FEBS Letters 583 (2009) 113–117

journal homepage: www.FEBSLetters.org

Mitochondrial dysfunction leads to reduced chronological lifespan

and increased apoptosis in yeast
An M. Aerts a, Piotr Zabrocki b, Gilmer Govaert a, Janick Mathys a, Didac Carmona-Gutierrez c,
Frank Madeo c, Joris Winderickx b, Bruno P.A. Cammue a,*, Karin Thevissen a
a
Centre of Microbial and Plant Genetics (CMPG), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium
b
Laboratory of Functional Biology (LFB), Katholieke Universiteit Leuven, B-3001 Heverlee, Belgium
c
IMB, Karl-Franzens University, 8010 Graz, Austria

a r t i c l e i n f o a b s t r a c t

Article history: We previously isolated a Saccharomyces cerevisiae mutant (HsTnII), which displays 40% reduced
Received 16 September 2008 chronological lifespan as compared to the wild type (WT). In this study, we found HsTnII cultures
Revised 6 November 2008 to be characterized by fragmented and dysfunctional mitochondria, and by increased initiation of
Accepted 19 November 2008
apoptosis during chronological aging as compared to WT. Expression of genes encoding subunits
Available online 4 December 2008
of mitochondrial electron transport chain and ATP synthase is significantly downregulated in
Edited by Vladimir Skulachev HsTnII, and as a consequence, HsTnII is not able to respire ethanol. All these data confirm the
importance of functional mitochondria and respiration in determining yeast chronological lifespan
Keywords:
and apoptosis.
Apoptosis Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Chronological lifespan
Microarray analysis
Mitochondrial function
Mitochondrial morphology
Respiration

1. Introduction
We recently isolated a transposon (Tn) Saccharomyces cerevisiae
mutant (HsTnII), having the Tn inserted in the 25 S rRNA gene,
which is hypersensitive towards the antifungal plant defensin
DmAMP1 as well as towards oxidative stress induced by hydrogen
peroxide [1]. Moreover, HsTnII is characterized by a 40% reduced
chronological lifespan (i.e. the survival time of a population of
non-dividing yeast cells) as compared to its wild type (WT). Since
the Tn insertion in HsTnII was not linked with any of these differ-
ent HsTnII phenotypes, it was concluded that HsTnII has a sponta-
neous mutation in a currently unidentified gene [1].
Chronologically aged yeast cultures die exhibiting typical mark-
ers of apoptosis or programmed cell death, including the accumu-
lation of reactive oxygen species (ROS) [2]. In general, long-living
yeast mutants accumulate less ROS and are characterized by a de-
layed initiation of apoptosis [2–4], whereas short-living yeast mu-
tants accumulate more ROS and are characterized by increased
apoptosis when chronologically aging [5]. To better characterize
the short-living Tn mutant HsTnII, we assessed the apoptotic fea-
tures of aging HsTnII cultures in this study and found that HsTnII
is characterized by increased initiation of apoptosis during chrono-
logical aging as compared to WT. Since mitochondrial function is
important for determining chronological lifespan in yeast [5], we
further investigated mitochondrial morphology and functionality
in HsTnII.
2. Materials and methods
2.1. Strains and growth conditions

Yeast strains used in this study are S. cerevisiae strains W303-1A

Abbreviations: DHE, dihydroethidium; DiOC6, 3,30 -dihexyloxacarbocyanine
iodide; CFU, colony-forming units; OD, optical density; ROS, reactive oxygen
species; TUNEL, Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End
Labeling; WT, wild type.
* Corresponding author. Fax: +32 16 32 19 66.
E-mail address: bruno.cammue@biw.kuleuven.be (B.P.A. Cammue).
(MATa leu2-3/112 ura3-1 trp1-1 his3-11/15) (WT) and Tn mutant
HsTnII [1]. The nuclear petite mutant yeast strain used is a S. cere-
visiae W303-1A pho85::HIS3 strain (MATa, leu2-3/112 ura3-1
trp1-1 his3-11, 15 ade2-1 can1-100 pho85::HIS3) [6] that will be
further abbreviated as Pho85. Media used were YPD (1% yeast

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.febslet.2008.11.028
114 A.M. Aerts et al. / FEBS Letters 583 (2009) 113–117

extract, 2% peptone, 2% glucose) and SC (0.8 g/l CSM, complete

amino acid supplement mixture, Bio 101 Systems; 6.5 g/l YNB,
yeast nitrogen base; 20 g/l glucose). Incubation temperature was
30 °C for all assays.

2.2. Chronological lifespan of yeast

Overnight yeast cultures in YPD were diluted in SC at optical

density (OD)600 = 0.2. Viability of the yeast cultures was analyzed
by counting the number of colony-forming units per ml (CFU/ml)
on YPD agar plates.

2.3. Apoptosis in yeast

Apoptotic markers, comprising ROS levels, phosphatidylserine

externalization and DNA fragmentation, of the above described
chronologically aging yeast cultures were quantified using fluores-
cence microscopy after staining with dihydroethidium (DHE)-
staining, Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick
End Labeling (TUNEL)-staining and FITC-labeled annexin V in com-
bination with propidium iodide-staining, respectively [5,7,8]. For
each staining, 1500 yeast cells were evaluated. Statistical analysis
was performed using paired t-test.

2.4. Visualization of mitochondrial morphology

Overnight yeast cultures in YPD were inoculated in SC at

OD600 = 0.025. At OD600 = 1 (exponential phase), cells were washed
with fresh medium and resuspended in PBS. Mitochondria were
visualized using 3,30 -dihexyloxacarbocyanine iodide (DiOC6)
(Acros Organics, Geel, Belgium) [5].

2.5. RNA extraction and microarray analysis

Total RNA from overnight yeast cultures in YPD was extracted

using RNApureTM (Gene Hunter Corporation, Nashville, USA). Fluo-
rescently labeled cDNA was prepared according to the standard
VIB-MAF labeling protocols (Array Express entries: P-MEXP-578
and P-MEXP-579) and used to hybridize VIB yeast 6K-v3 micro-
arrays according to the standard VIB-MAF hybridization protocol
(Array Express entry: P-MEXP-580). The in house developed yeast
array represents 6386 coding sequences of S. cerevisiae, as 70 bp
oligonucleotides spotted in duplicate (VIB MicroArray Facility, Bel-
gium). A dye swap design was applied. After hybridization, the
microarrays were washed, dried and scanned with a Generation
III scanner (GE Healthcare Life Sciences, Little Chalfont, England)
using the default scanner software. Data analysis and normaliza-
tion was performed using Spotfire DecisionSite software (Spotfire,
Sommerville, USA). Differentially expressed genes were identified
using the combination of a two-sample t-test (P 6 0.05) and a Fig. 1. HsTnII is characterized by increased apoptosis during chronological ageing.
fold-difference cut-off (P1.8) [9]. (A) Chronological lifespan of WT strain W303-1A (black squares) and HsTnII (open
squares). Yeast cultures were grown in SC medium and viability of these cultures
was analyzed by counting the number of CFU/ml. (B) Apoptotic features of WT (grey
2.6. Measurement of ethanol concentration bars) and HsTnII (white bars) after 2 and 6 days in SC were assessed by determining
the endogenous ROS levels via DHE staining, DNA fragmentation via TUNEL staining
Overnight yeast cultures in YPD were inoculated at OD600 = 0.2 and phosphatidylserine externalization and membrane integrity via annexin
V/propidium iodide co-staining. In each experiment, 1500 cells were evaluated
in 5 ml YPD or 250 ml beer wort and incubated for 4 days in a stir-
using fluorescence microscopy. *P < 0.05; **P < 0.01; ***P < 0.001.
red vessel. Samples (300 ll) of yeast cultures were assayed for eth-
anol content using the Ethanol UV-method kit (Roche, Basel,
Switzerland). 3. Results

2.7. H2O2 sensitivity assay
Fifteen microliters of 250 mM H2O2 was spotted on SC agar
plates, inoculated with 1/50 of an overnight yeast culture in YPD.
After 2 days of incubation at 30 °C, diameters of the inhibitory ha-
los were measured.
3.1. HsTnII is characterized by accelerated aging and increased
apoptosis
The transposon (Tn) mutant HsTnII is characterized by 40% re-
duced chronological lifespan in water as compared to its WT [1].
Accordingly, the survival of a chronologically aging HsTnII culture
 A.M. Aerts et al. / FEBS Letters 583 (2009) 113–117 115

in SC medium drastically dropped after 5 days of incubation as

compared to WT (Fig. 1A). Since chronological aging in yeast
reportedly leads to apoptosis [2,3], we assessed different apoptotic
features, including endogenous ROS levels, DNA fragmentation and
phosphatidylserine externalization [5,7,8], of chronologically aging
HsTnII and WT yeast cultures. After 2 days of aging, no significant
differences in apoptotic features between HsTnII and WT cultures
could be observed, except for ROS levels, which were about two-
fold lower in HsTnII as compared to WT (Fig. 1B, upper panel).
However, after 6 days of chronological aging (being shortly after
the survival drop of HsTnII) HsTnII cultures were characterized
by increased endogenous ROS levels, as we found 1.6-fold more
HsTnII cells accumulating ROS (92 ± 3%) as compared to WT cells
(59 ± 7%) (Fig. 1B, upper panel). Moreover, at this 6-day time-point,
the number of HsTnII cells characterized by DNA fragmentation
was four-fold higher (24 ± 1%) as compared to WT cells (6 ± 1%)
(Fig. 1B, central panel). In addition, phosphatidylserine externaliza-
tion, as visualized by strong fluorescence around the whole cir-
cumference of the cells upon staining with annexin V and
absence of propidium iodide-staining, was 1.8-fold higher in HsT-
nII cells (9 ± 1%) as compared to WT cells (5 ± 1%) (Fig. 1B, lower
panel). All these data point to accelerated chronological aging
and an early initiation of apoptosis in HsTnII.

3.2. Mitochondria of HsTnII are fragmented and dysfunctional

A direct correlation between reduced chronological lifespan and

dysfunctional mitochondria was previously reported [5,10–13].
Accordingly, we could show that HsTnII is hypersensitive to hydro-
gen peroxide (Fig. 2A) [1] and is respiratory-deficient since HsTnII
is unable to grow on glycerol-containing media (data not shown).
Both observations are indicative for dysfunctional mitochondria
[14]. To get more insight in the dysfunctional character of the HsT-
nII mitochondria, we visualized mitochondrial membranes in
exponential growing HsTnII and WT cultures using the dye DiOC6.
At a concentration of 20–100 ng/ml, this dye accumulates specifi-
cally at mitochondrial membranes in response to the electrochem-
ical potential across the inner membrane and can be observed by
fluorescence microscopy [15,16]. However, cells that have low
mitochondrial membrane potential will fail to accumulate DiOC6
at the indicated concentration. At 100 ng/ml DiOC6, staining was
greatly reduced in HsTnII cultures as compared to its WT
(Fig. 2B, panel I), pointing to a reduced mitochondrial membrane
potential in HsTnII cells. Using 10-fold higher DiOC6 concentra-
tions, a tubular mitochondrial morphology was observed in WT
cells, whereas excessive fission of tubular mitochondria into short
punctate structures, also referred to as mitochondrial fragmenta-
tion [17], was observed in HsTnII cells (Fig. 2B, panel II).
3.3. Mitochondria of HsTnII are respiratory-deficient
To get more mechanistic insight in the mitochondrial dysfunc-
tion of HsTnII, we performed a genome-wide expression analysis
on HsTnII and WT, this to identify differentially expressed genes
in HsTnII. To this end, HsTnII and its WT were grown overnight
in YPD and RNA was extracted for subsequent hybridization on a
microarray, representing the 6386 coding sequences of S. cerevisiae.
The number of genes that were significantly down- or upregulated
in HsTnII (t-test P 6 0.05 and P1.8-fold-difference) was calculated
(224 genes) (see Supplementary data file). Using GOstat software
[18], differentially expressed genes in HsTnII implicated in mito-
chondrial function were found to be statistically overrepresented
(36 genes) and represented the largest functional group retrieved
from the microarray data. Interestingly, genes implicated in mito-
chondrial function that were significantly downregulated in HsTnII
(being COR1, QCR7, RIP1, QCR8, QCR6, QCR10, COX4, COX7, COX5A,
Fig. 2. Mitochondrial function and morphology are compromised in HsTnII. (A)
Sensitivity of WT (left panel) and HsTnII (right panel) to 250 mM H2O2 was assessed
via growth inhibitory halo assay on SC agar plates. (B) – (I) DiOC6 mitochondrial
membrane straining of WT (left panels) and HsTnII (right panels) at standard DiOC6
concentration (100 ng/ml) and (II) 10-fold increased DiOC6 concentration (1 lg/ml).
ATP1 and ATP5) encode various subunits of the respiratory chain
complexes III and IV of the mitochondrial electron transport chain
and of the mitochondrial ATP synthase. These observations point
towards respiratory-deficient mitochondria in HsTnII, which was
indeed reflected by the above mentioned failure of HsTnII to grow
on glycerol-containing media.
To further document the respiratory defect of HsTnII mitochon-
dria, we assessed ethanol production during fermentation and its
utilization during respiration in HsTnII and WT yeast cultures in
batch growth. During fermentation, yeast produces ethanol as a
side-product, which can be reutilized during respiratory growth,
involving mitochondrial function. It was previously demonstrated
that respiratory-deficient yeast mutants do not metabolize the
product of fermentation, ethanol, as their secondary substrate
[19]. In the present study, we performed a batch fermentation in
a stirred vessel containing YPD and evaluated ethanol production
in WT and HsTnII cultures. Ethanol levels in both WT and HsTnII
cultures rose to 0.19 g/l during the first day of incubation (Fig. 3),
after which WT ethanol levels dropped to zero during the following
days. In contrast, HsTnII ethanol levels did not decrease up till 4
days of incubation. In this experiment, a comparison with the
116 A.M. Aerts et al. / FEBS Letters 583 (2009) 113–117
Fig. 3. HsTnII is unable to respire ethanol. WT cultures (gray triangles), HsTnII
cultures (open squares) and respiratory-deficient nuclear petite yeast mutant
Pho85 (black diamonds) were grown in a stirred 5 ml vessel containing YPD.
Ethanol levels at different time-points were measured using the Ethanol UV-
method kit (Roche).
respiratory-deficient nuclear petite yeast mutant Pho85 was made.
Like for HsTnII, ethanol levels in the Pho85 culture did not decrease
up till 4 days of incubation. Also in a more complex medium,
namely beer wort (250 ml working volume), we could observe this
phenomenon for HsTnII and WT (data not shown).
4. Discussion
In this study, we demonstrated that the Tn yeast mutant HsTnII,
which is characterized by accelerated aging [1], displayed in-
creased initiation of apoptosis during chronological aging. We
found HsTnII to be further characterized by fragmented mitochon-
dria, and this already in the exponential phase, whereas apoptotic
features were still neglectable at 2 days of incubation. This obser-
vation may imply that in HsTnII, mitochondrial fragmentation pre-
cedes apoptosis. The fragmented mitochondria of HsTnII are
dysfunctional as HsTnII is hypersensitive to oxidative stress and
characterized by impaired growth on glycerol. Moreover, mito-
chondrial membrane potential of HsTnII seemed reduced since
mitochondrial membranes of HsTnII could not be stained by DiOC6
at a standard concentration of 100 ng/ml [17]. A reduced mito-
chondrial membrane potential in yeast is often associated with
an increase in cytoplasmic ROS accumulation [16] and apoptosis
[20], corroborating our observation that initiation of apoptosis is
increased in HsTnII. Moreover, transcriptome analysis of HsTnII
showed downregulation of expression of genes encoding subunits
of the mitochondrial electron transport chain or mitochondrial ATP
synthase, pointing to a respiration defect of HsTnII. We could fur-
ther support this conclusion by demonstrating that ethanol utiliza-
tion was blocked in HsTnII during the respire-fermentative phase
of batch growth.
These data support the hypothesis that mitochondrial function,
more specifically respiration, is required in order to prolong chro-
nological lifespan in yeast. In this respect, it was previously dem-
onstrated that increasing the respiratory activity by calorie
restriction, mild uncoupling, reduced TOR signaling or deletion of
SCH9, decreases mitochondrial reactive oxygen release and in-
creases chronological lifespan in yeast [21–23]. Laovoie and White-
way further demonstrated that mitochondrial electron transport
chain genes were up-regulated in a Dsch9 mutant, and concomi-
tantly the oxygen consumption rate [23]. Vice versa, one report de-
scribes the link between a defective mitochondrial gene expression
in yeast, ROS-mediated inhibition of respiration and reduction of
yeast chronological lifespan [12]. In the present study, we further
evidenced the link between defective mitochondrial gene expres-
sion and reduced chronological lifespan in yeast, and hence, pro-
vided further evidence for the crucial role of respiration in
chronological lifespan extension in yeast.
From the ethanol productivity data presented in this study, it
seems that the increased productivity exhibited by HsTnII is the re-
sult of the inability to respire ethanol in the respire-fermentative
phase of batch growth. Indeed, during the first day of incubation
of the yeast cultures in YPD, ethanol levels in both WT and HsTnII
cultures rised to 0.19 g/l. However, starting from day 2, ethanol
levels in the WT culture dropped to zero, whereas ethanol levels
in HsTnII stayed high up till 4 days of incubation. Also for the respi-
ratory-deficient nuclear petite yeast mutant Pho85 ethanol levels
stayed high up till 4 days of incubation. The same scenario holds
true when HsTnII and WT are incubated in a more complex med-
ium, being beer wort. However, in contrast to HsTnII, nuclear petite
yeast cells are not characterized by fragmented mitochondria but
contain mitochondria with typical tubular morphology and periph-
eral distribution [24]. Moreover, when comparing the differential
transcript profiles obtained by microarray analysis of HsTnII (this
study) and a nuclear petite mutant [25], both grown in the same
conditions and from the same genetic background (W303-1A),
we found only 2.7% overlap in downregulated genes and 24% over-
lap in upregulated genes between both datasets, hence distin-
guishing nuclear petite yeast mutants and HsTnII. Because of this
interesting novel phenotype future experiments will be directed
at identifying the specific mutation in HsTnII.
Acknowledgements
This work was supported by FWO-Vlaanderen (research project
G.0440.07 to B.P.A.C. and G.0430.08 to J.W.). We are grateful for the
postdoctoral fellowships to A.M.A. (Research Council) and to K.T.
(Industrial Research Fund) both from K.U. Leuven, to P.Z. from
the EC-Marie Curie (MEIF-CT-2005-775 514281) and for the doc-
toral grant to G.G. from IWT-Vlaanderen. F.M. and D.C.G. are grate-
ful to the DFG and to the FWF for grants ‘‘Lipotoxicity” and S-9304-
B05. We thank Sofie Depraetere from the Centre for Malting and
Brewing Science (headed by Prof. F. Delvaux, K.U. Leuven, Belgium)
for beer wort supply and technical assistance for ethanol determi-
nation in beer wort.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.febslet.2008.11.028.
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