Received for publication, September 16, 2002, and in revised form, November 7, 2002
APH-1 and PEN-2 genes modulate the
function of nicastrin and the presenilins in Caenorhabditis
elegans. Preliminary studies in transfected mammalian
cells overexpressing tagged APH-1 proteins suggest that this genetic
interaction is mediated by a direct physical interaction. Using the
APH-1 protein encoded on human chromosome 1 (APH-11L; also
known as APH-1a) as an archetype, we report here that endogenous forms
of APH-1 are predominantly expressed in intracellular membrane
compartments, including the endoplasmic reticulum and
cis-Golgi. APH-1 proteins directly interact with immature
and mature forms of the presenilins and nicastrin within high molecular
weight complexes that display
- and
-secretase activity. Indeed
APH-1 proteins can bind to the nicastrin
312-369 loss of function
mutant, which does not undergo glycosylation maturation and is not
trafficking beyond the endoplasmic reticulum. The levels of
expression of endogenous APH-11L can be suppressed by
overexpression of any other members of the APH-1 family, suggesting that their abundance is coordinately regulated. Finally, although the
absence of APH-1 destabilizes the presenilins, in contrast to nicastrin
and PEN-2, APH-1 itself is only modestly destabilized in cells lacking
functional expression of presenilin 1 or presenilin 2. Taken together,
our data suggest that APH-1 proteins, and APH-11 in
particular, may have a role in the initial assembly and maturation of
presenilin·nicastrin complexes.
 |
INTRODUCTION |
Presenilin 1 (PS1)1 (1), presenilin 2 (PS2) (2), and nicastrin (3) are components of high molecular weight
protein complexes that are required for the intramembranous proteolysis of some type 1 transmembrane proteins, including the
-amyloid precursor protein (APP) (4), Notch (5-11), and ErbB-4 (12). Genetic
screens in Caenorhabditis elegans have identified two additional proteins, APH-1 (13) and PEN-2 (14), in which loss of
function mutations phenotypically modulate Notch signaling in a manner
similar to that of null mutants in nicastrin and the presenilins.
Preliminary studies in transfected cells overexpressing tagged APH-1
proteins suggest that the genetic interaction between APH-1 and the
presenilin-dependent cleavage of Notch and APP is mediated
by a direct physical interaction between APH-1 and nicastrin or the
presenilins (14). We report here that both the APH-1 homologue on human
chromosome 1 (termed APH-1a in Ref. 14, but here referred
to as APH-11 for clarity to avoid confusion with labeling
of alternate splice forms) and the APH-1 homologue on chromosome 15 (previously referred to as APH-1b; here termed
APH-115) are widely expressed in multiple tissues and that
the APH-11 transcript is present as several different
alternatively spliced forms (data not shown). We also report that
endogenous APH-11 directly interacts with both immature and
mature forms of the presenilins and nicastrin and that high molecular
weight complexes containing these proteins display
- and
-secretase activity. Overexpression of any one member of the APH-1
family results in suppression of the levels of expression of all the
endogenous APH-1 proteins, suggesting that their abundance is
coordinately regulated by binding to the same limiting factors,
presumably the presenilins, nicastrin, and/or PEN-2. In contrast to
nicastrin and PEN-2, APH-1 is only modestly destabilized in cells
lacking functional expression of PS1 or PS2. Our data suggest that the
presenilins, nicastrin, and APH-1 proteins physically interact with
each other within high molecular weight protein complexes and that
APH-11 may have a role in the maturation of these
PS1·nicastrin complexes.
 |
EXPERIMENTAL PROCEDURES |
APH-1 Expression Vectors--
cDNA clones encoding human
APH-11S, APH-11L, and APH-115
proteins were all generated by PCR from reverse-transcribed human brain
or leukocyte RNA. After complete sequencing of both strands, the
cDNAs were ligated into the expression vector,
pcDNA4/myc-His(C) (Invitrogen).
Generation of Stable HEK293 Cell Lines Expressing Human APH-1
Proteins--
HEK293 cells stably expressing APP-CTF99 were
transfected with the above plasmids or with empty vector (as a negative
control) using LipofectAMINE, and stable transfectant cells were
selected in 200 µg/ml zeocin. Stable cell lines were cultured in
Dulbecco's modified Eagle's medium (Invitrogen) containing
10% fetal bovine serum, 120 µg/ml zeocin, and 200 µg/ml G418.
Gradient Centrifugation--
Glycerol velocity gradient
centrifugation was performed as described previously (15). For
subcellular fractionation, membrane fractions were isolated from HEK293
cells or mouse brain homogenates as described previously (16) and were
applied to the top of an 11.5-ml 5-25% (v/v) linear iodixanol
gradient (Accurate). Following centrifugation, 0.8-ml fractions
were collected and analyzed by Western blotting. Specific marker
proteins of the ER (calnexin) and Golgi (GM130) were detected using
monoclonal antibodies (anti-calnexin, StressGen; anti-GM130, Sigma).
Immunoprecipitation--
HEK293 cells were lysed with 1%
digitonin in buffer containing 10% glycerol, followed by
centrifugation at 100,000 × g for 30 min at 4 °C
(15). All immunoprecipitation steps were performed at 4 °C. After
preclearing with protein G-Sepharose CL-4B (Amersham Biosciences) for 1 h, cell lysates were incubated with
antibody for 1 h. The immunoprecipitates were recovered by
overnight incubation with protein G-Sepharose CL-4B. The beads were
washed four times with 1% digitonin in the same buffer.
Immunocytochemistry--
For immunofluorescence microscopy, the
cells were plated onto 18-mm round coverslips coated with mouse
collagen, type 4 (BD Biosciences) in a 35-mm dish. After a brief
rinse in PBS, the cells were fixed with 4% paraformaldehyde in PBS at
room temperature for 20 min and permeabilized with 0.3% Triton X-100
in PBS for 5 min or 5% acetic acid in ethanol for 30 min at room
temperature. Cells were blocked with 10% calf serum in PBS for 30 min
and then incubated overnight with each primary antibody. The primary
antibodies used in this study were as follows: mouse monoclonal
antibodies anti-myc (Invitrogen); mouse monoclonal
antibodies directed to organelle-marker proteins BiP (an endoplasmic
reticulum marker) (StressGen, Victoria, BC, Canada), to GM130
(Transduction Laboratories, Lexington, KY), to TGN38 (Transduction
Laboratories), and to PS1 (NT1 from Paul M. Mathews); and rabbit
polyclonal antibody to PS1-NTF (Ab14) and to human APH-11
(O2C2). The cy2-conjugated goat anti-mouse IgG or cy3-conjugated goat
anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA)
were used as secondary antibodies. Nuclei were visualized by staining
with 2 µM ToPro3 (Molecular Probes). Specimens were
examined with a Bio-Rad laser scanning confocal imaging system
(Microradiance R2000/AG-2) equipped with the Lasersharp2000 software
(Bio-Rad). Images were processed using the Lasersharp2000
post-processing software and Adobe Photoshop (Adobe Systems).
Presenilin·
-Secretase Complex Purification--
Microsomal
membranes were lysed in 1% CHAPSO-containing IP buffer (25 mM Hepes, pH 7.4, 150 mM NaCl, and 2 mM EDTA plus a mixture of protease inhibitors (Sigma)). The
lysates were centrifuged at 45,000 rpm for 1 h, the supernatants
were immunoprecipitated with anti-PS1, anti-myc, or
anti-APH-11L antibodies, and the generation of
-stubs
and amyloid
-peptide was then performed as a cell-free assay system
by incubation at 37 °C for 2 h (
-stubs) or 4 h (amyloid
-peptide) (17-19). Control samples were kept on ice.
 |
RESULTS AND DISCUSSION |
In addition to the two APH-1 members reported on chromosome 1 (APH-11) and chromosome 15 (APH-115) (14), a
data base search and molecular cloning revealed a longer isoform of
APH-11 arising from alternative splicing of the transcript
from chromosome 1. Here we designate them as APH-11S (short
isoform) and APH-11L (long isoform), respectively (Fig.
1).

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|
Fig. 1.
Amino acid sequences of human APH-1
proteins. Peptide residues used for raising antisera are
underlined. The putative transmembrane domains are
shaded. The novel C-terminal residues in the long isoform of
APH-11 (APH-11L) are in italics. The
abundance of APH-11S and APH-11L is
similar.
|
|
Overexpression of exogenous presenilin or nicastrin proteins causes the
artifactual accumulation of immature forms of these proteins because of
the fact that the stoichiometry and abundance of components within the
mature presenilin complexes are highly regulated (15, 20, 21).
Consequently, we initially elected to investigate the biology of APH-1
in native cells expressing endogenous APH-1. Polyclonal sera were
raised against epitopes at the C terminus of APH-11 and of
APH-115. Analysis of Western blots expressing exogenous
APH-1 with or without myc tags in HEK293 cells revealed that
one antiserum (O2C2) specifically detected the longer isoform of
APH-11 but did not detect the shorter isoform of
APH-11 or APH-115 (Fig.
2, A and B). This
antibody also detected significant levels of expression of
APH-11 in brain and kidney. This endogenous APH-1,
immunoreactive band had an apparent molecular weight that is in good
agreement with that predicted from the primary amino acid sequence of
APH-11 and that is identical to that of the untagged
APH-11L transiently overexpressed in HEK293 cells as a
positive control. These results indicate that, in contrast to nicastrin
(22, 23) and the presenilins (24), it is unlikely that
APH-11L undergoes significant post-translational
modifications.
To discover the native conformation of the APH-1 proteins and to
examine the possibility that APH-1 proteins may form components of the
presenilin·nicastrin complexes, we used glycerol velocity gradient
centrifugation in cells expressing endogenous APH-11L. These studies revealed that, in digitonin-solubilized lysates, endogenous APH-11L co-fractionated with PS1 and nicastrin
(Fig. 3). However, the distribution of
APH-11L most closely resembled that of immature forms of
nicastrin, although there was substantial overlap with both the
distribution of mature nicastrin and PS1-NTF. As expected,
solubilization with Triton X-100 causes the nicastrin·PS1·APH-1 complex to be completely disassembled (data not shown). These results
are in agreement with the observation that APH-11L can be
co-immunoprecipitated with both immature and mature forms of nicastrin
(see below). These results also suggest that APH-11 is
likely to be a component of both the immature and the higher molecular
weight mature (functional) presenilin·nicastrin complexes (15).
Although it is not clear what the intermolecular binding interactions
are within the presenilin complexes, analysis of APH-11L co-immunoprecipitation products in cells expressing mutant nicastrin molecules suggests that APH-11L may preferentially bind
directly to immature nicastrin in the ER. Thus, in cells expressing the nicastrin
312-369 mutant, APH-11L co-immunoprecipitates
the mutant nicastrin molecule but not PS1 (Fig. 4B,
lane 9). Significantly, the nicastrin
312-369 mutant
blocks trafficking and maturation of nicastrin in the ER, inhibits
interaction of nicastrin with PS1, and causes loss of function of the
presenilin·nicastrin complex.
Several conclusions can be drawn from these results. First, APH-1
isoforms interact with both mature and immature forms of nicastrin and
the presenilins, and the overall abundance of APH-1 proteins is tightly
regulated (presumably because of competition for binding to the same,
limited-abundance components within the presenilin·nicastrin
complex). Second, our IP data, at least in cells overexpressing
individual APH-1 proteins, suggest that they may directly interact
physically with each other, in contrast to PS1 and PS2, which do not
physically interact but which are capable of displacing each other from
their native complexes, again by competing for the same shared limiting
cofactors (20). The absence of a 1:1 ratio of APH-11L to
APH-11S or APH-115 in the immunoprecipitation
products of cells overexpressing APH-1 proteins raises the possibility
that the co-precipitation may be artifactual. However, the fact that
overexpression of any one member of the APH-1 family reduces the level
of expression of the other APH-1 protein family members (by a
"displacement effect") provides an equally plausible
explanation for the absence of a 1:1 stoichiometry. This issue will be
best resolved when antibodies specific to APH-11S or
APH-115 become available, allowing analysis of endogenous
APH-11S or APH-115.
The presenilin proteins and nicastrin are components of high molecular
weight, membrane-bound protein complexes that co-purify in cell-free
systems with
-secretase and
-secretase activity (17, 26). To
ascertain whether the APH-1 proteins are also components of these
functionally active complexes, we generated functionally active
-/
-secretase complexes by immunopurification of the complexes
from cell-free membrane preparations, as described previously (17, 19,
26), using anti-PS1 (antibody NT1.1), anti-APH-11 (antibody
O2C2 in cells expressing endogenous APH-11L), or
anti-myc antibodies (in cells expressing
myc-tagged APH-1 proteins). In each instance, an aliquot of
the complexes was examined for its molecular components by Western
blotting, whereas the remainder was assayed for the presence of
-
and
-secretase catalytic activities by observing the generation of
both amyloid
-peptide (data not shown) and
-stubs (Fig.
5) as described previously (17, 19, 26).
These studies reveal that functionally active complexes were recovered
and contained APH-1 proteins regardless of whether they were purified
by anti-PS1 immunoprecipitation (Fig. 5, lane 1),
immunoprecipitation of the endogenous APH-11L (Fig. 5,
lanes 6 and 7), or immunoprecipitation of
exogenous APH-11L-myc,
APH-11S-myc, or
APH-115-myc in transfected cell lines (Fig. 5,
lanes 2-5).
The functional role of APH-1 in the nicastrin·presenilin complexes
cannot be deduced from its primary amino acid sequence. Absence of
APH-1, whether induced by null mutations or by RNA interference,
results in destabilization of PS1, PS2, nicastrin, and PEN-2 and in the
inhibition of Notch and APP processing (13, 14). Conversely, absence of
the presenilins destabilizes nicastrin (28,
29).2 The absence of
PS1 does reduce the endogenous levels of APH-11L (Fig.
8). This reduction is independent of age,
being equivalent in 2-month-old (Fig. 8), as well as 6-day-old and
3-month-old, PS1
/
mice. However, unexpectedly, in
PS1
/
mouse brain, the degree of reduction in endogenous
APH-11L is small compared with the reported reduction in
nicastrin levels (28, 29).2 The absence of PS2 has
minimal if any effect on APH-11L levels, presumably because
of the presence of adequate amounts of PS1 in the brain of
PS2
/
mice (Fig. 8).
Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc.M209499200
The abbreviations used are:
PS1, presenilin 1;
PS2, presenilin 2;
APP, amyloid precursor protein;
NTF, N-terminal fragment;
CTF, C-terminal fragment;
HEK, human embryonic
kidney;
ER, endoplasmic reticulum;
PBS, phosphate-buffered saline;
CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic
acid;
IP, immunoprecipitation.
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