Gene 323 (2003) 115 – 123
www.elsevier.com/locate/gene

A unifying model for functional difference and redundancyof presenilin-1 and -2 in cell apoptosis and differentiation
Tamotsu Hashimoto-Gotoh a,b,*, Atsushi Tsujimura a,b, Yoshihisa Watanabe b, Naoyuki Iwabe c,
Takashi Miyata c, Takeshi Tabira daDepartment of Genomic Medical Sciences, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japanb
Department of Biochemistry and Molecular Genetics, Research Institute for Neurological Diseases and Geriatrics,
Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japanc
Department of Biophysics, Faculty of Science, Kyoto University, Kyoto 606-8502, Japand
National Institute for Longevity Sciences, Aichi 474-8522, Japan
Received 6 June 2003; received in revised form 28 August 2003; accepted 10 September 2003
Received by T. Gojobori

Abstract
Mutations in genes encoding the highly homologous proteins presenilin-1 and -2 (PS1 and PS2) are linked to the early onset of
Alzheimer’s disease (AD). Here, we report that polyclonal antibodies against Xenopus PSh (PS2), but not PSa (PS1), suppress the in vitro
apoptotic activation of Xenopus egg extracts. To clarify the relationship between structural and functional differences in presenilins, wesearched for presenilin homologues in various living sources, and found that presenilins were divided into three distinct groups, named a-, hand g-types, based on the size of the large hydrophilic loop (HL) regions as follows: HLa/HLh/HLg = 4:3:6. No such size conservations were
found in the N-terminal (NT) hydrophilic regions. Phylogenetic studies revealed that the presenilin genes were duplicated independently in
different lineages of phyla/divisions, suggesting that there were functional requirements for and constraints on the generation andconservation of these HL sizes. On the basis of these findings, we propose a model postulating that both PS1 and PS2 can be differentiative or
apoptotic when they are proteolytically processed within the HL regions or not, respectively, and PS1 may be more sensitive than PS2 toauto-proteolytic cleavage due to the larger size of the HL region of the former. Furthermore, the model assumes that C-terminal fragments(CTF) stabilized by phosphorylation may inhibit both the activities due to the dominant-negative effect. The model explains not only thefunctional redundancy but also apparently conflicting observations reported so far for PS1 and PS2.
D 2003 Elsevier B.V. All rights reserved.
Keywords: APP; Alzheimer’s disease; h-amyloid; Dominant negative; Notch; g-Secretase; Processing

1. Introduction
Mutations in human genes encoding two highly homologous proteins, presenilin-1 and -2 (PS1 and PS2), have
Abbreviations: AD, Alzheimer’s disease; Ah, amyloid h-peptide; CTF,
C-terminal fragment; FAD, familial AD; FLM, full-length molecule; HL,
hydrophilic loop; MBT, midblastula transition; NT, N-terminus; NTF, Nterminal fragment; PBS, phosphate buffered saline (137 mM NaCl, 2.7 mM
KCl, 10 mM Na2HPO4, 2 mM KH2PO4 at pH 7.4); PS, presenilin.
* Corresponding author. Department of Biochemistry and Molecular
Genetics, Research Institute for Neurological Diseases and Geriatrics,
Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan. Tel.: +81-75-251-5798; fax: +81-75-2515799.
E-mail address: thg@koto.kpu-m.ac.jp (T. Hashimoto-Gotoh).
0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.gene.2003.09.010

been found in a number of pedigrees of familial Alzheimer’s disease (FAD) (Sherrington et al., 1995; Rogaev et al.,1995). It has been reported that presenilins are autocatalytic
aspartyl proteases that hydrolyze h-amyloid precursor protein (hAPP) and Notch protein, which are responsible for
AD genesis and Notch signaling, respectively (Wolfe et al.,1999). The proposed structure of presenilins possesses eighttransmembrane domains (TM I to TM VIII) with a largehydrophilic loop (HL) region flanked by TM VI and TM
VII (Li and Greenwald, 1998). The hydrophobic amino acidsequences in the TM domains are highly conserved between
human PS1 and PS2, as well as among various presenilin
homologues from different living sources. In particular, two
aspartate residues, one in TM VI and the other in TM VII,are conserved without exception among all the presenilin

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T. Hashimoto-Gotoh et al. / Gene 323 (2003) 115–123

homologues so far identified. These residues are thought tobe located at the active center of presenilins, and to be
responsible for hydrolyzing their own molecules into N- andC-terminal fragments (NTF and CTF), and in addition forhydrolyzing hAPP and Notch to yield amyloid h-peptide(Ah) and NICD peptides, which are responsible for apoptosis of neuronal cells and embryonic cell differentiation,
respectively (Steiner et al., 1999). The high structuralsimilarity between PS1 and PS2 suggests that their physiological functions may be redundant or overlapping at leastin part (Li and Greenwald, 1997).
On the other hand, several lines of evidence suggest that
there may be functional differences between PS1 and PS2.
For instance, (1) there is a strong bias in the frequency of
FAD mutations in PS1 and PS2, namely 24 vs. 1, (2) PS1
knock-out (KO-) mice are embryonic lethal or die just after
birth (Wong et al., 1997; Shen et al., 1997), while PS2 KOmice live without serious health problems (Herreman et al.,1999), and (3) PS1 gene expression is down-regulated prior
to apoptosis and tumor suppression (Roperch et al., 1998),
while over-expression of the PS2 gene induces apoptosis(Wolozin et al., 1996). These observations would support
the idea that PS1 and PS2 may play different biologicalroles (Lee et al., 1996).
In fact, we have observed that during Xenopus embryogenesis, PSa (PS1) and PSh (PS2) show different behaviors
at the levels of both gene expression and post-translational
modifications. Briefly, although both the PSa and PSh
mRNAs are present at high levels in oocytes, the former ismostly (>80%) degraded upon oocyte maturation while thelatter remains present at a high level until the midblastulatransition (MBT) (Tsujimura et al., 1997). Moreover, PSa
protein exists as processed molecules in oocytes and early
embryos, as in the brain, while PSh is present mainly (90%)
as a full-length molecule (FLM) in early embryos (Watanabe
et al., 2003). Furthermore, phosphorylation occurs efficiently(80%) on PSa but not PSh CTFs during early embryogenesis(Watanabe et al., 2003), at which time cells are potentiallyapoptotic (Newmeyer et al., 1994; Sible et al., 1997) and
Notch signaling is suppressed (Coffman et al., 1993). Finally, at MBT, the major species of PSa CTF is switched to theunphosphorylated one and the processed form of PShbecomes predominant over the unprocessed one (Watanabe
et al., 2003). The differential switching of gene expression
and post-translational modifications of PSa and PSh suggestthat PS1 and PS2 play different roles during embryogenesis.
In this work, we studied the influence of anti-PSa andanti-PSh antibodies on the in vitro activation of potential
apoptotic capability of Xenopus egg extracts, analyzed thephylogenetic trees of different types of presenilins, andlocalized the time of presenilin gene duplication on theuniversal evolutionary tree. We discuss the possible relationship between the functions and structural differences ofpresenilins and finally propose and mathematically analyze
a model for the differential involvement of PS1 and PS2 incell differentiation and apoptosis assuming a pivotal role of

CTF phosphorylation. This model explains both the differences and redundancy of ps1 and ps2 genes in biological
functions and FAD genesis.

2. Materials and methods
2.1. In vitro apoptosis assay using Xenopus laevis egg
extracts
Frog egg extracts were prepared according to Newmeyer
and Wilson (1991). Polyclonal antibodies against Xenopus
PSa and PSh, AbPSaCTF and AbPShCTF, have been
described (Watanabe et al., 2003). The cytoplasmic extracts
supplemented with mitochondrial fractions were divided
into four tubes containing phosphate buffered saline(PBS), rabbit IgG, AbPSaCTF or AbPShCTF kept at 4
jC for 30 min, and incubated at 15 jC for 3 h for apoptotic
activation. The in vitro apoptotic activity was measured
before (time 0) and after (3 h) apoptotic activation as
fluorescence generated by cleavage of DEVD-MCA (Peptide Institute, Osaka, Japan) by caspase-3 and other group II
caspases according to the manufacturer’s instructions.

3. Results and discussion
3.1. Apoptosis assay using Xenopus egg extracts
Since Xenopus embryos employ apoptosis to remove
damaged cells before but not after MBT (Hensey andGautier, 1997) and this capability can be detected in vitro
as apoptotic activation of Xenopus egg extracts (Newmeyer
et al., 1994), we examined the possible influence of antibodies against PSa and PSh on the apoptotic activation of
Xenopus egg extracts. We observed that AbPShCTF nearlycompletely suppressed the in vitro apoptotic activation of
Xenopus egg extracts, while AbPSaCTF did not (Fig. 1).
This observation suggesting that PSh may be required forthe apoptotic activation during Xenopus early embryogenesis appears to be consistent with a previous report on the
pro-apoptotic activity of mouse PS2 (Vito et al., 1996). On
the other hand, it should be emphasized that AbPSaCTF did
not inhibit apoptotic activity of Xenopus egg extracts,suggesting that PSa may be required for some function(s)other than apoptosis at least during early embryogenesis.
Since the major components of PSa and PSh in Xenopus
early embryos are processed and unprocessed molecules,
respectively (Watanabe et al., 2003), it is not clear whether
these results reflect functional differences between PSa andPSh themselves or conformational change of presenilins. If
PS1 and PS2 per se are functionally redundant, it is mostplausible that the functional differences may be due to
conformational differences. In any case, this may be thefirst direct indication of functional differences between thetwo presenilins obtained by comparative studies.

T. Hashimoto-Gotoh et al. / Gene 323 (2003) 115–123

117

1.37 – 1.63, irrespective of the living sources, while the NT
sizes vary between 0.15 to 1.46 relative to that of NT ofhuman PS1 (Table 1). In other words, the HL sizes can begiven approximately by integer ratios of HLa/HLh/
HLg = 4:3:6, whereas no such regularity exists in NT sizes.
These results may suggest that the HL rather than NT sizes
are biologically significant or functionally critical. Then,
we constructed phylogenetic trees to evaluate the functional constraints during evolution leading to conservationof the HL sizes, and estimated the approximate time ofgene duplication in presenilin genes during evolutionary
history.

Table 1
Typing of presenilin homologues from various organismsa
Sourceb

Presenilin Total Size ofNT reNT HL reHL

Fig. 1. Effect of anti-PSa and anti-PSh antibodies on in vitro apoptotic
activation of Xenopus egg extracts. Xenopus egg extracts were combinedwith mitochondrial fractions at 4 jC and divided into four tubes containing
PBS, rabbit IgG, AbPSaCTF or AbPShCTF. Fluorescence of free MCA
released from DEVD-MCA via cleavage by caspases was measured (Ex380
nm and Em460 nm) as described in Section 2. The caspase activities of therespective samples were expressed as fluorescence relative to that of thesample with PBS. The relative fluorescence value designated ‘‘preactivation’’ is for the sample with PBS (0.038) at time 0, and other preactivation samples with IgG, AbPSaCTF and AbPShCTF also gave values
within a similar range, namely 0.02 to 0.04.

3.2. Different types of presenilins
The major structural differences between PS1 and PS2
reside in the two large hydrophilic regions, namely the Nterminal (NT) and hydrophilic loop (HL) regions. If PS1
and PS2 play different roles during early embryogenesis, it
is likely that their functional differences may be ascribed toeither the NT or HL region. Therefore, we compared theseregions among various presenilin homologues. Although
the amino acid sequences of these hydrophilic regions areconserved only very poorly, the HL sizes relative to that ofhuman PS1 can be grouped into three distinct types, nameda, b and c, with the values of 0.94– 1.08, 0.75– 0.80 andgi/ci
numbersa type
Human
Frog
Fish
Threadworm
SpongeThale cress

PS1
PSa
PS1
SEL12
PSa
PSa

467
433
456
461
476
397

81
47
69
49
83
17

1.00
0.58
0.85
0.60
1.02
0.21

116
115
116
126
121
108

1.00
0.99
1.00
1.08
1.04
0.93

1709856609384945835645759133
our data
4567215

b type
Human
Frog
Fish
Threadworm

PS2
PSh
PS2
HOP1

448
448
449
358

87
90
85
13

1.07
1.11
1.05
0.16

102
99
97
95

0.78
0.77
0.74
0.72

17098586093851
69000197496294

c typeLancelet
Sea squirt
Snail
Fly
Threadworm
Thale cress
Rice

BfPS
PSg
PSg
DmPS
SPE4
PSg
PSg

525
504
582
541
465
453
478

93
63
118
103
16
12
31

1.15
0.78
1.46
1.27
0.20
0.15
0.38

174
171
207
181
193
183
179

1.37
1.47
1.63
1.43
1.52
1.44
1.41

17529693
0100144063
11066248
2194201
3881734
94544846539565

Not defined
Slime mold
Choanoflagellate
Trypanosome
Entamoeba

PS?
PS?
PS?
PS?

>284
>103
>187
>272

?
?
?
?

?
?
?
?

?
?
?
?

?
?
?
?

19215242
Our data
677102211823207a
Living organisms belonging to different classes or phyla/divisions arelisted. Sizes are given as the number of amino acid residues or relative values.
‘‘NT’’ and ‘‘HL’’ means the N-terminal hydrophilic region and thehydrophilic loop region, respectively. ‘‘reNT’’ and ‘‘reHL’’ are relative sizes
to NT and HL of human PS1, respectively. The ci (sea squirt) and gi (others)
numbers are for the JGI (http://genome.jgi-psf.org/ciona4/ciona4.home.html)
and NCBI (http://www.ncbi.nlm.nih.gov/) DNA databases, respectively.
b
Human, Homo sapiens; Frog, Xenopus laevis; Fish, Danio rerio;
Threadworm, Caenorhabditis elegans; Sponge, Ephydatia fluviatilis; Thalecress, Arabidobsis thaliana; Lancelet, Branchiostoma floridae; Sea squirt,
Ciona intestinalis; Snail, Helix lucorum; Fly, Drosophila melanogaster;Rice, Oryza sativa; Slime mold, Dictyostelium discoideum; choanoflagellate, Monosiga ovata; trypanosome, Trypanosoma brucei; entamoeba,
Entamoeba histolytica. In the last four species, the HL types could not be
defined due to the lack of information.

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T. Hashimoto-Gotoh et al. / Gene 323 (2003) 115–123

3.3. Presenilins in various living organisms
To date (May 9, 2003), we have identified, by searchingin the DNA databases as well our own data, 32 presenilin
homologues (E value V 1.4  10 9), including partial ones,in 21 species from 11 different phyla/divisions including
protozoa, plants and animals (Fig. 2). Although we havealso identified genes encoding presenilin-like proteins,
hereinafter termed pseudo-presenilins, in some archaebacterial species (for instance, gi:18160164 in Pyrobaculum
aerophilum), they were excluded from our studies eventhough the two conserved aspartate residues seemed to bepresent. This is because the homology with human presenilins is not sufficient (E value = 1.2  10 2) to expect
common biological functions, and in addition the numberof TM domains is fewer than eight in all cases (data notshown). Thus, we have confirmed the absence of presenilin
genes in at least 63 bacterial species, including archaebacteria and eubacteria, for which the entire genome sequences
are available. Moreover, it was found that fungi possess nopresenilin genes, irrespective of whether they are budding,fission or bisexual yeast such as Saccharomyces cerevisiae,
Schizosaccharomyces pombe and Neurospora crassa. Thiswas rather unexpected since, in general, genes possessed byboth animals and plants are also possessed by fungi, because
the Kingdom Fungi branched from the Kingdom Animalia
after the Kingdom Plantae did (Fig. 2). This suggests that
there are both presenilin-possessing and -lacking protozoa,
which are related to the ancestors of plants, animals andfungi. This is in fact the case, since presenilin genes were
found in protozoa close to animals and plants (Monosiga
ovata and Trypanosoma brucei, respectively) but not in
those close to fungi (Encephalitozoon cuniculi). Furthermore, it was surprising that presenilin gene-possessing
protozoa included not only multicellular protozoan species
such as cellular slime molds (Dictyostelium discoideum) but
also unicellular species such as entamoeba (Entamoeba
histolytica), choanoflagellates (M. ovata) and trypanosomes(T. brucei). On the other hand, the absence of presenilin
homologues was confirmed in other unicellular protozoa
such as malaria parasites (Plasmodium falciparum). Consequently, it is plausible that the first presenilin gene may havebeen generated by the recruitment of some gene similar topseudo-presenilins very early in protozoan history (around
1.0 to 1.2 billion years ago), and later transmitted toancestral protozoans that evolved into animals and plants,

but not to those that evolved into fungi (Fig. 2). This isconsistent with the idea that T. brucei, M. ovata and E.
cuniculi may be ancestral species of plants, animals andfungi, respectively (data not shown).
3.4. Phylogenetic examination of presenilins
To estimate the approximate time of the gene duplication of presenilins on the universal evolutionary tree, thephylogenetic trees of presenilins were constructed by using
two methods (Fig. 3). The tree constructed using the NJ
method (Saitou and Nei, 1987) suggests that presenilin
genes were duplicated independently in vertebrates andplants (Fig. 3A). On the other hand, the tree constructedusing the ML method (Yang, 1994; Katoh et al., 2001)
suggested that another duplication event occurred twice
independently in nematodes (Fig. 3B). In any case, boththe methods suggested that the a- and h-types of presenilins were generated independently in different phyla/divisions. These results strongly suggest that there werefunctional requirements for and constraints on the generation and conservation of certain sizes of HL regions inpresenilins.
3.5. Alternative proteolytic processing model for presenilins
To explain the results obtained from our studies aswell as results previously reported by others, we constructed a simple model, named the alternative proteolytic
processing model, which involves both different andredundant functions of PS1 and PS2. We postulate that
presenilins with larger HL sizes would be more sensitive
to autocatalytic proteolysis in this region (Shirotani et al.,2000). Thus, PS1 and PS2 may exist in general mainly asprocessed and unprocessed molecules, respectively, although the level of these molecules in either presenilin
depends on the tissue and developmental stage. NTF +CTF and FLM are further postulated to promote differentiation and apoptosis, respectively. When CTF is phosphorylated, it is stabilized but its counterpart, NTF, isdegraded so as to maintain the level of NTF at a ratio of
1:1 to that of unphosphorylated CTF (Lau et al., 2002).
Consequently, an excess of phosphorylated CTF may act
as a dominant-negative inhibitor against both NTF + CTF
and FLM (Vito et al., 1996). The model can then besummarized as in Fig. 4.

Fig. 2. Presence/absence, and generation and duplication times of presenilin genes. The evolutionary tree was constructed according to Miyata et al. (data notshown). Symbols; B and on the tree indicate approximate times of generation and duplication of presenilin genes, respectively. Open arrows above the open
star indicate the directions of presenilin gene transmissions at the branching points of Kingdoms from Protozoa to Plantae, Animalia and Fungi. Note that
presenilin genes were not transmitted to the ancestral species of fungi. Species in which presenilin genes were found are marked by ‘‘a’’, ‘‘h’’, ‘‘g’’ and/or ‘‘?’’in circles, which indicate the presence of a-, h-, g- or undefined type of presenilins, respectively. Species in which the absence of presenilins was confirmed are
indicated by ‘‘  ’’. The appearance of Domain Eucarya or Kingdom Protozoa (1.0 – 1.2 billion years ago), Cambrian explosion (530 million years ago) andmammalian adaptive radiation (80 million years ago) are shown by gray ovals. Species whose entire genome sequences are available are in gray boxes. The
branching point of P. falciparum is a tentative proposal.

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T. Hashimoto-Gotoh et al. / Gene 323 (2003) 115–123

Fig. 3. Molecular phylogenetic trees inferred by the neighbor joining method (Saitou and Nei, 1987) under the JTT-model (A) and by the genetic algorithmbased maximum likelihood method (Katoh et al., 2001) (B). (A) The evolutionary rate heterogeneity among sites was taken into account, assuming Yang’s
discrete gamma model (Yang, 1994) and optimized shape parameter alpha of 0.74; highly homologous sites without gap sites were selected for the comparison.
Two presenilins of the threadworm, namely HOP1 and SPE4, were omitted because their high evolutionary rates might have caused a long-branch attraction(LBA) artifact. (B) The evolutionary rate variation among sites was corrected by assuming the gamma distribution with optimized shape parameter alpha of
0.92; gap sites were excluded from the comparison. Using either method, it was revealed that presenilin genes could have been duplicated at least twice (A) or
four times (B), independently in different phyla/divisions as indicated by . The numbers at each branching node represent the bootstrap probability. ‘‘a’’, ‘‘h’’and ‘‘g’’ are as in Table 1.

If this model is adapted to Xenopus early embryos(Watanabe et al., 2003), one can analyze the model mathematically as follows. During early embryogenesis, both celldifferentiation and apoptosis should be suppressed. Then,
we obtain

and CTFs are phosphorylated with the efficiencies of pa andpb, for PSa and PSh, respectively, we obtainaPCTF ¼ pa ka abPCTF ¼ pb kb bðaPCTF þ bPCTF Þ > ðaNþC þ bNþC Þ þ ðaFLM þ bFLM Þ;
in which aPCTF and bPCTF are the amounts of PSa and PSh
CTFs phosphorylated in a cell, aN + C and bN + C are theamounts of NTF + CTF complex, and aFLM and bFLM are theamounts of FLM, respectively. Providing that the initialamounts of presenilin molecules are given by a and b,
presenilins are processed with the efficiencies of ka and kb

aFLM ¼ ð1  ka ÞabFLM ¼ ð1  kb ÞbaNþC ¼ ka að1  pa ÞbNþC ¼ kb bð1  pb Þ

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121

Fig. 4. Alternative proteolytic processing model for different and redundant involvement of PS1 and PS2. After autocatalytic processing in the HL region,
presenilins are converted to NTF + CTF complexes with the efficiency of k (a pathway), and those not processed remain as FLM with the efficiency of 1  k (hpathway). PS1 and PS2 enter both the pathways but preferentially the a and h pathways, respectively, as indicated by PS1>PS2 and PS2>PS1 due to the HL
size differences (see Section 3.5). Then, the CTF is phosphorylated with the efficiency of p, yielding a stable form of CTF (PCTF), without forming a complex
with its counterpart NTF, the latter of which is then degraded. NTF + CTF and FLM are postulated to be pro-differentiative and pro-apoptotic, respectively.
Therefore, it is likely that Notch and hAPP may be preferentially cleaved by NTF + CTF and FLM, respectively. PCTF blocks the overall activities of
NTF + CTF and FLM with equal probabilities as a dominant negative inhibitor.

Fig. 5. Phosphorylation efficiency ( pa) of PSa CTF as a function of the ratio of PSa to PSh protein synthesis (n) for regulating the choice among differentiation(Df), apoptosis (Ap) and proliferation (Pf) states in Xenopus early embryos. Above the curve given by pa=(n + 1)/2n, where 1 < n, embryonic cells will be
proliferative, as shown in the light gray area (Pf). Below the curves given by pa = 1  0.8/n, where 0.8 < n < 2.6, and pa=(n + 1)/2n (n>2.6), cells will bedifferentiative as shown in the dark gray area (Df). In-between, cells are potentially apoptotic as shown in the closed area (Ap). The relative rate of PSa to PShprotein synthesis should be between 1 and 2.6 for switching Xenopus embryonic states among Df, Ap and Pf by PSa CTF phosphorylation.

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T. Hashimoto-Gotoh et al. / Gene 323 (2003) 115–123

By introducing these, we obtainðpa ka a þ pb kb bÞ > ða þ bÞ=2:
If one introduces ka = 1, kb = 0.1 and pb = 0 as observed inearly embryonic stages 2 to 6 in Xenopus laevis (Fig. 2 in
Watanabe et al., 2003), we obtainpa > ðn þ 1Þ=2n;where n is given by a/b.
On the other hand, if the balance between cell differentiation and apoptosis is determined by the ratio between
NTF + CTF and FLM activities, the differentiation andapoptosis states will be represented bynilins. Concerning the g-type presenilins, it has beenreported that alternative splicing sites result in shorter HLvariants in DmPS (Boulianne et al., 1997) and BfPS (Martinez-Mir et al., 2001). According to our model, it is crucial
that PS1 synthesis should be more efficient than that of PS2
and that CTF phosphorylation should be highly efficient inorder to avoid apoptosis. It is of interest to see what would
fully enhance phosphorylation of presenilin CTFs in nerve
cells, as progesterone does in Xenopus oocytes and whether
some other steroid hormones such as estrogen would do so.

4. Note added in proofðaNþC þ bNþC Þ < ðaFLM þ bFLM Þ;

After submission of this paper, in the newest disclosureof Rice Genbank (http://www.tigr.org/tdb/e2k1/osa1/), we
found another possible presenilin (8360.m03641), whichshowed significant homology to the a-type presenilin of
A. thaliana.

respectively. Then, the border between apoptosis and differentiation will be defined by

AcknowledgementsðaNþC þ bNþC Þ > ðaFLM þ bFLM Þandðka a þ kb bÞ  ðpa ka a þ pb kb bÞ ¼ ð1  ka Þa þ ð1  kb Þb;that is,We thank Drs. H. Suga and K. Kuma for collecting DNA
sequence data, and Dr. T. Mizuno for valuable discussions.

pa ¼ 1  0:8=n;where the activities of wild-type NTF + CTF and FLM are
assumed to be equivalent per molecule for opposite directions, though this may well be altered by mutations such as
found in FAD pedigrees. The results are diagrammed inFig. 5.
The model developed above may explain the bias ofmutation frequencies of FAD pedigrees, in terms of loss- andgain-of-function mutations in ps1 and ps2 genes, respectively, since the effective n values will be reduced in either caseby a decrease of PS1 or an increase of PS2 activity.
Similarly, this would also explain why PS1 KO-mice arelethal while PS2 KO-mice can survive. Furthermore, thismodel predicts that the PS2 expression level must be lower
than that of PS1 in order to regulate the distribution of cells
among the differentiative, apoptotic and proliferative/staticstates by the degree of CTF phosphorylation. In the case of
Xenopus early embryos (Watanabe et al., 2003), pa was 0.89
and n values should be between 1.3 and 2.6. Although it is
not clear at the moment how NTF + CTF and FLM induce
differentiation and apoptosis, respectively, the model
appears to be compatible with the idea that they may
preferentially cleave Notch and hAPP, respectively, although the model does not exclude the possibilities that
other substrates may be involved in intramembranous cleavage by presenilins (Lammich et al., 2002) or that presenilins
may require other components in order to exhibit theirproteolytic activities (Herreman et al., 2003). In this context,
it is of interest to point out that Arabidobsis thaliana, whichshows no apoptotic phenotype, possesses no h-type prese-

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