Review

Senescence and apoptosis in yeast mother cell-specic aging and in higher cells:
A short review
Peter Laun, Gino Heeren, Mark Rinnerthaler, Raphaela Rid, Sonja Kssler,
Lore Koller, Michael Breitenbach
Department of Cell Biology, Division of Genetics, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria
a r t i c l e i n f o a b s t r a c t
Article history:
Received 4 January 2008
Received in revised form 12 February 2008
Accepted 13 February 2008
Available online 4 March 2008
It is our intention to give the reader a short overview of the relationship between apoptosis and senescence in
yeast mother cell-specic aging. We are studying yeast as an aging model because we want to learn something of
the basic biology of senescence and apoptosis even from a unicellular eukaryotic model system, using its
unrivalled ease of genetic analysis. Consequently, we will discuss also some aspects of apoptosis in metazoa and
the relevance of yeast apoptosis and aging research for cellular (Hayick type) and organismic aging of
multicellular higher organisms. In particular, we will discuss the occurrence and relevance of apoptotic
phenotypes for the aging process. We want to ask the question whether apoptosis (or parts of the apoptotic
process) are a possible cause of aging or vice versa and want to investigate the role of the cellular stress response
system in both of these processes. Studying the current literature, it appears that little is known for sure in this
eld and our reviewwill therefore be, for a large part, more like a memorandumor a programfor future research.
2008 Elsevier B.V. All rights reserved.
Keywords:
Yeast apoptosis
Replicative lifespan
Chronological lifespan
S. cerevisiae
Translational control
Eukaryotic ribosome
AIF
YCA1
NUC1
NMA111
Modern apoptosis research starts with the seminal paper of Kerr
[1] which drew the attention of the scientic world to the previously
ignored fact that the ordered and programmed removal of cells of
multicellular organisms under a variety of physiological conditions is
as important as the production of these cells. In this early paper a clear
distinction was made between apoptosis, a smooth and mostly
unnoticed process which occurs without inammation, and necrosis,
a process usually accompanied with inammation. At least three
rather different biological purposes of the apoptotic process of cellular
suicide seem to exist. First, cells commit suicide during the process of
morphogenesis early in life. Secondly, cells that have accumulated
damage beyond a certain threshold are removed by apoptosis and are
replaced thereby ensuring proper functioning of organs in adult life.
Thirdly, senescent cells also undergo apoptosis, a process that up to
now has been studied mainly in cultured mammalian cells at the
Hayick limit (replicative senescence).
A prominent example for morphogenic apoptosis in the embryo
which has found its way into numerous textbooks is the formation of
digits of the mouse paw during embryogenesis [2]. In this work,
apoptotic cells were recognized through acridine orange staining and
by electron microscopy analyzing apoptotic bodies which were either
free or engulfed by professional phagocytes. Mouse embryos
completely devoid of professional phagocytes as a consequence of a
transcription factor mutation in homozygous PU.1 null mice showed a
defect in the process of engulfment which was partly taken over by
mesenchymal cells. Later [3] apoptotic markers like DNA fragmenta-
tion shown by the TUNEL test were shown to occur in the system, and
the signaling function of externalized phosphatidyl serine and other
possible surface markers was studied in detail [4,5], revealing that
externalized phosphatidyl serine signals both to macrophages which
subsequently engulf the apoptotic cells, and to down-regulate the
inammatory response in the tissue [6]. Mutants in known apoptotic
genes that were studied revealed mostly a mild phenotype but
occasionally an embryonic or perinatal lethal phenotype as shown by
the DNAse II alpha homozygous knock-out mutant [79]. The
embryonic lethal phenotype in this case is probably not due to lack
of apoptotic functions but due to a defect in the essential day-job
function of the DNAse II alpha, for instance in haematopoiesis.
It is remarkable that the signals and the mechanism of removal of
apoptosed cells seem to have been conserved between widely
divergent animal species, like Drosophila melanogaster, nematodes
and vertebrates. What is also conserved to a certain degree are the
genes and proteins that are needed for the execution of the
programmed cell death process. Many of the CED (cell death) genes
of Caenorhabditis elegans have been shown to fulll biochemical
Biochimica et Biophysica Acta 1783 (2008) 13281334
Corresponding author.
E-mail address: michael.breitenbach@sbg.ac.at (M. Breitenbach).
0167-4889/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbamcr.2008.02.008
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
j our nal homepage: www. el sevi er. com/ l ocat e/ bbamcr
functions conserved in mammals and also show a certain degree of
sequence conservation [10]. On the other hand, the multiple stimuli
(pathways) converging into apoptotic processes and the presumed
multiplicity of apoptotic mechanisms is less well known and poses a
challenge to current research. For instance, little is known about the
signals (intracellular and extracellular) leading into the embryonic
morphogenic apoptosis just described and about the conservation of
pathways between the embryonic and other forms of apoptosis. An
interesting hint can be seen in the fact that exposing C. elegans to
oxidative stress during development leads to an increase in the
number of cells that undergo apoptosis. Although a correlation of
apoptosis with senescence and aging has been found in some systems
(see below), in C. elegans the genetic analysis of both processes,
apoptosis and aging, has revealed little overlap between the two
processes so far [10]. This is to say that compromising the develop-
mental apoptosis through mutation has, surprisingly, not diminished
the tness and fecundity of the organism [11,12], but has also not led
to a change in lifespan [13]. Conversely, and interestingly, prohibiting
developmental apoptosis through null mutations in pro-apoptotic
genes leads to embryonic lethality in D. melanogaster and in the
mouse [14,15]. The worm system has arguably the best genetics of
aging in the formof a large number of mutants that can be ordered in
a signaling pathway, the IGFR pathway, which also governs the
formation of dauer larvae. However, these mutations have no
inuence on developmental apoptosis. Although apoptosis occurs in
more than 10% of all cells during development, many of them in the
nervous system, and apoptosis can be induced through external
oxidative stress, apoptotic markers are reportedly absent from
senescent worms.
Apoptosis plays a major role in several distinct processes of
immune regulation. To give an example, the down-regulation of
expanded clones of B- and T-cells when a challenge with microbial
pathogens is no longer present; and the elimination of anti-self clones
from the immune repertoire of the organism. These processes work
through activation-induced cell death [16] and involve binding of a
relevant ligand to the surface receptor of immune cells, Fas. This
system is obviously independent of external oxidative stress and it is
not completely clear if the well known mitochondrial branch of the
apoptotic mechanism is a necessary component of this form of
apoptosis [16].
Apoptosis in multicellular animals is well documented as an anti-
aging mechanism, because it can remove damaged cells from tissues,
inducing the replacement of these cells by homeostatic mechanisms,
and thereby leads to tissue repair [17]. Many different forms of
molecular damage can lead cells into this process. The most generally
accepted mechanism of damage that is at work here is the oxidative
damage exerted primarily by reactive oxygen species (ROS) produced
as by-products of the mitochondrial respiratory chain. This kind of
oxidative damage is found in aged and apoptotic cells in all model
systems known, including yeast. A huge body of published evidence
exists describing the origin (mainly from complex I and III), chemical
reactivity, reaction products and forms of cellular damage (genotoxic
and cytotoxic) which the primary oxygen radical, superoxide, can
induce directly or indirectly in the living cell (summarized in [18]).
These processes can be mimicked by exposing the cell to oxidants, like
hydrogen peroxide or to a large number of other damaging agents.
Much less is known about the possible role in apoptosis of
extramitochondrial sources of ROS (for instance, NADPH oxidases;
[19]).
However, an additional large number of primary triggers different
from the mitochondrial oxidative damage just described and leading
ultimately also into the apoptotic process have been published.
Frequently, as a consequence of these other defects, the cell
secondarily creates oxidative stress. One example is replication stress
[20,21].
The relationship of cell division cycle checkpoints and cell
division cycle regulation with the entry into apoptotic pathways is
an interesting and unnished eld of apoptosis research. Cell cycle
arrest at a checkpoint (often studied at the DNA damage
checkpoint in G2) in principle can lead to two scenarios depending
on the duration of cell cycle arrest: i) The cell can return into a
normal cell cycle after DNA repair; or ii) the cell can switch on the
apoptosis program [22,23]. However, another avenue into apoptosis
is opened by loss of a checkpoint function [24,25]. This is
Fig. 1. The picture shows in part: (A) exponentially growing young yeast cells of strain W303, a commonly used haploid MATa strain, which are included here to show the size and
morphology difference to old cells. (B) A typical lifespan determination of the same wild-type strain. The number of budding cycles that each of a set of 50 virgin cells undergoes
before it stops dividing was determined by micromanipulation and counting of budding cycles. (C) M is the terminal mother cell after 15 cell cycles. Note the enormous size as
compared to young cells and the surface changes. D14 is the second but last daughter that did not completely separate fromthe mother and did not give rise to newliving cells. Also,
the surface of D14 is folded or wrinkled. The budding of the granddaughter D14-1 stopped at an early stage of the cell cycle. The budding of last daughter cell, D15, also stopped at an
early stage of the cell cycle. Reprinted from [28], with permission from Elsevier.
1329 P. Laun et al. / Biochimica et Biophysica Acta 1783 (2008) 13281334
intriguing, because the apoptotic nal stage senescent yeast mother
cells observed by us [26] often die in the middle of an ongoing cell
cycle [27] as judged by their still attached daughter cell (of variable
size) or by the initiation of a new cell cycle while the old cell
cycle is still not nished [28] (Fig. 1). Although not studied or
published in detail up to now, the occurrence of such processes in
very old yeast mother cells logically would lead to aneuploid cells
among the daughters of these mother cells. This is a striking
parallel to many human cancer cells which also have lost cell cycle
controls and which also are often aneuploids.
It is remarkable that the correlation of apoptosis with oxidative
stress seems to be general. Even in those cases where the primary
cause of apoptosis is unrelated to oxidative stress (for instance during
faulty response to DNA damage), the destruction program which is
turned on, includes production of ROS [21,29].
Apoptosis can act as a quality control in germ cells. Both in the
nematode and in mammals, a very high proportion of the female germ
cells undergo apoptosis and only a tiny fraction survive [30]. It has
been speculated that this surprising fact serves the purpose of quality
control eliminating any haploids with errors due to premeiotic DNA
synthesis, faulty segregation at the meiotic divisions and, perhaps also
heteroplasmic mitochondrial mutations [31]. A similar quality control
was observed in mammalian male meiosis [32].
We now want to give an overview of the key topic of this short
review article, which is the correlation of cellular senescence with
apoptosis.
Very few papers dealing explicitly with the relation of aging and
apoptosis have been published so far and most of them are
speculative. Their main point is twofold: i) caloric restriction which
is the only method of lifespan elongation that works in all known
model systems of aging, likewise inuences lifespan and apoptosis
(compare [33] and the literature references therein); ii) genes in the
insulin like growth factor receptor pathway have a strong inuence on
lifespan in C. elegans, are conserved in mammals, and are in the
mammalian system known for their control of apoptosis [33]. More
recently, an elongation of the lifespan has been shown for mouse
mutants in the same pathway [34]. Papa and Skulachev [35] discuss
the mitochondrial origin of oxygen radicals, and their inuence on
both processes, aging and apoptosis. However, as mentioned else-
where in this short review article, in many cases manipulating an
apoptotic gene has not resulted in the expected lifespan elongation
and, vice versa, manipulating a longevity gene has not resulted in the
expected effect on apoptosis (compare for instance, [13] and Table 1).
Currently available evidence shows that both in replicative aging of
yeast mother cells [26] and in the replicative in vitro (Hayick-type)
aging of primary human cells (HUVEC; [36]), these cells in their nal
stage display markers of apoptosis: inversion of the plasma membrane
as analyzed by binding of annexinV without general perforation of the
plasma membrane, an increase in ROS as detected by dihydrorhoda-
mine, and fragmentation of nuclear DNA as analyzed by the TUNEL
method. Not all human cell cultures show this phenotype, as
established lines of broblasts do not [36]. In the eld of aging
research of higher organisms it is an open question to what degree
Hayick-type aging of cell culture cells in vitro might be relevant for
the aging process of the organism as a whole. The discrepancy
between the in vivo and the in vitro situation might be summarized in
three points: i) the cell culture mediumdoes not exactly reproduce the
in vivo situation and the neighboring cells of the niche are absent; ii)
the cells studied are not stem cells which arguably may be the most
relevant cells to study; iii) the lifespan of postmitotic cells of some
organs (for instance of the central nervous system) might be limiting
for the survival and function of the respective organ, a situation which
is not mimicked in the cell culture system. It is highly relevant to
answer the questions whether apoptotic cells can be found in healthy
centenarians, whether an exhaustion of a stem cell population is
indeed found, and whether the exhaustion of a stem cell population
takes place (if it takes place) by way of cellular senescence and
apoptosis. These questions are presently mostly answered in a
negative way. To give an example, there seems to be no exhaustion
of the satellite cells of skeletal muscle and no apoptotic markers are
observed in biopsies. However, in certain diseases, like Duchenne
muscular dystrophy, the stage of exhaustion of stem cells is indeed
reached. Clearly, further progress of stem cell research is needed to
answer these questions and it is to be expected that in the future the
role of stem cell exhaustion and apoptosis in organismic aging will be
claried.
Table 1
Genes involved in the yeast apoptotic pathway with a short description according to Incyte BioBase [63] and the change in replicative and chronological lifespan of the corresponding
deletion mutant compared to wild type strain
Name ORF Description [63] Chronological
lifespan [60]
Replicative
lifespan
Proapoptotic
YCA1[64,65] YOR197W Putative cysteine protease similar to mammalian caspases, involved in regulation of apoptosis upon hydrogen
peroxide treatment
Increased n. a.
NDI1[66,67] YML120C NADH:ubiquinone oxidoreductase, transfers electrons from NADH to ubiquinone in the respiratory chain but
does not pump protons, in contrast to the higher eukaryotic multisubunit respiratory complex I; phosphorylated;
homolog of human AMID
Increased n. a.
AIF1 [53,64] YNR074C Mitochondrial cell death effector that translocates to the nucleus in response to apoptotic stimuli, homolog of
mammalian Apoptosis-Inducing Factor, putative reductase
Increased n. a.
NUC1[64,68] YJL208C Major mitochondrial nuclease, has RNAse and DNA endo- and exonucleolytic activities; has roles in mitochondrial
recombination, apoptosis and maintenance of polyploidy
Increased no change
NMA111 [69,70] YNL123W Protein of unknown function which may contribute to lipid homeostasis and/or apoptosis; sequence similarity
to the mammalian Omi/HtrA2 family of serine proteases
Increased n. a.
CPR3 [71] YML078W Mitochondrial peptidyl-prolyl cis-trans isomerase (cyclophilin), catalyzes the cis-trans isomerization of peptide
bonds N-terminal to proline residues; involved in protein refolding after import into mitochondria
Increased n. a.
DNM1[72,73] YLL001W Dynamin-related GTPase required for mitochondrial ssion and the maintenance of mitochondrial morphology,
assembles on the cytoplasmic face of mitochondrial tubules at sites at which division will occur;
also participates in endocytosis
Increased increased
FIS1[7274] YIL065C Mitochondrial outer membrane protein involved in membrane ssion, required for localization of Dnm1p
and Mdv1p during mitochondrial division; mediates ethanol-induced apoptosis and ethanol-induced
mitochondrial fragmentation
Decreased increased
Antiapoptotic
BIR1 [70] YJR089W Essential chromosomal passenger protein involved in coordinating cell cycle events for proper chromosome
segregation; C-terminal region binds Sli15p, and the middle region, upon phosphorylation, localizes Cbf2p to the
spindle at anaphase
n. a. n. a.
1330 P. Laun et al. / Biochimica et Biophysica Acta 1783 (2008) 13281334
The situation of aging yeast mother cells is simpler than the
scenario just described. Bttner et al. [37] have given a nice overview
of the physiological situations in which yeast cells display apoptotic
phenotypes. Both of the aging processes of yeast cells, chronological
aging and mother cell-specic aging, have been described to lead to
apoptosis of the terminally senescent cells. This is remarkable because
the physiological situation of terminal mother cells is very different
from chronologically aged cells. The aging of yeast mother cells
depends on the number of cell generations that a mother cell has
undergone and not on calendar time (Fig. 2). The distribution function
of lifespans of a cohort of yeast cells is described by the Gompertz law.
The aging process occurs in the presence of nutrients and has nothing
to do with starvation. The old mother cells, like the senescent HUVEC,
are much larger than young cells, display a longer cell cycle time and a
slowed down translational activity of the ribosomes, their nuclei
appear fuzzy (not compact like in young cells), DHR staining indicates
that the cells have produced an oxidizing internal milieu even if
external oxidative stress is absent, and their actin cytoskeleton looks
collapsed and appears clumpy [26,38]. On the other hand, chron-
ologically aged (stationary) yeast cells are reproductively still young,
but undergo a process of senescence due to the lack of nutrients. From
stationary phase cultures two quite different cell populations can be
isolated on density gradients [39]. One population of cells in a
stationary culture acquires a long chronological lifespan through a
process of differentiation involving changes of the cell wall, synthesis
of reserve carbohydrates like trehalose and glycogen and quiescence
of the genome, resembling a true G0 state. This cell population
consists of the daughter cells resulting from the last cell division cycle
which the culture has performed. The other population of cells in the
stationary cultures shows adherent daughter cells in unnished cell
cycles, is much shorter lived than the rst one, creates oxygen radicals
and dies much earlier than the rst one. Eventually, both populations
at very different times reach apoptosis. It has been speculated that the
postmitotic aging just described and resulting in apoptotic death
might be an adaptive trait that was positively selected for during
evolution [37,40] because it supports the survival of the yeast strain in
times of lack of nutrients. This was further elaborated by Longo and
Skulachev who hypothesized that most aging processes (also those
that occur in higher organisms) have an adaptive value and are
altruistic [41]. However this and similar hypotheses concerning
multicellular organisms have been challenged on the grounds of
evolutionary theory, in our opinion with convincing arguments [42
44], two of which are as follows: i) The vast majority of individuals in a
wild population never reach senescence; therefore, the process of
aging cannot have been selected for; and ii) the force of biological
selection is at work until progeny has been procreated and can survive
on their own.
Turning to yeast mother cell-specic aging, we can say that the
nal demise of the senescent mother cells is almost certainly of no
positive value for the growing yeast strain or for the growing yeast
colony. Wild type laboratory yeast strains display a median replicative
lifespan of about 2030 generations and a maximumlifespan of about
4060 generations [38]. The fraction of cells which is senescent in a
population of yeast cells is exceedingly small (1/2
n
) where n is the
number of generations after which the cell dies. The nutrients
supplied by the death and lysis of the old mother cell are therefore
negligible. We have to look for other more convincing reasons why
those old mother cells undergo apoptosis.
The key observation was that cell division cycles of budding yeast
are asymmetric, producing one daughter that is rejuvenated and has
reset the clock to zero, and one mother cell which in every cell cycle
acquires not only an additional bud scar, but also a gradual change in
the parameters mentioned above (cell size etc.). This cell division
asymmetry is similar to the asymmetric division of a stem cell in the
human body, which produces one new stem cell and one cell that has
undergone the rst step to cell differentiation. A further key
observation is that life and metabolism inevitably produce waste
and the waste must be either cleanly removed or at least asymme-
trically segregated between mother and daughter cell if the species is
to survive over evolutionary time spans. Waste in the case of the yeast
cell (but also in higher cells) is not exclusively but predominantly the
oxidation products of cellular components. There are a myriad of
compounds that occur in vivo and could be monitored because they
are waste products that do not occur in newborn cells. One method
that has been used frequently for quantitation and for uorescent in
situ microscopy is the following: The carbonyl groups which exist on
proteins due to oxidative reactions (protein carbonyls) are con-
jugated with p-nitro phenylhydrazine and then the hydrazones are
decorated with standardized antibodies directed to p-nitro phenylhy-
drazone (the oxy-blot technique) [45,46]. In dividing yeast cells it is
found that both soluble cytoplasmic proteins and mitochondrial
proteins are oxidized during aging and under stress that can induce
apoptosis [47]. Oxidation is not generally found on every protein but is
observed on a small number of specic proteins and with a strong bias
in the mother cell, not in the daughter cell [4749]. The mechanism of
this asymmetric segregation or asymmetric scavenging which occurs
also in E. coli and in higher cells [49] is presently not known. However,
manipulating the asymmetric segregation through mutation of the
yeast SIR2 gene, leads to the expected shortening of the lifespan [48].
In one scenario that looks plausible to us, survival and evolutionary
success of a (single-celled) species primarily depends on the ability to
asymmetrically segregate damage in cell division cycles. A side effect
of this phenomenon is the gradual accumulation of damage in mother
cells, leading to the activation of programmed cell death once a
Fig. 2. Yeast mother cell specic aging is characterized by a limited capacity to produce
daughter cells. The lifespan is determined by the number of cell cycles a mother cell has
undergone, not by calendar time, and in a population of cells the lifespan distribution
follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full
lifespan characteristic for the strain. Reprinted from [62] with permission fromElsevier.
1331 P. Laun et al. / Biochimica et Biophysica Acta 1783 (2008) 13281334
threshold is reached and nally to senescence and cell death. This kind
of cell death clearly is evolutionarily neutral and was therefore not
selected against; however it is not altruistic. What appears as a
genetic program of aging is in reality the genetic program(s) of stress
response, because the accumulation of damage means that the aging
cells build up stress, most importantly, oxidative stress. Part of this
stress response program is identical with the apoptotic program
which in a rudimentary form existed in evolution already before the
rst multicellular organisms appeared and which still exists today in
many single celled organisms. However, not every aspect of apoptosis
exists in monocellular organisms. To give and example, the highly
important engulfment part of the apoptotic program [50] and the
Fas-receptor are not found, but extrinsic pathways (alpha factor; [51])
as well as the intrinsic mitochondrial pathway into apoptosis are
surprisingly well conserved. This includes mitochondrial components
that are translocated to the nucleus (AIF, endo-G) and a growing
number of genes and gene functions which were previously thought
to be specic for the apoptotic process in metazoan eukaryotes, are
now known to exist in yeast too. In addition, the genetic system of
yeast which is probably the most highly advanced genetic system
existing, enabled the researchers to discover new apoptotic genes,
which are highly conserved but previously unknown as apoptotic in
higher cells. One prominent example is CDC48 [52]. Another one is AIF,
which was found in both systems [53]. A list of yeast genes which
directly or indirectly are involved in the apoptotic mechanism is given
in other review articles in this volume.
Another example is ER stress related to the unfolded protein
response and, closely related, the stress created by defects in the
ribosome and protein synthesis machinery. There is also indirect
evidence for a switch in ribosome specicity associated with the
switch of a cell to the apoptotic mode, which includes a change in gene
expression at the transcriptional but also at the translational level. As
this translational road into apoptosis has not been reviewed in detail
before, we will now briey elaborate on this emerging topic.
Recently, modulation of ribosome function during the apoptotic
response was described [54]. The authors show that apoptosis is
dependent on synthesis of apoptosis-specic proteins through a
switch in the protein synthesis machinery.
Similar switching of the ribosome is observed from yeast to
mammals in recent reports for the aging process [55,56] and for apop-
tosis [54,57]. The translational switch in the course of the unfolded
proteinresponse in mammalian cells leads to differential translation of
ATF4 and Bip/GRP78 mRNAs, both encoding anti-apototic proteins, via
PERK kinase and eIF2 phosphorylation. The kinases that mediate
translational response to stress (starvation stress and ER stress) are
counteracted by kinases that mediate nutrient signaling and growth
signals. The TOR (Target of Rapamycin) kinase is a highly conserved,
central controller of cell growth. Inyeast and human cells, TOR is found
in two distinct multisubunit complexes, the rapamycin sensitive
TORC1 completely and the rapamycin insensitive TORC2 complex. In
regulation of translation, TORC1 controls protein synthesis and rib-
sosome biogenesis. Via phosphorylation of S6 kinase and eIF-4B
binding proteins (4E-BP), two key regulators of translation initiation,
TORC1 orchestrates increased translation of mRNAs encoding growth
control genes, while repressing genes encoding components of the
apoptotic machinery. Thus, active TORC1 favors translation of mRNAs
that drive cellular growth, whichis supported by the timely expression
of antiapoptotic proteins, to ameliorate the cellular stress derived from
increased protein synthesis, i.e. activation of the UPR pathway. Aber-
rant activation of the TOR pathway induces apoptosis [57] which is a
crucial event in the early response to malignant development.
Ribosomal proteins (RP) have been shown to regulate activity of
the prototypic apoptotic protein p53. Ribosomal proteins L23, L11, and
L5 regulate P53 activity by abrogating Mdm2-induced degradation
and RPL26 modulates p53 translation [58]. A variant ribosmal protein,
S27L, is pro-apoptotic and biosynthesized under the direct control by
p53 [58]. We have recently shown in yeast that mutants of RPL10 and
RPS6 mediate differential translation as shown in 2D gel analysis
(unpublished results). It will be interesting to learn whether these RPs
are involved in a translational switch between growth and stress
induced apoptosis.
We return now to the questions asked in the beginning of this
review article.
i) Is apoptosis a cause or a consequence of senescence?
ii) Is the apoptotic killing of yeast cells an adaptive trait?
One simple approach to start answering the rst question is to
determine the mother cell specic lifespan of deletion mutants
(available through the EUROSCARF deletion collection) corresponding
to the apoptotic yeast genes identied so far. A research program is
under way to systematically determine the mother cell-specic
lifespans of all 4800 mutants, and about 1/9 of these mutants are
published [59]. All 4800 mutants have been studied for their inuence
on chronological aging [60]. There is in general very little concordance
between the mother cell-specic and chronological lifespan data
obtained with the 4800 deletion mutants [61]. We have now re-
examined these published data and compared them with the data
obtained in our laboratory for mutants in apoptotic genes. There is one
important caveat: practically all apoptotic genes of yeast have a second
function (we like to call this the dayjob) in growing cells. This second
function inyeast is in many cases not essential for life. However, even if
it is not, we cannot separate the two functions through specic
mutations (certainly not when dealing with deletion mutations). The
nal phenotype, inparticular if it shortens the lifespan, could be due to
the loss of the apoptotic function or, alternatively, due to the loss of the
dayjob function. In Table 1 we are presenting a short list of genes that
were found to be directly involved in the apoptotic process and their
effect on mother cell specic as well as chronological aging. One
essential yeast gene that has been shown to be involved in apoptosis is
included in the list but the somewhat more labor-intensive lifespan
experiment with this mutant has not been done.
Another way to gain information related to question i) is to look at
the apoptotic phenotypes of long-lived mutants. For example, a
mutation of YGR076C leading to a 60% (and statistically signicant)
elongation of the mother cell-specic lifespan (unpublished work)
does not prevent yeast apoptosis to occur; the apoptotic phenotypes
just appear at a later cell generation, according to the longer lifespan.
Taken together, these data rather seem to support the notion that
apoptosis is a consequence (not a cause) of mother cell specic aging,
however, more systematic data are needed before this question can be
denitely answered.
The answer to the second question posed above (is apoptotic
killing of yeast cells an adaptive trait?) seems to be: Yes, for
chronological aging. The argument is that a substantial part of a
stationary population dies and lyses either in spent medium or in
water and the nutrients liberated can be used by surviving cells.
However, the answer is no for mother cell-specic (replicative)
aging, because senescence occurs in spite of adequate food supply and
the fraction of cells dying is very small.
Acknowledgements
We are grateful to the Austrian Science Fund FWF (Vienna, Austria)
for grants S9302-B05 (to M.B.) and to the EC (Brussels, Europe) for
project MIMAGE (contract no. 512020; to M.B.).
References
[1] J.F. Kerr, A.H. Wyllie, A.R. Currie, Apoptosis: a basic biological phenomenon with
wide-ranging implications in tissue kinetics, Br. J. Cancer 26 (1972) 239257.
[2] W. Wood, M. Turmaine, R. Weber, V. Camp, R.A. Maki, S.R. McKercher, P. Martin,
Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless
PU.1 null mouse embryos, Development 127 (2000) 52455252.
1332 P. Laun et al. / Biochimica et Biophysica Acta 1783 (2008) 13281334
[3] M.A. Fernandez-Teran, J.R. Hinchliffe, M.A. Ros, Birth and death of cells in limb
development: a mapping study, Dev. Dyn. 235 (2006) 25212537.
[4] Y. Hamon, O. Chambenoit, G. Chimini, ABCA1 and the engulfment of apoptotic
cells, Biochim. Biophys. Acta 1585 (2002) 6471.
[5] P.M. Henson, D.A. Hume, Apoptotic cell removal in development and tissue
homeostasis, Trends Immunol. 27 (2006) 244250.
[6] M. Cvetanovic, J.E. Mitchell, V. Patel, B.S. Avner, Y. Su, P.T. van der Saag, P.L. Witte, S.
Fiore, J.S. Levine, D.S. Ucker, Specic recognition of apoptotic cells reveals a
ubiquitous and unconventional innate immunity, J. Biol. Chem. 281 (2006)
2005520067.
[7] C.J. Evans, R.J. Aguilera, DNase II: genes, enzymes and function, Gene 322 (2003)
115.
[8] K. Kawane, H. Fukuyama, G. Kondoh, J. Takeda, Y. Ohsawa, Y. Uchiyama, S. Nagata,
Requirement of DNase II for denitive erythropoiesis in the mouse fetal liver,
Science 292 (2001) 15461549.
[9] K. Kawane, H. Fukuyama, H. Yoshida, H. Nagase, Y. Ohsawa, Y. Uchiyama, K. Okada,
T. Iida, S. Nagata, Impaired thymic development in mouse embryos decient in
apoptotic DNA degradation, Nat. Immunol. 4 (2003) 138144.
[10] M.O. Hengartner, Genetic control of programmed cell death and aging in the
nematode Caenorhabditis elegans, Exp. Gerontol. 32 (1997) 363374.
[11] H.M. Ellis, H.R. Horvitz, Genetic control of programmed cell death in the nematode
C. elegans, Cell 44 (1986) 817829.
[12] G. Lettre, M.O. Hengartner, Developmental apoptosis in C. elegans: a complex
CEDnario, Nat. Rev. Mol. Cell Biol. 7 (2006) 97108.
[13] B. Conradt, D. Xue, Programmed cell death, WormBook (2005) 113.
[14] T. Lindsten, A.J. Ross, A. King, W.X. Zong, J.C. Rathmell, H.A. Shiels, E. Ulrich, K.G.
Waymire, P. Mahar, K. Frauwirth, Y. Chen, M. Wei, V.M. Eng, D.M. Adelman, M.C.
Simon, A. Ma, J.A. Golden, G. Evan, S.J. Korsmeyer, G.R. MacGregor, C.B. Thompson,
The combined functions of proapoptotic Bcl-2 family members bak and bax are
essential for normal development of multiple tissues, Mol. Cell 6 (2000)
13891399.
[15] K. White, M.E. Grether, J.M. Abrams, L. Young, K. Farrell, H. Steller, Genetic control
of programmed cell death in Drosophila, Science 264 (1994) 677683.
[16] R.C. Budd, Activation-induced cell death, Curr. Opin. Immunol. 13 (2001) 356362.
[17] E. Schmitt, C. Paquet, M. Beauchemin, R. Bertrand, DNA-damage response network
at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis, J.
Zhejiang. Univ. Sci. B 8 (2007) 377397.
[18] B. Halliwell, J. Gutteridge, Free Radicals in Biology and Medicine, 4th ed.Oxford
University Press, New York, 2007.
[19] K.H. Krause, Aging: a revisited theory based on free radicals generated by NOX
family NADPH oxidases, Exp. Gerontol. 42 (2007) 256262.
[20] W.C. Burhans, M. Weinberger, DNA replication stress, genome instability and
aging, Nucleic Acids Res. 35 (2007) 75457556.
[21] M. Weinberger, L. Feng, A. Paul, D.L. Smith Jr., R.D. Hontz, J.S. Smith, M. Vujcic, K.K.
Singh, J.A. Huberman, W.C. Burhans, DNA replication stress is a determinant of
chronological lifespan in budding yeast, PLoS ONE 2 (2007) e748.
[22] E. Jacotot, K.F. Ferri, G. Kroemer, Apoptosis and cell cycle: distinct checkpoints
with overlapping upstream control, Pathol. Biol. (Paris) 48 (2000) 271279.
[23] J.A. Pietenpol, Z.A. Stewart, Cell cycle checkpoint signaling: cell cycle arrest versus
apoptosis, Toxicology 181182 (2002) 475481.
[24] Y. Luo, S.K. Rockow-Magnone, P.E. Kroeger, L. Frost, Z. Chen, E.K. Han, S.C. Ng, R.L.
Simmer, V.L. Giranda, Blocking Chk1 expression induces apoptosis and abrogates
the G2 checkpoint mechanism, Neoplasia 3 (2001) 411419.
[25] Y.B. Wang, Y. Lou, Z.F. Luo, D.F. Zhang, Y.Z. Wang, Induction of apoptosis and cell
cycle arrest by polyvinylpyrrolidone K-30 and protective effect of alpha-
tocopherol, Biochem. Biophys. Res. Commun. 308 (2003) 878884.
[26] P. Laun, A. Pichova, F. Madeo, J. Fuchs, A. Ellinger, S. Kohlwein, I. Dawes, K.U.
Frohlich, M. Breitenbach, Aged mother cells of Saccharomyces cerevisiae show
markers of oxidative stress and apoptosis, Mol. Microbiol. 39 (2001)
11661173.
[27] A. Pichova, D. Vondrakova, M. Breitenbach, Mutants in the Saccharomyces
cerevisiae RAS2 gene inuence life span, cytoskeleton, and regulation of mitosis,
Can. J. Microbiol. 43 (1997) 774781.
[28] R. Nestelbacher, P. Laun, M. Breitenbach, Images in experimental gerontology. A
senescent yeast mother cell, Exp. Gerontol. 34 (1999) 895896.
[29] M.A. Marchetti, M. Weinberger, Y. Murakami, W.C. Burhans, J.A. Huberman,
Production of reactive oxygen species in response to replication stress and
inappropriate mitosis in ssion yeast, J. Cell Sci. 119 (2006) 124131.
[30] M. De Felici, A.M. Lobascio, F.G. Klinger, Cell death in fetal oocytes: Many players
for multiple pathways, Autophagy 4 (2007).
[31] L.M. Cree, D.C. Samuels, S.C. de Sousa Lopes, H.K. Rajasimha, P. Wonnapinij, J.R.
Mann, H.H. Dahl, P.F. Chinnery, A reduction of mitochondrial DNA molecules
during embryogenesis explains the rapid segregation of genotypes, Nat. Genet. 40
(2008) 249254.
[32] T. Odorisio, T.A. Rodriguez, E.P. Evans, A.R. Clarke, P.S. Burgoyne, The meiotic
checkpoint monitoring synapsis eliminates spermatocytes via p53-independent
apoptosis, Nat. Genet. 18 (1998) 257261.
[33] Y. Zhang, B. Herman, Ageing and apoptosis, Mech. Ageing Dev. 123 (2002)
245260.
[34] C. Selman, S. Lingard, A.I. Choudhury, R.L. Batterham, M. Claret, M. Clements, F.
Ramadani, K. Okkenhaug, E. Schuster, E. Blanc, M.D. Piper, H. Al-Qassab, J.R.
Speakman, D. Carmignac, I.C. Robinson, J.M. Thornton, D. Gems, L. Partridge, D.J.
Withers, Evidence for lifespan extension and delayed age-related biomarkers in
insulin receptor substrate 1 null mice, FASEB J. (2007).
[35] S. Papa, V.P. Skulachev, Reactive oxygen species, mitochondria, apoptosis and
aging, Mol. Cell. Biochem. 174 (1997) 305319.
[36] M. Wagner, B. Hampel, D. Bernhard, M. Hala, W. Zwerschke, P. Jansen-Durr,
Replicative senescence of human endothelial cells in vitro involves G1 arrest,
polyploidization and senescence-associated apoptosis, Exp. Gerontol. 36 (2001)
13271347.
[37] S. Buttner, T. Eisenberg, E. Herker, D. Carmona-Gutierrez, G. Kroemer, F. Madeo,
Why yeast cells can undergo apoptosis: death in times of peace, love, and war, J.
Cell Biol. 175 (2006) 521525.
[38] M. Breitenbach, F. Madeo, P. Laun, G. Heeren, S. Jarolim, K.-U. Frhlich, S. Wissing,
A. Pichova, Yeast as a model for ageing and apoptosis research, in: T. Nystrm, H.D.
Osiewacz (Eds.), Topics in Current Genetics, Model Systems in Aging, vol. 3,
Springer Heidelberg, Heidelberg, 2003, pp. 6197, 552.
[39] C. Allen, S. Buttner, A.D. Aragon, J.A. Thomas, O. Meirelles, J.E. Jaetao, D. Benn, S.W.
Ruby, M. Veenhuis, F. Madeo, M. Werner-Washburne, Isolation of quiescent and
nonquiescent cells from yeast stationary-phase cultures, J. Cell Biol. 174 (2006)
89100.
[40] V.P. Skulachev, Programmed death in yeast as adaptation? FEBS Lett. 528 (2002)
2326.
[41] V.D. Longo, J. Mitteldorf, V.P. Skulachev, Programmed and altruistic ageing, Nat.
Rev. Genet. 6 (2005) 866872.
[42] T.B. Kirkwood, C.J. Proctor, Somatic mutations and ageing in silico, Mech. Ageing
Dev. 124 (2003) 8592.
[43] G.J. Lithgow, T.B. Kirkwood, Mechanisms and evolution of aging, Science 273
(1996) 80.
[44] J. Vijg, Somatic mutations and aging: a re-evaluation, Mutat. Res. 447 (2000)
117135.
[45] T. Nystrom, Role of oxidative carbonylation in protein quality control and
senescence, EMBO J. 24 (2005) 13111317.
[46] J.R. Requena, R.L. Levine, E.R. Stadtman, Recent advances in the analysis of
oxidized proteins, Amino Acids 25 (2003) 221226.
[47] H. Aguilaniu, L. Gustafsson, M. Rigoulet, T. Nystrom, Asymmetric inheritance of
oxidatively damaged proteins during cytokinesis, Science 299 (2003)
17511753.
[48] N. Erjavec, L. Larsson, J. Grantham, T. Nystrom, Accelerated aging and failure to
segregate damaged proteins in Sir2 mutants can be suppressed by overproducing
the protein aggregation-remodeling factor Hsp104p, Genes Dev. 21 (2007)
24102421.
[49] N. Erjavec, T. Nystrom, Sir2p-dependent protein segregation gives rise to a
superior reactive oxygen species management in the progeny of Saccharomyces
cerevisiae, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 1087710881.
[50] R.J. Krieser, K. White, Engulfment mechanism of apoptotic cells, Curr. Opin. Cell
Biol. 14 (2002) 734738.
[51] F.F. Severin, A.A. Hyman, Pheromone induces programmed cell death in S.
cerevisiae, Curr. Biol. 12 (2002) R233R235.
[52] F. Madeo, E. Frohlich, K.U. Frohlich, A yeast mutant showing diagnostic markers of
early and late apoptosis, J. Cell Biol. 139 (1997) 729734.
[53] S. Wissing, P. Ludovico, E. Herker, S. Buttner, S.M. Engelhardt, T. Decker, A. Link, A.
Proksch, F. Rodrigues, M. Corte-Real, K.U. Frohlich, J. Manns, C. Cande, S.J. Sigrist, G.
Kroemer, F. Madeo, An AIF orthologue regulates apoptosis in yeast, J. Cell Biol. 166
(2004) 969974.
[54] M. Holcik, N. Sonenberg, Translational control in stress and apoptosis, Nat. Rev.,
Mol, Cell Biol 6 (2005) 318327.
[55] K.K. Singh, Mitochondria damage checkpoint in apoptosis and genome stability,
FEMS Yeast Res. 5 (2004) 127132.
[56] P. Syntichaki, K. Troulinaki, N. Tavernarakis, eIF4E function in somatic cells
modulates ageing in Caenorhabditis elegans, Nature 445 (2007) 922926.
[57] K. Thedieck, P. Polak, M.L. Kim, K.D. Molle, A. Cohen, P. Jeno, C. Arrieumerlou, M.N.
Hall, PRAS40 and PRR5-like protein are new mTOR interactors that regulate
apoptosis, PLoS ONE 2 (2007) e1217.
[58] H. He, Y. Sun, Ribosomal protein S27L is a direct p53 target that regulates
apoptosis, Oncogene 26 (2007) 27072716.
[59] M. Kaeberlein, R.W. Powers III, K.K. Steffen, E.A. Westman, D. Hu, N. Dang, E.O.
Kerr, K.T. Kirkland, S. Fields, B.K. Kennedy, Regulation of yeast replicative
life span by TOR and Sch9 in response to nutrients, Science 310 (2005)
11931196.
[60] R.W. Powers, M. Kaeberlein, S.D. Caldwell, B.K. Kennedy, S. Fields, Extension of
chronological life span in yeast by decreased TOR pathway signaling, Genes Dev.
20 (2006) 174184.
[61] P. Laun, M. Rinnerthaler, E. Bogengruber, G. Heeren, M. Breitenbach, Yeast as a
model for chronological and reproductive aging a comparison, Exp. Gerontol. 41
(2006) 12081212.
[62] S. Michal Jazwinski, N.K. Egilmez, J.B. Chen, Replication control and cellular life
span, Exp. Gerontol. 24 (1989) 423436.
[63] P.E. Hodges, P.M. Carrico, J.D. Hogan, K.E. O'Neill, J.J. Owen, M. Mangan, B.P. Davis,
J.E. Brooks, J.I. Garrels, Annotating the human proteome: the Human Proteome
Survey Database (HumanPSD) and an in-depth target database for G protein-
coupled receptors (GPCR-PD) from Incyte Genomics, Nucleic Acids Res. 30 (2002)
137141.
[64] S. Buttner, T. Eisenberg, D. Carmona-Gutierrez, D. Ruli, H. Knauer, C. Ruckenstuhl,
C. Sigrist, S. Wissing, M. Kollroser, K.U. Frohlich, S. Sigrist, F. Madeo, Endonuclease
G regulates budding yeast life and death, Mol. Cell 25 (2007) 233246.
[65] F. Madeo, E. Herker, C. Maldener, S. Wissing, S. Lachelt, M. Herlan, M. Fehr, K.
Lauber, S.J. Sigrist, S. Wesselborg, K.U. Frohlich, A caspase-related protease
regulates apoptosis in yeast, Mol. Cell 9 (2002) 911917.
[66] W. Li, L. Sun, Q. Liang, J. Wang, W. Mo, B. Zhou, Yeast AMID homologue Ndi1p
displays respiration-restricted apoptotic activity and is involved in chronological
aging, Mol. Biol. Cell 17 (2006) 18021811.
1333 P. Laun et al. / Biochimica et Biophysica Acta 1783 (2008) 13281334
[67] J.S. Park, Y.F. Li, Y. Bai, Yeast NDI1 improves oxidative phosphorylation capacity
and increases protection against oxidative stress and cell death in cells carrying a
Leber's hereditary optic neuropathy mutation, Biochim. Biophys. Acta 1772 (2007)
533542.
[68] S. Buttner, D. Carmona-Gutierrez, I. Vitale, M. Castedo, D. Ruli, T. Eisenberg, G.
Kroemer, F. Madeo, Depletion of endonuclease G selectively kills polyploid cells,
Cell Cycle 6 (2007) 10721076.
[69] B. Fahrenkrog, U. Sauder, U. Aebi, The S. cerevisiae HtrA-like protein Nma111p is a
nuclear serine protease that mediates yeast apoptosis, J. Cell Sci. 117 (2004) 115126.
[70] D. Walter, S. Wissing, F. Madeo, B. Fahrenkrog, The inhibitor-of-apoptosis protein
Bir1p protects against apoptosis in S. cerevisiae and is a substrate for the yeast
homologue of Omi/HtrA2, J. Cell Sci. 119 (2006) 18431851.
[71] R.J. Braun, H. Zischka, F. Madeo, T. Eisenberg, S. Wissing, S. Buttner, S.M.
Engelhardt, D. Buringer, M. Uefng, Crucial mitochondrial impairment upon
CDC48 mutation in apoptotic yeast, J. Biol. Chem. 281 (2006) 2575725767.
[72] Y. Fannjiang, W.C. Cheng, S.J. Lee, B. Qi, J. Pevsner, J.M. McCaffery, R.B. Hill, G.
Basanez, J.M. Hardwick, Mitochondrial ssion proteins regulate programmed cell
death in yeast, Genes Dev. 18 (2004) 27852797.
[73] C.Q. Scheckhuber, N. Erjavec, A. Tinazli, A. Hamann, T. Nystrom, H.D. Osiewacz,
Reducing mitochondrial ssion results in increased life span and tness of two
fungal ageing models, Nat. Cell Biol. 9 (2007) 99105.
[74] H. Kitagaki, Y. Araki, K. Funato, H. Shimoi, Ethanol-induced death in yeast exhibits
features of apoptosis mediated by mitochondrial ssion pathway, FEBS Lett. 581
(2007) 29352942.
1334 P. Laun et al. / Biochimica et Biophysica Acta 1783 (2008) 13281334