THE JOURNAL OF BIOLOGICAL CHEMISTRY© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 280, No. 13, Issue of April 1, pp. 12967–12975, 2005
Printed in U.S.A.

Aph-1 Contributes to the Stabilization and Trafficking of the
␥-Secretase Complex through Mechanisms Involving Intermolecularand Intramolecular Interactions*
Received for publication, August 26, 2004, and in revised form, January 3, 2005
Published, JBC Papers in Press, January 11, 2005, DOI 10.1074/jbc.M409829200

Manabu Niimura‡, Noriko Isoo‡, Nobumasa Takasugi‡, Makiko Tsuruoka‡, Kumiko Ui-Tei§,
Kaoru Saigo§, Yuichi Morohashi‡, Taisuke Tomita‡¶储, and Takeshi Iwatsubo‡储
From the ‡Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences and the§Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan

Mutations in presenilin (PS)1 genes account for the majorityof early onset familial Alzheimer’s disease cases, causing an
overproduction of those amyloid ␤ peptides (A␤) ending at* This work was supported by grants-in-aid from the Ministry of
Education, Science, Culture and Sports for the 21st Century Center of
Excellence Program and by the Program for Promotion of Fundamental
Studies in Health Sciences of the Pharmaceuticals and Medical Devices
Agency (PMDA), Japan. The costs of publication of this article weredefrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
¶ Recipient of a postdoctoral fellowship for research abroad from theJapan Society for the Promotion of Science.
储 To whom correspondence may be addressed: Dept. of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Tel.: 81-3-5841-4877; Fax: 81-3-5841-4708; E-mail: taisuke@
mol.f.u-tokyo.ac.jp or iwatsubo@mol.f.u-tokyo.ac.jp.
1
The abbreviations used are: PS, presenilin; A␤, amyloid ␤ peptide;␤APP, ␤-amyloid precursor protein; CDS, coding sequence; CHAPSO,
3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate; CHX, cycloheximide; dsRNA, double-stranded RNA; ER, endoplasThis paper is available on line at http://www.jbc.org

position 42 (A␤42) that most readily form amyloid deposits (1).
A␤ is derived from the ␤-amyloid precursor protein (␤APP)
through sequential cleavages by ␤- and ␥-secretases (2). Genetic and biochemical studies suggest that PS is essential forthe ␥-secretase-mediated intramembrane cleavage of ␤APP.
Additional type 1 transmembrane proteins (e.g. notch, ErbB4,
and CD44) also are cleaved by ␥-secretase to release intracellulardomains that harbor biological activities (3). These data suggestthat PS-dependent ␥-cleavage is involved in a novel mode of intramembrane proteolysis-dependent signal transduction.
PS is a highly conserved, polytopic integral membrane protein that spans the membrane eight times and undergoes endoproteolysis to generate amino- and carboxyl-terminal fragments (reviewed in Ref. 4). The endoproteolytic fragments of
PS form a heterodimer and are then incorporated into a highly
stabilized, high molecular weight (HMW) protein complex,
whereas the PS holoprotein forms a low molecular weight complex and is rapidly degraded (5, 6). We have shown by a systematic mutational analysis that the stabilization and HMW
complex formation of PS are required for ␥-secretase activity,
which is mediated by the carboxyl terminus of PS (7–9). PS
carries two highly conserved intramembrane aspartates thatare required for the ␥-secretase activity (10). Moreover, solubilized ␥-secretase activity is recovered in HMW fractions associated with PS fragments, and transition state analogue
␥-secretase inhibitors covalently label PS fragments but not PS
holoproteins (11–13). These results suggest that the stabilized
HMW PS complex harbors ␥-secretase activity and that thepaired intramembrane aspartates in PS fragments serve as theactive center of the ␥-secretase, which functions as a novel typeof membrane-bound aspartic protease.
The molecular composition and the mechanism of assemblyof the ␥-secretase complex have remained elusive. However,recent studies have identified several putative PS complex
components. Nicastrin (Nct) is a type 1 transmembrane protein
harboring a large ectodomain that is highly glycosylated in
mammalian cells (14). Fully glycosylated Nct is selectively
associated with PS fragments, and genetic studies indicate arequirement of Nct for ␥-secretase activity (15). Genetic studies
in Caenorhabditis elegans and Drosophila melanogaster haverevealed two additional polytopic membrane proteins, Aph-1
and Pen-2, that also are required for the ␥-secretase activity
mic reticulum; FL, full-length or holoprotein; GFP, green fluorescentprotein; HMW, high molecular weight; mt, mutant; Nct, nicastrin;
RNAi, double-stranded RNA-mediated interference; SC100, thecarboxyl-terminal 99 amino acid fragment of ␤APP fused to a signal
peptide of rat preproenkephalin cDNA; TGN, trans-Golgi network;
TMD, transmembrane domain; UTR, untranslated region; wt,
wild-type.

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␥-Secretase cleaves type I transmembrane proteins,
including ␤-amyloid precursor protein and Notch, andrequires the formation of a protein complex comprisedof presenilin, nicastrin, Aph-1, and Pen-2 for its activity.
Aph-1 is implicated in the stabilization of this complex,
although its precise mechanistic role remains unknown.
Substitution of the first glycine within the transmembrane GXXXG motif of Aph-1 causes a loss-of-function
phenotype in Caenorhabditis elegans. Here, using an
untranslated region-targeted RNA interference/rescue
strategy in Drosophila Schneider 2 cells, we show that
Aph-1 contributes to the assembly of the ␥-secretase
complex by multiple mechanisms involving intermolecular and intramolecular interactions depending on or
independent of the conserved glycines. Aph-1 binds tonicastrin forming an early subcomplex independent of
the conserved glycines within the endoplasmic reticulum.
Certain mutations in the conserved GXXXG motif affect theinteraction of the Aph-1䡠nicastrin subcomplex with presenilin that mediates trafficking of the presenilin䡠Aph-1䡠nicastrin tripartite complex to the Golgi. The same mutations
decrease the stability of Aph-1 polypeptides themselves,possibly by affecting intramolecular associations throughthe transmembrane domains. Our data suggest that theproper assembly of the Aph-1䡠nicastrin subcomplex withpresenilin is the prerequisite for the trafficking as well as
the enzymatic activity of the ␥-secretase complex and that
Aph-1 functions as a stabilizing scaffold in the assembly ofthis complex.

12968Aph-1 Function in ␥-Secretase Complex Formation

MATERIALS AND METHODS

Construction of Expression Plasmids—Expression plasmids for Psn,
Nct-V5, Aph-1-FLAG, hemagglutinin-Pen-2, and the carboxyl-terminal
99-amino acid fragment of ␤APP fused to a signal peptide of rat preproenkephalin cDNA (SC100) were generated as described previously(9, 19). cDNAs encoding Aph-1/G112D, Aph-1/G112F, Aph-1/G112A,
Aph-1/G112L, Aph-1/G116D, Aph-1/G116A and Aph-1/G116L were generated by the long-PCR protocol. All constructs were sequenced using
Thermosequenase (Amersham Biosciences) on an automated sequencer(Li-Cor, Lincoln, NE).
Cell Culture and Transfection—Maintenance of Drosophila Schneider (S2) cells, production of the double-stranded RNA (dsRNA), andtransient transfection were done as described previously (9, 19). Shortinterfering RNA was designed by the siDirect program (33, 34) using
the primers 5⬘-GCUUUUGUAUAACAUUAUAAA-3⬘ (sense) and 5⬘UAUAAUGUUAUACAAAAGCUA-3⬘ (antisense). 0.1 nmol of RNA duplex (per well in a 6-well plate) was transfected into S2 cells in shortinterfering RNA experiments. Stable transformants in S2 cells weregenerated by transfection of cDNAs together with pCoHygro or pCoBlast vector (Invitrogen) (ratio of transfected cDNAs was 2:0.1 ␮g)using Cellfectin and selected in S2 medium containing hygromycin(Wako) or blastcidin (Invitrogen) at 250 or 5 ␮g/ml, respectively.
Antibodies and Immunochemical Analyses—Two rabbit polyclonal

FIG. 1. UTR-targeted RNAi in S2 cells. Immunoblot analysis of S2cells stably expressing SC100 transfected with dsRNAs against GFP,
Aph-1 CDS, or UTR with (⫹) or without (⫺) co-transfection of a cDNA
encoding wild-type Aph-1 tagged with FLAG (as indicated at the top).
Cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with each antibody (as indicated at the left). Note that thedecrease in the levels of Psn fragments (arrowheads) as well as theaccumulation of the ␤APP carboxyl-terminal stub (␣-stub-like) was
observed in cells transfected with dsRNA against the CDS or the UTR
regions of Aph-1. Exogenous Aph-1 was expressed in UTR-targeted
knockdown cells, whereas CDS-targeted RNAi abolished the expressionof exogenous proteins.
antibodies to Psn, anti-GDN1 and anti-GDL1, were generated as described (9). The rabbit polyclonal antibody against the carboxyl-terminal region of Drosophila Nct, SdN2, was raised against a KLH-conjugated synthetic peptide (CRSEVLFEDLPA) and affinity-purified. The
rabbit polyclonal antibodies C4 against the cytoplasmic carboxyl terminus of human ␤APP, MLO7 against GM130, and JSEE1 against syntaxin 5 were kindly provided by Drs. Y. Ihara (University of Tokyo) and
M. Lowe (The University of Manchester), respectively. The other antibodies were purchased from Invitrogen (anti-V5 monoclonal), Sigma(anti-V5 polyclonal, anti-FLAG M2 monoclonal, and anti-FLAG polyclonal), Stressgen (anti-KDEL), Transduction Laboratories (anti-Rab8,
anti-syntaxin 6, and anti-adaptin-␥), and Roche Diagnostics (anti-HA
3F10 monoclonal). Sample preparations and analyses, including immunoblot analysis and co-immunoprecipitation, were performed as described previously (7–9, 19, 35– 40).
RESULTS

Establishment of a Complementation Assay by 3⬘-UTR-targeted RNAi Knock-down in Drosophila S2 Cells—To minimize
the effect of endogenous Aph-1 proteins in the analyses of theexogenous mutant Aph-1, we generated short interfering RNAs
targeted to the 3⬘-UTR of the Drosophila Aph-1 gene using acomputer-based prediction program (33, 34). UTR-targeted
RNAi in S2 cells expressing SC100, the carboxyl-terminal 99amino acid fragment of ␤APP fused to a signal peptide of rat
preproenkephalin cDNA, decreased the levels of Psn fragments(i.e. amino-terminal fragment or carboxyl-terminal fragment)
in a similar manner to that observed in cells treated withcoding sequence (CDS)-targeted RNAi (Fig. 1). Furthermore, arobust accumulation of APP carboxyl-terminal stubs (i.e. the␣-stub-like; see Fig. 1) and a significant reduction in A␤ secretion (24.9 ⫾ 1% of those with GFP-RNAi) were observed (datanot shown; see Fig. 2B). These data suggest that the UTRtargeted RNAi against the Aph-1 gene caused a reduction in
the levels of endogenous Aph-1 proteins, thereby leading to thebreakdown of the ␥-secretase complex and inhibition of its
activity. We next expressed the wild-type Aph-1 protein that is
encoded by an expression plasmid containing the CDS of Aph-1
with a vector-derived artificial UTR. As expected, exogenous
proteins were expressed in cells despite UTR-targeted RNAi,
whereas these proteins were abolished by the CDS-targeted
RNAi (Fig. 1). These data were consistent with the previous
reports demonstrating that transitive RNAi is absent in Drosophila (41, 42). Moreover, in UTR knockdown cells, the levelsof Psn fragments as well as the ␥-secretase activity were completely restored by the exogenous wild-type (wt) Aph-1 protein(Fig. 2, A and B). These data suggested that the complementation assay in cells treated with UTR-targeted RNAi enables
us to analyze the activity and metabolism of exogenous Aph-1
proteins under the Aph-1-null phenotype in Drosophila S2cells.

Downloaded from www.jbc.org at University of Tokyo Library, on October 17, 2010(16, 17). We and others have shown that overexpression of
Aph-1 together with Nct facilitates the formation of a stabilized
but ␥-secretase-inactive HMW complex containing PS holoproteins and that Pen-2 subsequently facilitates the formation of
PS fragments, conferring ␥-secretase activity (18 –22). Biochemical studies support the notion that a membrane protein
complex comprised of these four proteins represents the minimal framework of ␥-secretase (23, 24). Recently, it was reported
that Aph-1 preferentially interacts with immature Nct to form
an intermediate subcomplex during the early biosynthetic process of ␥-secretase in mammalian cells (25–27), suggesting that
the Aph-1䡠Nct subcomplex functions as a stabilization factor forthe ␥-secretase complex.
To investigate the mechanistic roles of Aph-1 in the formation of the PS complex, we focused on the conserved glycine
residues within transmembrane domain 4 (TMD4) that form a
GXXXG motif. In C. elegans, a glycine to aspartate substitutionof the initial glycine residue of this motif causes the aph1(or28) allele, which is a recessive, maternal effect mutation(16). Aph-1 mutant worms lack the anterior pharynx through adefect in Notch signaling, suggesting that this glycine is obligatory for Aph-1 function. It was shown that the GXXXG motifof mammalian Aph-1a is critical for the assembly and activityof ␥-secretase (28, 29). However, the specific protein-protein
interactions and stoichiometry of individual components aswell as the molecular basis for the stabilizing effect of Aph-1,
including the mechanistic role of the GXXXG motif, remain
unknown. Notably, the amount of exogenous ␥-secretase component proteins that can acquire proper functions is limited,
because they need to compete with the endogenous proteins forbinding with other components in a proper stoichiometry andacquire stability to form a functional ␥-secretase complex (30,31). Furthermore, the presence of multiple homologues/isoforms of Aph-1 in the mammalian genome that form different
complexes containing either PS1 or PS2 (32) may confound theresults of overexpression experiments of Aph-1. To overcome
these technical obstacles, we developed a complementation assay using 3⬘-untranslated region (UTR)-targeted RNAi knockdown followed by transfection of the coding sequence of mutant
Aph-1 with artificial UTR derived from an expression vector in
Drosophila S2 cells, the latter harboring single presenilin (Psn)or Aph-1 genes, respectively, to minimize the effect of endogenous proteins. Using this system, we analyzed the role of Aph-1
in ␥-secretase complex formation, as well as the effect of mutations in the GXXXG motif on the function and metabolismof Aph-1.

Aph-1 Function in ␥-Secretase Complex Formation

Certain Mutations of the Conserved Glycines in TMD4 of
Aph-1 Abolish the Formation of the Active ␥-Secretase Complex—Amino acid sequence alignment of Aph-1 in different
organisms showed that all known Aph-1 proteins contain ahighly conserved amino acid motif within the proximal portionof TMD4, which is comprised of the GLXXGXX(S/H)(G/A) sequence (16, 28). The substitution of the first glycine residue of
this sequence to aspartate (G122D) has been linked to a lossof-function mutation in C. elegans (16). To gain insight into therole of this glycine residue in the metabolism of the ␥-secretase
complex, we transiently transfected Aph-1/G112D, which ismutated at an equivalent residue to the G122D mutant of
C. elegans, into S2 cells deficient in endogenous Aph-1. Immunoblot analysis revealed that Aph-1/G112D failed to restore
Psn fragments, suggesting that substitution of the first glycineof the GXXXG motif abolished the formation of ␥-secretase
complex (Fig. 2A). Conserved residues in TMD4 comprise two
GXXXG motifs, known as a transmembrane helix-helix packing motif (43, 44). Previous data from a TOXCAT assay thatinvestigated TMD association in biological membranes using
artificial TMD fused with transcription factors indicated thatthe replacement of glycine with small side-chain amino acids(e.g. alanine or serine) in the GXXXG motif facilitated theoligomerization of TMDs, whereas bulky amino acids (e.g. tyrosine or phenylalanine) or hydrophilic residues destabilized
the TMD association (45). Furthermore, large aliphatic residues such as leucine, isoleucine, or valine are disfavored at thepositions of the glycines within the GXXXG motif, althoughthese are the most frequent residues in TMDs (46). To elucidate
the roles of glycines in the GXXXG motif of TMD4 of Aph-1, we
further transfected Aph-1 harboring G112A, G112F, G112L,

G116D, G116A, or G116L substitutions into the Aph-1 knockdown cells. Immunoblot analysis revealed that G112F, G112L,
G116D, or G116L mutant (mt) Aph-1 failed to rescue the Psn
fragments just as G112D mt Aph1 failed, whereas mt Aph-1
harboring the alanine substitution (i.e. G112A or G116A) restored the levels of Psn fragments to a similar extent as those
restored by wt Aph-1 (Fig. 2A). In addition, the steady-statelevels of Aph-1 polypeptides were lower in G112D, G112F,
G112L, G116D, or G116L mutants as compared with those in
wt or alanine mt Aph-1. To further characterize the effect of theglycine mutations of Aph-1 on ␥-secretase activity, we analyzed
the metabolism of SC100 and quantitated the levels of A␤
secreted from knockdown cells transiently expressing SC100.
Mutants that were unable to rescue the Psn fragments (e.g.
G112D) also failed to diminish the accumulation of the ␣-stublike by knockdown of Aph-1, whereas alanine mutants rescued
these fragments in a similar manner as that of wt Aph-1 (Fig,
2A). Similarly, overexpression of wt Aph-1 as well as the alanine mutants led to a complete rescue of A␤ secretion (Fig. 2B)(e.g. 109.0 ⫾ 1.0% for wt Aph-1, 107.9 ⫾ 1.8% for G112A, and111.2 ⫾ 2.5% for G116A, respectively, in A␤40 secretion compared with those with GFP-RNAi). In contrast, the levels ofsecreted A␤ from Aph-1 knockdown cells transfected with mt
Aph-1 (i.e. G112D, G112F, G112L, G116D, and G116L) were
not recovered (25.3 ⫾ 0.9, 25.8 ⫾ 1.4, 26.7 ⫾ 1.2, 23.4 ⫾ 0.9, and24.0 ⫾ 0.8%, respectively, in A␤40 secretion as compared tothose with GFP-RNAi), suggesting that these mutations causea loss-of-function in ␥-secretase activity. These results suggestthat the small size and hydrophobic nature of the conserved
glycine residues within TMD4 of Aph-1 are important for thefunction of Aph-1 to facilitate the generation of the active
␥-secretase complex.
Certain Mutations of the Conserved Glycines of Aph-1 Abolish the Formation of a Functional ␥-Secretase Complex throughAssembly with Psn or Pen-2 but Not with Nct—We next established S2 cell lines stably expressing wt or mt Aph-1 (i.e.
G112D, G112F, G112L, G116D, or G116L) together with Nct
and Pen-2 (wt or mt ANPen cells, respectively). Overexpressionof wt Aph-1 together with other components resulted in asignificant increase in the levels of Psn fragments as described
previously (19). However, the levels of the Psn fragments, Nct,and Pen-2, in mt ANPen cells were lower than those in wt
ANPen cells (Fig. 3A, left, lysate) as observed in transient
rescue experiments in Aph-1 knockdown cells (Fig. 2A). To gain
more insight into the roles of these mutations of the conserved
glycines, we performed co-immunoprecipitation assays in ANPen stable cell lines (Fig. 3A, right). Endogenous Psn as well as
exogenously transfected Nct or Pen-2 were co-precipitated with
FLAG-tagged wt Aph-1, consistent with our previous results(19). However, none of the glycine mt Aph-1 interacted withendogenous Psn or transfected Pen-2, whereas mt Aph-1
polypeptides were stably associated with Nct regardless of theglycine mutations. We further treated ANPen cells by UTRtargeted RNAi and confirmed that overexpression of mt Aph-1
failed to restore the levels of Psn under these conditions (datanot shown). To examine whether Psn interacts with mt Aph-1,
we transiently transfected Psn into mt ANPen stable cell lines
together with UTR-targeted RNAi to knockdown endogenous
Aph-1. Despite a robust accumulation of the Psn holoprotein(also called full length or FL) upon overexpression of Psn with
UTR-targeted Aph-1 RNAi, the levels of Psn fragments, Nct,Aph-1, or Pen-2 were not altered in mt ANPen cells, whereas
Psn fragments were increased in wt ANPen cells upon thetransfection of Psn (Fig. 3B, left, input; compare the wt columns
without (⫺) and with (⫹) Psn). We then performed co-immunoprecipitation assays on lysates of these cells; for these exper-

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FIG. 2. Effect of glycine substitutions in the GXXXG motif of
Aph-1 on ␥-secretase complex formation and ␥-secretase activity. A, immunoblot analysis of S2 cells transiently transfected with
cDNAs encoding wt or mt Aph-1 (as indicated at the top) together withdsRNAs. Cell lysates were analyzed by immunoblotting with each antibody (as indicated at the left). B, enzyme-linked immunosorbent assay
quantitation of A␤40 (open columns) and A␤42 (filled columns) secreted
from knockdown SC100 cell lines expressing wt or mt Aph-1. Bars
represent the mean ⫾ S.E. in four independent experiments, and thenames of transfected Aph-1 cDNAs and dsRNAs are indicated below thecolumns. A␤ secretion was significantly increased exclusively in knockdown cells expressing wt Aph-1 (asterisk represents statistically significant compared with mock-transfected knockdown cell (p ⬍ 0.001) asanalyzed by Dunnett test) but not mt Aph-1.

12969

12970Aph-1 Function in ␥-Secretase Complex Formation

iments we prepared lysates that contained equivalent levels of
mt Aph-1 (⬃5-fold total proteins) as compared with those of
Aph-1 in wt ANPen cells treated with UTR-targeted RNAi
based on an estimation by immunoblot analysis of input samples (Fig. 3B, left). For co-immunoprecipitation experiments,
we used wt ANPen cells treated with UTR-targeted RNAi without (⫺) transfection of Psn (Fig. 3B left, input) instead of thosetransfected with (⫹) Psn to keep the cellular Psn at moderatelevels. Although the levels of exogenous Psn FL in samples
from mt ANPen cells were higher than the levels of endogenous
Psn FL in wt ANPen cells, and the levels of the Psn fragments
were similar (Fig. 3B, left, input), neither Psn (FL or fragments) nor Pen-2 polypeptides were co-immunoprecipitatedwith mt Aph-1. In sharp contrast, Nct was again co-precipitated with Aph-1 regardless of the mutations, suggesting theformation of a subcomplex (Fig. 3B right). Finally, we performed immunoprecipitation analysis in ANPen cells treatedwith RNAi for Psn or for Psn and Pen-2 and found that Aph-1
interacts with Nct to form a subcomplex regardless of thepresence of Psn or Pen-2 (Fig. 3C). These results suggest that
the GXXXG motif of TMD4 in Aph-1 might play an essential
role in the interaction of Aph-1 with Psn and with Pen-2, but
not for Nct, in the assembly of a functional ␥-secretase complex.
Certain Mutations of the Conserved Glycines of the GXXXG
Motif Affect the Transport of the Aph-1䡠Nct Subcomplex fromthe ER to the Golgi Depending on the Interaction with Psn
through the GXXXG Motif—Subcellular fractionation studies
in mammalian cells have shown that the ␥-secretase-active
protein complexes that contain PS fragments as well as mature
Nct are distributed in ER and the Golgi/trans-Golgi network(TGN), whereas the ␥-inactive complexes harboring the PS
holoprotein are distributed exclusively in the ER (8, 36, 47).
Subcellular fractionation and localization analyses indicatethat overexpressed mammalian Aph-1aL polypeptides are localized predominantly to ER and pre-Golgi compartments (31).
Moreover, Aph-1 forms a subcomplex with an immature form of
Nct (25). However, complex N-glycosylated, mature Nct is preferentially associated with the active ␥-secretase complex thatharbors the PS fragments, Aph-1 and Pen-2. These data suggest that the intracellular trafficking of Aph-1 is strictly controlled. However, the subcellular localization and trafficking of
Psn complex components in Drosophila cells have not been
extensively examined, although the targeting of Psn to theplasma membrane was reported (48, 49). To determine thesubcellular localization of the Psn complex as well as the effectof certain mutations of the conserved glycines on the subcellular localization of Aph-1, we separated the membrane fractionsof ANPen cells by discontinuous iodixanol gradients (Fig. 4A).
Bip/Hsc 70-3, an ER resident protein that is positive for antiKDEL, was separated in the bottom fraction (fractions 10 and

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FIG. 3. Effects of glycine mutationof Aph-1 on the interaction of ␥-secretase complex components. A, co-immunoprecipitation analysis of 1% CHAPSOsolubilized fractions from ANPen cells
stably expressing wt or mt Aph-1 (as indicated at the top of each section) without
RNAi knockdown of endogenous Aph-1.
Cell lysates containing equal levels of wt
or mt Aph-1 proteins (cell lysates show
the steady-state level of wt Aph-1 in ANPen cells; left section, labeled lysate) wereprecipitated by preimmune serum (PI) or
anti-FLAG antibody and then analyzed
by immunoblotting (right section, labeled
IPed (immunoprecipitated) with antiFLAG) with each antibody as indicated.
B, co-immunoprecipitation analysis of 1%
CHAPSO-solubilized fractions from wt or
mt ANPen cells transiently transfectedwith dsRNA for UTR sequences of Aph-1
with (⫹) or without (⫺) the concomitanttransfection of Psn cDNA. Immunoblot
analysis of input samples for immunoprecipitation is shown in the left section (labeled input). Note that co-immunoprecipitation of Psn with mt Aph-1 is notobserved in mt ANPen cells expressing
Psn (⫹), whereas lower level of Psn FL
present in wt ANPen cells without (⫺)
transfection of Psn (left section) is boundto wt Aph-1 (right section). C, co-immunoprecipitation analysis in multiple RNAi
(i.e. Psn, Psn, and Pen-2)-treated ANPen
cells. HA, hemagglutinin.

Aph-1 Function in ␥-Secretase Complex Formation

12971

11), whereas Golgi/TGN marker proteins (i.e. GM130, syntaxin
5, Rab8, or adaptin-␥) were detected chiefly in the middle
fractions (fractions 3–9), which were used as markers for separation of the ER and Golgi proteins, respectively (data notshown). Psn FL was exclusively detected in the ER fraction. In
contrast, Psn fragments were distributed widely in ER to Golgi
fractions. Nct, wt Aph-1, and Pen-2 in ANPen cells also weredetected widely in ER and Golgi fractions, exhibiting fractionation patterns very similar to those of the Psn fragments.
These data suggest that endoproteolyzed forms of Psn, which
are associated with other Psn components, are able to exit fromthe ER to the Golgi in a similar manner as that in mammaliancells (37, 47). We next examined the subcellular localization of
mt Aph-1 polypeptides (i.e. G112D, G112F, or G116D). Thesemutant proteins were retained exclusively in the bottom fraction, suggesting that mt Aph-1 as well as the subcomplex
consisting of mt Aph-1 and Nct are unable to be transported
from the ER (Fig. 4B). To determine the dependence of thetrafficking of Aph-1 on its interaction with other components,
we fractionated the membranes from ANPen cells transfectedwith dsRNAs for Psn, Nct, or Pen-2 by discontinuous iodixanol
gradients (Fig. 5A). We found that abrogation of the expressionof Psn or Nct disrupted the export of Aph-1 from the ER,
whereas Pen-2 RNAi had no effect on the subcellular localization of Aph-1 (in the ER and the Golgi). Furthermore, we
observed the retention of Aph-1 within the ER fraction in themembranes from cells lacking Psn and Pen-2, suggesting that
Aph-1 trafficking requires both Psn and Nct. Finally, we analyzed the subcellular localization of endogenous Nct in cells
deficient in other ␥-secretase components to determine
whether Nct itself has a trafficking activity for other components (Fig. 5B). Unlike in mammalian cells, endogenous as well
as exogenous Nct polypeptides were detected as a ⬃100-kDa
single band that disappeared upon RNAi treatment against
Drosophila Nct CDS (data not shown). Moreover, endogenous
Nct was clearly detected in Psn knockdown cells similarly as
mammalian Nct in PS knock-out cells (50), whereas the migration pattern of Nct was similar to that in mammalian cells.
This suggests that Nct stability is independent of Psn (seebelow) and that the glycosylation pattern of Drosophila Nct isdifferent from that in mammalians or, alternatively, that theresolution of SDS-PAGE is not enough to separate the differently glycosylated Drosophila Nct. The majority of endogenous

FIG. 5. Subcellular localization of Aph-1 and Nct in S2 cells
treated by multiple RNAi knockdown. A, analysis of subcellular
localization of Aph-1 (arrowheads) and the Psn holoprotein (arrow) instable cells treated with RNAi. Note that the loss of Psn or Nct expression, but not that of Pen-2, caused the retention of Aph-1 protein in ER
fractions. B, analysis of subcellular localization of endogenous Nct(arrowheads) in S2 cells treated with RNAi, showing that the trafficking of Nct into Golgi/TGN fraction required the expression of Psn andAph-1. Separation, numbering, and analysis of the fractions were performed as in Fig. 4.

Nct was fractionated in the Golgi/TGN fraction, whereas depletion of Psn caused a marked accumulation of Nct in the ER
fraction, similar to that described in mammalian cells (50).
Furthermore, Aph-1 RNAi also caused an ER retention of endogenous Nct, whereas RNAi depletion of Pen-2 had no effecton the trafficking of Nct. This suggested that Aph-1, in addition
to Psn, is required for the trafficking of Nct. Taken together,
these data indicate that Aph-1 forms a subcomplex with Nct inthe ER independently of the conserved glycines, the latterbeing required for the interaction with Psn as well as for theformation of an active ␥-secretase complex and for the trafficking of the subcomplex to Golgi/TGN.

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FIG. 4. Subcellular localization of
␥-secretase complex components including mutant Aph-1 in S2 cells. A,
analysis of subcellular localization of the␥-secretase complex in ANPen cells. Total
membrane proteins from ANPen cells
were separated by 2.5–30% discontinuous
iodixanol gradients and numbered as
fraction 1 (lightest) to fraction 11 (heaviest), as indicated at the top of the panel.
20 ␮l of each fraction was separated by
SDS-PAGE and analyzed by immunoblotting. Fractions enriched in ER or Golgi
vesicles were revealed by immunoblottingwith anti-KDEL, anti-GM130, anti-syntaxin 5, anti-Rab8 or anti-adaptin-␥ antibodies, respectively, as underlined above
the panel. B, analysis of subcellular localization of mutant Aph-1 in stable cells.
Total membranes from ANPen cells stably expressing Aph-1 (indicated at the leftof the panel) were fractionated and analyzed as in panel A. HA, hemagglutinin.

12972

Aph-1 Function in ␥-Secretase Complex Formation

The Stability of Aph-1 Is Dependent on the Conserved Glycines but Not on Association with Other Components—Stabilization of the components of the Psn complex is the prerequisitefor ␥-secretase activity (5, 7, 19). Pen-2 RNAi caused the accumulation of a stable Psn䡠Nct䡠Aph-1 HMW complex, and thepersistence of the Pen-2 protein depends on the presence ofother components, suggesting that Pen-2 is a highly unstable
protein and is not involved in the stabilization of the ␥-secretase complex (19, 23, 51). Furthermore, the accumulation of PS
fragments as well as holoproteins was abolished by RNAi for
Nct or Aph-1, suggesting that Nct and Aph-1 are required forthe stability of PS (19, 32, 50 –53). To investigate the effect ofglycine mutations on the stability of Aph-1 as a polypeptide, we
treated wt and mt ANPen cells by cycloheximide (CHX) (Fig.
6A). Immunoblot analysis revealed that all three mt Aph-1
polypeptides were rapidly degraded during 4 h of chase incontrast to the persistence of wt Aph-1 (t1⁄2 ⫽ ⬎12 h for wt, 2.1 h
for G112D, 3.5 h for G112F, 4.3 h for G112L, 2.3 h for G116D,
and 2.7 h for G116L, respectively, calculated by densitometricanalysis), suggesting that the conserved glycines are required
for the stability of Aph-1 itself. Because mt Aph-1 is capable ofinteracting with Nct but not with Psn, we investigated thestability of Nct in cells stably expressing mt Aph-1. Consistent
with the result that the steady-state levels of Nct were decreased in mt ANPen cells (Fig. 3A), the stability of Nct was
lower than that in wt ANPen cells (Fig, 6B). Thus, the stabilityof Nct appears to be dependent on that of Aph-1. Furthermore,
we investigated the dependence of the stability of Aph-1
polypeptides on other components in ANPen cells using RNAi.
The levels of Aph-1 were constant during CHX chase in theabsence of any other component proteins (i.e. Psn, Nct, and
Pen-2) (Fig. 6C). These data suggest that Aph-1 per se is ahighly stable membrane protein. We next analyzed the stability of Nct, a binding partner of Aph-1 within the subcomplex.
As expected from the results in mt ANPen cells, Nct was
short-lived in Aph-1 depleted cells (t1⁄2 ⫽ 6.8 h), whereas RNAi
for Psn and/or Pen-2 had no effect on the stability of Nct (t1⁄2 ⫽⬍12 h) (Fig. 6D). Notably, stable Psn fragments were completely lost, and the Psn holoprotein was rapidly degraded (t1⁄2⬍ 2 h) in Aph-1 depleted cells, whereas a fraction of Nct
proteins persisted, suggesting that Nct is unable to stabilize
Psn in the absence of Aph-1. Taken together, we conclude thatthe interaction with Aph-1, which itself is a highly stable protein, confers stability to all the other components of thePsn complex.

DISCUSSION

The RNAi technique made it possible to analyze the functions of proteins, including mammalian Nct and Aph-1 (32, 54),
under null or partial loss-of-function backgrounds in cultured
cells. However, it is necessary to establish stable knockdown
cell lines for the analysis of proteins that have long half-lives,
e.g. the ␥-secretase complex components. Moreover, the lowtransfection efficiency as well as the technical limitations inintroducing long dsRNAs into mammalian cells renders simultaneous RNAi knockdown of multiple molecules highly difficult. In contrast, the high transfection efficiency and the propagation of RNAi in Drosophila cells enable the simultaneous
suppression of multiple gene expression. The lack of transitive
RNAi in Drosophila cells makes it possible to target RNAi
against the UTR sequences also (41, 42). Taking advantage ofthese merits we utilized two novel RNAi strategies; the firstwas UTR-targeted RNAi followed by the rescue by transfectionof CDS, the second was suppression of up to three different
genes by multiple RNAi, and we then examined the role ofAph-1 on Psn complex formation and its metabolism in Drosophila S2 cell lines in the absence of endogenous Aph-1 andother components. We have shown the following. (i) Aph-1 is ahighly stable protein that is required for the assembly andstabilization of the Psn complex. (ii) Mutations in the GXXXG
motif of Aph-1 do not affect the formation of the Aph-1䡠Nctsubcomplex. (iii) Mutations in the GXXXG motif decrease thestability of the Aph-1 polypeptide itself (Fig. 7). Recent reportssuggest that the ␥-secretase components, including Pen-2, areassembled within the ER and that subsequently the complex istransported to the Golgi in mammalian cells (55, 56). Although
the process of assembly and the localization of Drosophila
Pen-2 await further analysis, our results from the Drosophila
S2 cells described above are in agreement with the previous
findings indicating that the GXXXG motif in Aph-1a is required
for the assembly and activity of ␥-secretase in mammalian cells
and yeast (28, 29), and they extend considerably the previous
data in such a way as to provide further insight into themechanistic roles of Aph-1 in the trafficking and stability of the␥-secretase complex.
Stabilization of the PS complex is the prerequisite for␥-secretase activity (5, 7, 8). The high stability of Aph-1 per se,
as well as its association with other components, may renderthe ␥-secretase complex highly resistant to degradation andable to perform proteolytic activity. The molecular basis for the

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FIG. 6. Stability of wild-type or glycine mutant Aph-1 and Nct polypeptides in ANPen cells. A, analysis of the half-lives of Psn and wt
or mt Aph-1 in stable ANPen cells incubated in culture media containing CHX (30 ␮g/ml). Lysates prepared after various incubation periods (0 – 4h) were analyzed by immunoblotting with anti-GDN1 (for Psn) or anti-FLAG (for Aph-1) antibodies. Note that all of the glycine mutant Aph-1
polypeptides were degraded rapidly compared with wt during the CHX chase period. B, Nct stability in wt or mt ANPen cells treated with CHX,
showing the destabilization of Nct polypeptides upon the overexpression of mt Aph-1. C, lack of decremental effects on the stability of Aph-1 by
the depletion of other ␥-secretase components. Cell lysates of ANPen cells transfected with dsRNA for GFP or Psn, Nct, and Pen-2 were analyzed
by immunoblotting for Aph-1 (arrowheads) as in panel A. D, Nct stability in knockdown cells. ANPen cells transfected with dsRNAs (indicated at
the left of the panel) were analyzed by immunoblotting for Nct (arrowheads) as in panel A. Note that the stability of Nct was dependent on Aph-1
expression, whereas Aph-1 was highly stable in the absence of any other component.

Aph-1 Function in ␥-Secretase Complex Formation

12973

FIG. 7. Schematic representations on
the possible role of Aph-1 in the trafficking and maturation of ␥-secretase
complex. A, Aph-1 polypeptides (open cylinders; cylinders represent TMDs) aretightly folded in a manner dependent on the
GXXXG motif and form a stable subcomplex
with Nct (striped cylinder) within the ER.
This stable subcomplex interacts with nascent Psn (cylinders with gradation) throughthe hydrophobic interface formed by the
TMDs of Aph-1 and Nct. This trimeric intermediate, further assembled with Pen-2 (dotted cylinders), is sorted out from the ER and
activated. B, mt Aph-1 proteins are folded
loosely and degraded rapidly within ER, although they are able to interact with Nct.

ment outside the amino-terminal fragment region of Aph-1
that harbors the TMD4 (60). Thus, the GXXXG motif may be
important for the direct interaction of Aph-1 with Psn. Thiswas somewhat different from the previous observations indicating that GXXXG mutations disrupt the co-immunoprecipitation of Aph-1 and Nct in mammalian cells (28, 29). The
reason for this discrepancy is unknown, but one possible explanation would be that the use of 1% CHAPSO as a detergent inour study had a lesser effect on the sensitive but specific interaction with mt Aph-1 and Nct (24, 26, 61). Another factor might
be the species difference between mammalian and Drosophila
cells; it is noteworthy that two PS (PS1 and PS2) and two(human) or three (rodent) Aph-1 family genes, as well as theirsplice variants, have been documented in mammals (15). It has
been shown that these distinct mammalian Aph-1 proteins
form different complexes that contain either PS1 or PS2, suggesting that each mammalian Aph-1 homologue/isoform has
different biological activity, although only the carboxyl-terminal 20 amino acids are different between Aph-1aS and Aph-1aL(32). The observation that Aph-1aS or Aph-1b transfected in
mammalian cultured cells replaced the endogenous Aph-1aL
suggests that overexpression of a single Aph-1 homologue/isoform in mammalian cells may compromose the function andmetabolism of the others (31), although the effect of overexpression of a single homologue on the assembly of distinct
␥-secretase complexes awaits further determination. Moreover,co-expression of other components in mammalian cells andyeast restored the complex formation and activity; G122D mutant Aph-1aL (an equivalent of G112D in Drosophila) rescued
␥-secretase complex formation in yeast (29). Taken together,
the knockdown/rescue paradigm used in this study in Drosophila cells (with the latter harboring single Psn and Aph-1 genes)
may provide the most clear-cut results in the analysis of therole of each component in the assembly of the ␥-secretase
complex.
The subdomain in Aph-1 that is required for its interaction
with Nct remains unknown, although the carboxyl-terminal
half of Aph-1 has been shown to be associated with Nct inmammalian cells (60). It may be noteworthy that Aph-1 harbors a highly conserved serine cluster within the TMD2, as well
as a couple of hydrophilic residues located in the middle of
TMDs that have been reported as being determinants of thestability and specificity of TMD association (62– 65). Glycine
mutations of Aph-1 disrupted the interaction of the Aph-1䡠Nct

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stability of Aph-1, however, remains unclear. Substitutions ofconserved glycines in the GXXXG motif, which is known as aninteraction motif for TMDs (44), disrupted the stability as wellas the interaction of Aph-1 with Psn and Pen-2, although thestability of Aph-1 was independent of the intermolecular interactions (Fig. 6). We cannot completely exclude the possibility
that impairment in the proper structure of the TMD4 caused byamino acid substitutions led to a loss-of-function effect by disrupting the stability of the entire molecule as well as theassembly of TMDs. However, the substitution to leucine, but
not to alanine, of these conserved glycines abolished the functions (e.g. molecular assembly and stability) of Aph-1. This
result is consistent with the recent finding that substitution toleucine, the most frequent residue in TMD, at the glycine
residue disrupts GXXXG-mediated interaction (46). Thus, interms of the stabilization of the Aph-1 polypeptide, we suggestthat the GXXXG motif itself may play a role in the intramolecular interaction via TMDs to correctly fold and stabilize Aph-1(Fig. 7B). The GXXXG motif forms helix-helix interactions withan extended C␣–H䡠䡠䡠O hydrogen bond within the lipid bilayer(57). Intriguingly, a recent report indicates that substitution toalanine at either of the glycines in the GXXXG motif disrupts
TMD homodimerization, whereas the alanine mutant is still
capable of facilitating heterodimerization with a TMD containing a (small)XXX(small) motif (e.g. GXXXS, GXXXS) (58). Considering our result that Aph-1/G112A or Aph-1/G116A retain
its functions, it is possible that the GXXXG motif of TMD4 of
Aph-1 is involved in the intramolecular heterodimeric TMD
association. The additional, GXXXG-like, (small)XXX(small)
motifs within TMD1 (15GPPFA19), TMD2 (45SLLIS49), andTMD3 (65GVVFS69) of Aph-1, which contain relatively conserved glycines or small residues and have similar topology
within the TMDs, might be candidates for being intramolecularinteraction domains for the GXXXG sequence in TMD4.
The molecular stoichiometry and interaction domains of individual components in the ␥-secretase complex, especially in
its active state in situ, still remain unknown. Aph-1 forms asubcomplex with Nct in the absence of Psn and Pen-2 in Drosophila cells in a similar manner to that observed in mammalian cells (Fig. 3C) (59). GXXXG-mutant Aph-1 retained thecapacity to bind Nct but failed to associate with Psn and Pen-2.
Indeed, the interaction of Nct/Aph-1 regardless of the mutations in TMD4 is consistent with the recent data showing that
Nct interacts with the Aph-1 “lower” carboxyl-terminal frag-12974Aph-1 Function in ␥-Secretase Complex Formation

Acknowledgments—We thank Drs. K. Maruyama for SC100 cDNA,
Y. Ihara for the anti-C4 antibody, M. Lowe for the MLO7 and JSEE1
antibodies, and the Takeda Pharmaceutical Company for A␤ ELISA.
We also thank Y. Takahashi, S. Tanaka, I. Hayashi, C. Sato,
M. Shinohara, and N. Watanabe for helpful discussions and technicalassistance and Dr. D. M. A. Mann for invaluable comments on the
manuscript.

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subcomplex with Psn, raising the possibility that Aph-1 may
interact directly with Psn through the GXXXG motif in TMD4.
In agreement with this notion, Aph-1 depletion completely
diminished the stability of Psn, whereas Nct was relatively
stable in the absence of Aph-1 (Fig. 6). However, the abrogationof Nct expression also resulted in a complete depletion of stable
Psn polypeptides, whereas the expression levels and stability of
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Aph-1 together with PS and Nct restored the loss-of-function
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Aph-1 is overcome by a high level expression of mutant Aph-1
protein together with other components, especially Nct (Fig. 6;29). Thus, it would be possible that the helix-helix interface
formed by TMDs of the Aph-1䡠Nct subcomplex, including TMD4of Aph-1 and Nct TMD, may provide the interaction surface
with Psn, leading to the subsequent stabilization and eventual
activation of the complex (Fig. 7).
The intracellular compartment(s) wherein the individualcomponents assemble to form the active ␥-secretase complex isan open question. The mutant Aph-1 polypeptides were retained in the ER fraction, which was similar to the localizationof wt Aph-1 in cells depleted of Psn or Nct. In agreement withthis observation, subcellular localization of Aph-2䡠Nct was altered in the embryos mutated in sel-12 or hop-1 as well as in
aph-1 in C. elegans (16), and it has been shown that the proper
trafficking of Nct requires the expression of PS in mammaliancells (15). Of particular interest is the observation that the
carboxyl-terminal aromatic/hydrophobic amino acids of PS thatare essential to the formation of a ␥-secretase-active HMW PS
complex through the interaction with Nct (7, 67), are required
for the efficient export of PS from the ER to the intermediate
compartment (ERGIC) (68). Moreover, recent report indicatesthat the PALP motif at the carboxyl terminus of PS harbors an
ER retention signal (67). These findings altogether suggestthat PS might act as a regulator for trafficking of the early␥-secretase subcomplex harboring Nct and Aph-1 from the ER.
In contrast, it has been documented that Pen-2 primarily localizes to the Golgi (50) and that stabilized Psn holoproteinsinduced by Pen-2 depletion in S2 cells are chiefly fractionatedin the Golgi fraction together with Aph-1 (Fig. 5). In sum, theproper intracellular trafficking of the Aph-1䡠Nct subcomplex
requires an interaction with Psn, which in turn facilitates thesorting of the ␥-secretase to the Golgi.
Our present study underscores the importance of the helixhelix interactions through TMDs of ␥-secretase components inthe assembly and activation of the complex. Further genetic,
biochemical, and cell biological studies on the roles of ␥-secretase components will facilitate clarification of the remaining
important problems (e.g. the molecular mechanism of intramembrane cleavage, the binding domains mediating interactions of individual components, and the three-dimensional
structure of the active complex) in ␥-secretase research.

Aph-1 Function in ␥-Secretase Complex Formation

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