretion in a manner distinct from Rab6
. Cells were
cotransfected with phCK751 alone or in combination with plasmids
encoding Myc-tagged Rab8
or Rab8
,
essentially as described in the studies with Rab6. All cultures were
incubated without changing the medium for 48 h. Upon harvesting the
cultures, levels of s-APP (counts/min) and A
(picograms) were
determined in aliquots of the conditioned medium by Western blot assay
using
IgG or ELISA, respectively (see ``Experimental
Procedures''). The values for total s-APP (panel A) and
A
(panel B) in 2 ml of medium were normalized to the
total intracellular
APP
in the cell monolayer. In panel C, the values for total extracellular A
were
expressed as a direct ratio to total extracellular s-APP. The results
shown are means (± S.E.) of separate determinations performed on
three parallel cultures.
To determine whether the higher end
point values for accumulated extracellular s-APP in cultures expressing
Rab6 reflected an increased rate of secretion of s-APP,
samples of medium from individual cultures overexpressing
APP
with either Rab6
or Rab6
were collected at multiple time points over a 24-h period (Fig. 3A). The results of this analysis demonstrated an
increased rate of s-APP accumulation in the cultures expressing
Rab6
compared to cultures expressing Rab6
.
The increased deposition of s-APP into the medium could not be
attributed to differential expression of
APP
, since
the intracellular levels of mature (fully glycosylated)
APP
(Fig. 3B) and immature
APP
(Fig. 3C) were comparable in
cultures expressing both the wild-type and mutant Rab6 proteins.
Consistent with the results obtained in the 48-h coexpression
experiment (Fig. 1), the 24-h end point values for extracellular
s-APP, normalized to total intracellular
APP
, were
increased approximately 2-fold in the cultures expressing
Rab6
versus Rab6
(Fig. 3D), while the values for A
were not
increased (Fig. 3E).
Figure 3:
Rate of secretion of s-APP is increased in
cells coexpressing APP
with Rab6
versus Rab6
. Beginning 18 h after transfection,
fresh medium (2 ml) was added to each culture and 50-µl aliquots
were removed at the designated intervals for quantitation of total
s-APP (panel A). After the last samples were removed, the cell
monolayers were harvested and the intracellular levels of mature
APP
(125-130 kDa) (panel B) and
immature
APP
(108-110 kDa) (panel C)
were determined. The values for total s-APP (panel D) and
A
(panel E) in the conditioned medium at 24-h are also
expressed as normalized values (ratio to the total intracellular
APP
). Values for s-APP and
APP are derived from
Western blot assays employing
I-IgG and are expressed as
total phosphorimager units/culture. Values for total A
(picograms)
are derived from ELISA. Each value is a mean (± S.E.) of
determinations performed on three parallel
cultures.
The s-APP secreted by 293 cells
expressing wild-type APP
is predominantly the
form (i.e. s-APP
) released upon cleavage by
-secretase(52) . In light of the contrasting effects of
Rab6
on levels of s-APP and A
, we were interested
in determining how expression of this Rab6 mutant might affect
secretion of the immediate product generated by
-secretase
cleavage (i.e. s-APP
). To facilitate this analysis, 293
cells were cotransfected with plasmids encoding either Rab6
or Rab6
together with the Swedish variant of
APP
(i.e. SW
APP
), which
contains a dual amino acid change (Lys
Asn/Met
Leu) known to promote increased processing
of the protein along the A
pathway (52, 53, 54) . Using antibodies that
discriminate between
and
forms of s-APP, we observed that
the amount of s-APP
(normalized to intracellular SW
APP
) secreted into the medium was almost
doubled in cultures expressing Rab6
(Fig. 4A). In contrast, the normalized value for
extracellular s-APP
was modestly reduced in the same cultures (Fig. 4B). These reciprocal changes were reflected in a
3-fold increase in the direct ratio of the
and
forms of
s-APP in the medium from cultures coexpressing SW
APP
with Rab6
versus Rab6
(Fig. 4C). Although
Rab6
caused a small decline in the level of
extracellular s-APP
in this experiment, we were unable to detect a
significant decrease in intracellular s-APP
(referred to as c-APP
in Fig. 4D). This suggests that
expression of Rab6
does not interfere directly with
transport steps required for SW
APP
to gain
access to the subcellular compartment containing
-secretase.
Figure 4:
Differential changes in s-APP versus s-APP
and A
in 293 cells coexpressing the
Swedish variant of
APP
with Rab6
.
Cells were cotransfected with pohCK751sw alone or in combination with
plasmids encoding Myc-tagged Rab6
or Rab6
,
as indicated. All cultures were incubated without changing the medium
for 48 h. Upon harvesting the cultures, levels of total A
(panel E) or A
(panel F) in
the conditioned medium were determined by ELISA. Heparin-Sepharose
pellets containing the total s-APP precleared from the samples of
medium prior to ELISA were eluted with SDS sample buffer, and levels of
extracellular s-APP
(panel A) and s-APP
(panel
B) were determined by Western blot assay using
IgG
(see ``Experimental Procedures''). Intracellular s-APP
(referred to as c-APP
) was also measured in lysates of
the cell monolayers (panel D). The results of the foregoing
assays were normalized to total intracellular SW
APP
. In panel C, the values for
s-APP
and s-APP
in the medium are also expressed as a direct
ratio. All values are means (± S.E.) of separate determinations
performed on three parallel cultures.
In
accord with the earlier studies of wild-type APP (Fig. 1B), coexpression of SW
APP
with Rab6
resulted in a 35-40% decrease in
the amount of A
that accumulated in the conditioned medium after
48 h (Fig. 4E). This decrease was similar in magnitude
to the decline in s-APP
in the same cultures (Fig. 4B). It is now recognized that A
exists in a
number of isoforms that include alternate carboxyl termini: e.g. A
and
A
(55, 56) . Although
A
is the more abundant species,
A
has attracted particular attention since it
is prone to form insoluble amyloid fibrils (57) and appears to
be a major constituent in amyloid deposits in Alzheimer's
disease(58, 59) . Thus, in addition to assaying total
A
(mostly A
), we employed an ELISA that
specifically measures A
in medium from the
cultures expressing SW
APP
(Fig. 4F). In contrast to the modest but
significant decline in total A
observed in cells expressing
Rab6
versus Rab6
(Fig. 4E), there was no detectable difference in
the extracellular A
in the same cultures (Fig. 4F).
Fig. 5depicts the results of a
separate time-course study in which we confirmed that the increased
extracellular s-APP/s-APP
ratio in medium from cultures
coexpressing Rab6
and SW
APP
arises as a result of differential effects of the Rab6 mutant on
the rates of secretion of the
and
forms of s-APP (Fig. 5, A-C). The marked increase in the rate of
sAPP
secretion in the cultures expressing Rab6
versus Rab6
was reflected in a 3-fold
elevation in the total extracellular s-APP
, normalized to
intracellular
APP
, at the 12-h end point (Fig. 5D). In contrast, the cultures expressing
Rab6
showed a slight decrease in the normalized value
for s-APP
(Fig. 5E) and no significant change in
A
(Fig. 5F).
Figure 5:
Differential changes in rates of secretion
of s-APP versus s-APP
in 293 cells coexpressing SW
APP
with Rab6
versus Rab6
. Beginning 18 h after transfection, fresh medium
(2 ml) was added to each culture and aliquots of medium were removed at
the designated intervals for quantitation of s-APP
(panel
A) and s-APP
(panel B) by Western blot assay using
I-IgG. The direct ratios of the
and
forms of
s-APP measured in the medium at 12 h are depicted in panel C.
Values for total extracellular s-APP
, s-APP
(phosphorimager
units) and A
(picograms) at the 12-h end point were normalized to
the total intracellular SW
APP
(phosphorimager units) and are depicted in panels
D-F. Each value is a mean (± S.E.) of separate
determinations performed on three parallel cultures. As in the case of
wild-type
APP
(Fig. 3), immunoblot assays
performed on the cell monolayers harvested at the 12-h end point
indicated that the intracellular levels of mature and immature SW
APP
were comparable in the cultures
expressing Rab6
and Rab6
(not
shown).
The results described in this report demonstrate that
overexpression of a GTP-binding-defective Rab6 mutant in 293 cells
results in a marked enhancement of secretion s-APP, while
extracellular accumulation of products derived from the amyloidogenic
processing pathway (i.e. s-APP
and A
) is either
modestly inhibited or unaffected. Extensive studies have documented the
localization of Rab6 in the trans-Golgi cisternae and trans-Golgi network in mammalian
cells(30, 31, 32) . Therefore, our findings
indicate that intra-Golgi transport events regulated by Rab6
selectively influence the routing of
APP
into the
-secretase pathway. While previous studies have shown that
s-APP
and s-APP
are sorted differently in polarized MDCK
cells (52) and that secretion of s-APP
and A
respond
in opposite ways to stimulation of protein kinase
C(60, 61, 62, 63, 64) , the
present report provides the first genetic evidence implicating a
specific protein (Rab6) and a specific subcellular compartment (the trans-Golgi network) in the branching of the
and
-secretase pathways for
APP processing.
Our studies with
Rab8 underscore the specificity of the results obtained with the Rab6
mutant. In contrast to the stimulatory effect of Rab6 on s-APP
secretion, Rab8
had a small
inhibitory effect on the same pathway. The decreased secretion of
s-APP
caused by the Rab8 mutant in 293 cells is consistent with
the proposed role of Rab8 in transport of proteins between the Golgi
apparatus and the basolateral plasma membrane in polarized epithelial
cells(50) . However, because the effects of Rab8
were relatively minor in comparison to the complete block in
s-APP secretion previously observed when ER
Golgi transport was
disrupted by Rab1B
(27) , we speculate that
post-Golgi secretory transport in non-polarized cells probably involves
members of the Rab family in addition to Rab8.
In addition to
providing mechanistic insights into APP processing, the finding
that the dominant-negative N126I mutation in Rab6 stimulates rather
than inhibits secretion of s-APP
augments the only other mutagenic
analysis of Rab6 function performed in intact cells. Specifically,
Martinez et al.(33) showed that overexpression of an activating Rab6 mutant, Rab6
, caused a delay in constitutive protein secretion, with a corresponding
accumulation of transport markers in late Golgi compartments. The
latter findings were interpreted as indicating that Rab6 acts either as
an inhibitor of anterograde transport through the trans-Golgi
compartment or as a positive regulator of retrograde transport from
post-Golgi vesicles back to the trans-Golgi network or Golgi
cisternae. Our results support this model of Rab6 function, since it
would predict that a dominant-negative Rab6 mutant such as
Rab6
might facilitate the anterograde flow of
glycoproteins into the distal portion of the constitutive secretory
pathway where
-secretase is thought to reside.
Based on the
foregoing model of Rab6 function in intra-Golgi trafficking, we propose
that by impairing retrograde transport or facilitating anterograde
transport within the trans-Golgi compartment, Rab6 increases the flow of
APP into the constitutive
-secretory pathway. The absence of a parallel stimulatory effect
on secretion of s-APP
and A
suggests that the pool of
APP destined for processing by
-secretase is sorted into a
distinct trans-Golgi or endosomal compartment prior to the
transport steps mediated by Rab6. This proposal is consistent with
studies indicating that
-secretase operates in an acidic
compartment (16, 17, 18, 19, 65) . An
alternative model of amyloidogenic processing envisions that
APP
is cleaved by
-secretase and
-secretase in endosomes or
lysosomes after it escapes cleavage by
-secretase and is
reinternalized from the plasma membrane. Support for this model is
derived from studies showing that intact
APP exists at the cell
surface and is reinternalized in clathrin-coated endocytic vesicles (12, 65) . However, it seems unlikely that this is the
major pathway for biogenesis of A
in 293 cells, since we failed to
observe a coordinate increase in s-APP
and A
production in
cells where transport of
APP to the cell surface was enhanced
2.5-3-fold.
The general experimental approach described herein
might be particularly useful in future studies aimed at elucidating the
molecular mechanisms underlying the biogenesis of distinct forms of
A. Specifically, the mutagenesis of various Rab proteins localized
in different segments of the exocytic and endocytic pathways might
provide insight into the question of whether A
and A
arise through the action of
unique
-secretase activities residing in separate subcellular
compartments. While the differential effect of Rab6
on
A
versus A
observed in the present study (Fig. 4) could be consistent
with the latter possibility, the results are inconclusive because the
effects of Rab6
on total A
were small and somewhat
variable (e.g. no decrease was observed in the short term
studies depicted in Fig. 3and Fig. 5). Thus, it will be
of considerable interest to see whether more definitive differences in
production of the A
isoforms will be detected as these studies are
expanded to include additional members of the Rab protein family.