Maintenance of Mitochondrial Morphology by Autophagy and Its Role in High Glucose Effects on Chronological Lifespan of Saccharomyces cerevisiae.
ABSTRACT In Saccharomyces cerevisiae, mitochondrial morphology changes when cells are shifted between nonfermentative and fermentative carbon sources. Here, we show that cells of S. cerevisiae grown in different glucose concentrations display different mitochondrial morphologies. The morphology of mitochondria in the cells growing in 0.5% glucose was similar to that of mitochondria in respiring cells. However, the mitochondria of cells growing in higher glucose concentrations (2% and 4%) became fragmented after growth in these media, due to the production of acetic acid; however, the fragmentation was not due to intracellular acidification. From a screen of mutants involved in sensing and utilizing nutrients, cells lacking TOR1 had reduced mitochondrial fragmentation, and autophagy was found to be essential for this reduction. Mitochondrial fragmentation in cells grown in high glucose was reversible by transferring them into conditioned medium from a culture grown on 0.5% glucose. Similarly, the chronological lifespan of cells grown in high glucose medium was reduced, and this phenotype could be reversed when cells were transferred to low glucose conditioned medium. These data indicate that chronological lifespan seems correlated with mitochondrial morphology of yeast cells and that both phenotypes can be influenced by factors from conditioned medium of cultures grown in low glucose medium.
- [Show abstract] [Hide abstract]
ABSTRACT: Although kidney is a target organ of arsenic cytotoxicity, the underlying mechanisms of arsenic-induced nephrotoxicity remain poorly understood. As tetramethylpyrazine (TMP) has recently been found to be a renal protectant in multiple kidney injuries, we hypothesize that TMP could suppress arsenic nephrotoxicity. In this study, human renal proximal tubular epithelial cell line HK-2 was used to elucidate the precise mechanisms of arsenic nephrotoxicity as well as the protective mechanism of TMP in these cells. Sodium arsenite exposure dramatically increased cellular reactive oxygen species (ROS) production, decreased levels of cellular glutathione (GSH), decreased cytochrome c oxidase activity and mitochondrial membrane potential, which indicated mitochondrial dysfunction. On the other hand, sodium arsenite activated pro-inflammatory signals, including β-catenin, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor alpha and cyclooxygenase-2 (COX-2). Small molecule inhibitors of NF-κB and p38 MAPK blocked arsenic-induced COX-2 expression, suggesting arsenic-induced COX-2 up-regulation was NF-κB- and p38 MAPK-dependent. Finally, sodium arsenite induced autophagy in HK-2 cells at early phase (6 h) and the subsequent apoptosis at 24 h. Treatment by TMP or by the antioxidant N-acetylcysteine decreased arsenic-induced ROS production, enhanced GSH levels, prevented mitochondria dysfunction and suppressed the activation of pro-inflammatory signals and the development of autophagy and apoptosis. Our results suggested that TMP may be used as a new potential therapeutic agent to prevent arsenic-induced nephrotoxicity by suppressing these pathological processes.Archive für Toxikologie 06/2014; · 5.08 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The target of rapamycin (TOR) is an important signaling pathway on a hierarchical network of interacting pathways regulating central biological processes, such as cell growth, stress response and aging. Several lines of evidence suggest a functional link between TOR signaling and sphingolipid metabolism. Here, we report that the TORC1-Sch9p pathway is activated in cells lacking Isc1p, the yeast orthologue of mammalian neutral sphingomyelinase 2. The deletion of TOR1 or SCH9 abolishes the premature aging, oxidative stress sensitivity and mitochondrial dysfunctions displayed by isc1Δ cells and this is correlated with the suppression of the autophagic flux defect exhibited by the mutant strain. The protective effect of TOR1 deletion, as opposed to that of SCH9 deletion, is not associated with the attenuation of Hog1p hyperphosphorylation, which was previously implicated in isc1Δ phenotypes. Our data support a model in which Isc1p regulates mitochondrial function and chronological lifespan in yeast through the TORC1-Sch9p pathway although Isc1p and TORC1 also seem to act through independent pathways, as isc1Δtor1Δ phenotypes are intermediate to those displayed by isc1Δ and tor1Δ cells. We also provide evidence that TORC1 downstream effectors, the type 2A protein phosphatase Sit4p and the AGC protein kinase Sch9p, integrate nutrient and stress signals from TORC1 with ceramide signaling derived from Isc1p to regulate mitochondrial function and lifespan in yeast. Overall, our results show that TORC1-Sch9p axis is deregulated in Isc1p-deficient cells, contributing to mitochondrial dysfunction, enhanced oxidative stress sensitivity and premature aging of isc1Δ cells.Microbial Cell. 01/2014; 1(1):21-36.
Hindawi Publishing Corporation
Oxidative Medicine and Cellular Longevity
Volume 2013, Article ID 636287, 13 pages
Maintenance of Mitochondrial Morphology by Autophagy
and Its Role in High Glucose Effects on Chronological Lifespan
of Saccharomyces cerevisiae
May T. Aung-Htut,1Yuen T. Lam,1Yu-Leng Lim,1Mark Rinnerthaler,2Cristy L. Gelling,1
Hongyuan Yang,1Michael Breitenbach,2and Ian W. Dawes1
1School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia
2Department of Genetics, University of Salzburg, Hellbrunnerstrare 34, 5020 Salzburg, Austria
Correspondence should be addressed to Ian W. Dawes; email@example.com
Received 13 May 2013; Accepted 21 June 2013
Academic Editor: Joris Winderickx
Copyright © 2013 May T. Aung-Htut et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
In Saccharomyces cerevisiae, mitochondrial morphology changes when cells are shifted between nonfermentative and fermentative
carbon sources. Here, we show that cells of S. cerevisiae grown in different glucose concentrations display different mitochondrial
cells. However, the mitochondria of cells growing in higher glucose concentrations (2% and 4%) became fragmented after growth
in these media, due to the production of acetic acid; however, the fragmentation was not due to intracellular acidification. From a
screen of mutants involved in sensing and utilizing nutrients, cells lacking TOR1 had reduced mitochondrial fragmentation, and
autophagy was found to be essential for this reduction. Mitochondrial fragmentation in cells grown in high glucose was reversible
by transferring them into conditioned medium from a culture grown on 0.5% glucose. Similarly, the chronological lifespan of cells
grown in high glucose medium was reduced, and this phenotype could be reversed when cells were transferred to low glucose
conditioned medium. These data indicate that chronological lifespan seems correlated with mitochondrial morphology of yeast
cells and that both phenotypes can be influenced by factors from conditioned medium of cultures grown in low glucose medium.
Mitochondria are important organelles whose primary func-
tion is to synthesize ATP, but they also play important roles
in many cellular processes including apoptosis and aging [1–
4]. Due to their dynamic nature, the number and shape of
mitochondria in a cell are variable depending on the growth
conditions of the cell [5–7].
In Saccharomyces cerevisiae, the morphology of mito-
chondria is under the influence of the availability of oxygen
and the nature of the carbon source for growth. Under
anaerobic conditions, very small mitochondria known as
promitochondria are observed. These are devoid of respi-
ratory pigments and import ATP to perform the remain-
ing essential metabolic functions . On the other hand,
enlarged tubular structures are found in aerobically grown
cells . S. cerevisiae cells respire in the absence of glucose,
and these cells have a similar mitochondrial morphology
to those observed in stationary phase cells where many
small, round mitochondria are the dominant form [6, 10].
High glucose concentrations promote calcium and mitogen
protein kinase-mediated activation of mitochondrial fission
and stimulate reactive oxygen species production .
Restriction of glucose intake extends the cellular lifespan
in a manner similar to caloric restriction [12, 13]. Alterna-
extends both replicative and chronological lifespan (CLS)
in S. cerevisiae [14, 15]. One of the downstream processes
under regulation by the TOR pathway is autophagy, which is
Autophagy is conserved in all eukaryotic cells [17, 18] and it
2Oxidative Medicine and Cellular Longevity
is important during starvation because it not only removes
damaged organelles, but it also provides nutrients by recy-
cling cellular constituents [19–21]. There is also increasing
in Caenorhabditis elegans, Drosophila melanogaster, and S.
cerevisiae [22–24], especially during caloric restriction .
In neonatal rat ventricular myocytes, a high glucose con-
centration induced cell death via mitochondrial fragmenta-
tion possibly due to increased production of reactive oxygen
species (ROS) . Despite this interest in ageing, nutrients,
and mitochondrial morphology, it remains to be determined
whether there is any correlation between mitochondrial
morphology and chronological ageing in S. cerevisiae over a
wide range of glucose concentrations. We, therefore, inves-
tigated the mitochondrial morphology of yeast cells grown
in different concentrations of glucose and sought to identify
functions that are important in maintaining mitochondrial
structure at elevated levels of glucose.
2. Materials and Methods
visiae strains used were derived from BY4743 (MATa/MAT훼
2.1. Yeast Strains, Media, and Growth Conditions. All S. cere-
ura3Δ0/ura3Δ0). Yeast strains were grown aerobically at
yeast nitrogen base without amino acids and ammonium
sulfate, 0.5% ammonium sulfate and 0.79g/L amino acids
mixture) supplemented with the indicated concentration
of carbon source. The concentrations of amino acids used
were according to . For visualization of mitochondria,
strains were transformed with the plasmid pUC35-ACO1-
GFP and pUC35-CIT1-Dsred (gift from Professor Trevor.
Lithgow, Monash University, Melbourne, VIC, Australia).
Yeast strains harboring the plasmid pUC35-ACO1-GFP
were grown in SC medium lacking uracil. For antibiotic
selection, nourseothricin (ClonNAT, Werner BioAgents) or
hygromycin B (Sigma-Aldrich) were added to a final con-
centration of 100mg/L and 300mg/L, respectively. Starter
cultures were prepared by inoculating a single colony into
starter culture was then diluted to OD6000.1–0.15 in 2mL
nation or 10mL in a 50mL tube for CLS and incubated at
examination and 100휇L for serial dilution and spotting on a
Conditioned medium was prepared by growing the cells in
SC medium containing different concentrations of glucose
for 48h and collecting the supernatant by centrifuging at
his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 met15Δ0/MET15 LYS2/lys2Δ0
cose) or in synthetic complete medium SC (0.17% Difco
30∘C in YPD (1% yeast extract, 2% peptone, and 2% glu-
1.5mL SC medium and incubating overnight at 30∘C. The
fresh SC medium in a 10mL tube for microscopic exami-
30∘C with shaking throughout the experiment. At the indi-
1800×g for 5min. For media exchange experiments, the cells
morphology of mitochondria was observed 2h after media
cated intervals, 20휇L culture was removed for microscopic
YPD plate. Five 휇L of undiluted culture and of each dilution
were grown for 6h before pelleting and resuspending in the
indicated conditioned media unless otherwise stated. The
2.2. Measurement of Oxygen and Glucose Consumption. The
rate of oxygen consumption was monitored using a standard
3mL Clark-type oxygen electrode. The system was con-
nected to a PowerLab data acquisition and analysis system
was transferred to the chamber maintained at 30∘C with
constant stirring, and oxygen content was monitored for at
least 10min. For glucose consumption, the concentration of
after inoculation using an automated glucose analyzer (YSI
2300 STAT Plus Glucose & Lactate Analyzer).
using an Olympus BX60 fluorescence microscope at 100×
were acquired using IP lab software, and Adobe Photoshop
was used to adjust the image size and the brightness and
chondrial fragmentation within a population, the cells were
grown for three days. An aliquot of sample was examined
at indicated time points. The percentage of cells showing
no visible tubular mitochondrial structure was determined
by direct microscopic examination. For each assay at least
350 cells were counted, and the data are the mean of three
2.3. Microscopy. Mitochondrial morphology was observed
magnification. An aliquot 5–10휇L of sample was taken at
2.4. Gas Chromatography-Mass Spectrometry (GC-MS) Anal-
ysis. The GC-MS analysis was carried out using the Thermo
Scientific DSQ II Single Quadrupole GC/MS at the Bio-
analytical Mass Spectrometry Facility (BMSF), University
of New South Wales. The samples were analyzed by GC-
MS with the split injection mode and split ratio of 1:10.
Carrier gas was helium at a constant flow rate of 1.5mL/min.
temperature was held at 70∘C for 1min and then ramped
electron ionization and the mass spectra recorded within 41–
The inlet temperature was maintained at 240∘C. GC oven
to 140∘C at 15∘C/min where it was held for a further min.
2.5. Measurement of Intracellular pH. Intracellular pH was
determined by the method of Brett et al. . Strain BY4743
was transformed with pCB901YpHc containing the pHluorin
gene (gift from Professor Rajini Rao, Johns Hopkins Uni-
versity School of Medicine, Baltimore) and pUG35 (non-
pH sensitive GFP). The cells were grown in different glucose
signals were collected at two different channels: Alexa Fluor
488 (488nm) and AmCyan (405nm). A calibration curve
of the ratio of fluorescent intensities of 405/488nm versus
pH was obtained as follows. Sample (50휇L) was diluted in
1mL of medium containing 50mM MES, 50mM HEPES,
50mM KCl, 50mM NaCl, 0.2M ammonium acetate, 10mM
NaN3, 10mM 2-deoxyglucose, 75휇M monensin, and 10휇M
nigericin, titrated to eight different pH values within the
Oxidative Medicine and Cellular Longevity3
range of 5.0–8.0. The background was subtracted using the
cells with pUG35, and the value of 405/488 was calculated
using FlowJo software for each individual pH. The intracel-
lular pH of the cells growing in different concentrations of
glucose was estimated by comparing the ratio of fluorescent
intensities of 405/488nm obtained for the cells with the
2.6. Measurement of Intracellular Acetate. The intracellular
acetate concentration was measured for cells of the wild
type and the Δatg1 mutant grown in different concentrations
of glucose for 24 hours using the commercial kit from R-
Biopharm (Cat. no. 10148261035) according to the manufac-
2.7. Dihydroethidium (DHE) Staining and FACS Analysis.
Staining with DHE (Molecular Probes) was performed as
described by . Briefly, cells (500휇L) were stained with
20,000–30,000 cells per sample. All analyses were performed
5휇g/mL final concentration of DHE for 10min and analyzed
3.1. Mitochondrial Morphology Changes in Response to Differ-
ent Glucose Concentrations. In order to determine whether
increasing glucose concentration also has an effect on mito-
pan in S. cerevisiae, we monitored the changes in mitochon-
drial morphology in S. cerevisiae cells growing in calorie-
restricted (0.5%) and high glucose conditions (2% and 4%).
S. cerevisiae cells were transformed with an aconitase-GFP
has been verified in .
To ensure that mitochondrial morphology was examined
at a similar growth phase, the growth of wild-type cells
expressing ACO1-GFP in the different levels of glucose was
monitored (Figure 1). A similar growth rate was observed for
all glucose conditions and cells reached stationary phase at
a comparable time. The final yields of these three levels of
glucose culture were also similar.
Having determined the growth states of the cultures
in the three glucose conditions, changes of mitochondrial
morphology were examined. Cells were grown in synthetic
medium (SC) containing 0.5%, 2%, or 4% glucose, and after
17h growth, as cells entered the diauxic shift, mitochondrial
morphology was examined using fluorescence microscopy.
Remarkable differences in mitochondrial morphology were
observed in response to changing glucose concentrations
(Figure 2(a)). Under the standard laboratory condition with
2% glucose as a carbon source, mitochondria appeared as
elongated tubular structures. However, in media containing
0.5% glucose, mitochondria displayed a highly branched,
short-rodmorphology similar to that observed in cells grow-
ing by respiration in ethanol medium . In the highest level
thread structure with very few connections and branches.
012 24364860 72
Absorbance at 600nm
Figure 1: Growth of BY4743 overexpressing ACO1-GFP plasmid in
SC medium supplemented with different concentrations of glucose.
An overnight culture of cells in SC medium was diluted to an initial
absorbance at 600nm of 0.1 in fresh SC medium containing the
glucose concentration indicated and the cultures incubated at 30∘C
are from a single experiment.
Observations using a CIT1-DsRed construct instead of the
ACO1-GFP construct also produced the same result, indicat-
fusion (data not shown).
The difference in mitochondrial morphologies between
cells grown in 2% glucose and 4% glucose was independent
of osmotic stress, since addition of an equivalent molar
the mitochondrial appearance (data not shown).
Having observed the characteristic mitochondrial mor-
phology associated with glucose levels, we monitored the
change of mitochondrial morphology in cells grown in
0.5%, 2% or 4% glucose media for 24, 48 and 72 hours
(Figure 2(a)). Furthermore, to assess the structural changes
of mitochondria, the percentage of cells in the population
displaying total mitochondrial fragmentation, in which only
punctate mitochondria with complete absence of tubular
mitochondria within an individual cell, was determined
Cells grown in 2% or 4% glucose displayed an increased
heterogeneity in mitochondrial morphology with time,
showing a progression towards punctate fragmented struc-
tures over 72 hours (Figure 2(a)). After 24 hours of growth,
the culture grown in 4% glucose had the highest number of
cells with totally fragmented mitochondria (10%) followed
by those grown in 2% glucose (7%) and 0.5% glucose (2%)
4Oxidative Medicine and Cellular Longevity
Rate of oxygen consumption (nmol/min/OD)
0 1224 3648 60 72
Figure 2: Mitochondrial morphologies of S. cerevisiae grown in different concentrations of glucose. (a) BY4743 wild-type cells transformed
with an ACO1-GFP fusion construct were grown for 72 hours in three different concentrations of glucose (0.5%, 2%, and 4%), and the
morphology of mitochondria was observed at the indicated times using a fluorescent microscope. The micrographs shown are representative
and at 12 hours intervals throughout the 72 hours time course. Data are the mean of three separate cultures in parallel. Error bars indicate the
Oxidative Medicine and Cellular Longevity5
Table 1: The percentage of wild-type and mutant cells showing completely fragmented mitochondrial morphology in cultures incubated in
three different concentrations of glucose (0.5%, 2%, and 4%) at the times indicated.
Wild type + rapamycin1
Δatg1 + rapamycin1
caused by FIS1 deficiency, but which also causes a failure to suppress cell growth during amino acid deprivation .
1Wild-type cells or mutants were grown in the presence of 10nM rapamycin.
Δuth1 + rapamycin1
∗The fis1Δ strain used in these experiments has been shown to also carry a mutation in the WHI2 gene which rescues the mitochondrial respiratory defect
Incubation time (h)
Glucose concentration (%)
Figure 3: Glucose consumption of S. cerevisiae growing in 0.5%,
2%, and 4% glucose. Glucose in the culture medium was measured
at intervals after incubation of the cells in SC medium containing
glucose at 0.5% (closed squares), 2% (closed circles), or 4% (open
circles). The measurements were performed on three separate
cultures grown in parallel, and error bars indicate the SEM.
(Table 1). The percentages of cells with totally fragmented
mitochondria grown in 4% and 2% glucose increased to 64%
and 66%, respectively, after 72 hours. However, cells under
caloric restriction showed an average of less than 3% of the
population with total mitochondrial fragmentation at that
We monitored respiratory rate under the above condi-
tions to determine whether this affected the morphology
of mitochondria. Maximal respiratory activity was observed
in cells after 12 hours of growth in 0.5% glucose medium
(Figure 2(b)). This respiration peak coincided with the
presence of highly branched mitochondrial morphology
observed in 0.5% glucose-grown cells. However, respiratory
activity in these cells decreased from 24 hours to a low
level at 72 hours, yet the highly branched mitochondrial
morphology was maintained throughout the 72 hours time
course. Therefore, a high rate of respiration was not required
to maintain the highly branched mitochondrial morphology
in these cells. On the other hand, the respiratory activity of
4% glucose-grown cells was relatively low and underwent a
gradual decrease throughout the 72h incubation. Since total
mitochondrial fragmentation was observed in 4% glucose-
grown cells as early as 24 hours of growth, decreasing the
respiratory activity could not be the cause of the onset of
mitochondrial fragmentation in the presence of high glucose
Glucose concentrations were measured in the super-
natant collected from the different media at intervals
24 hours of growth in medium originally supplemented with
0.5% and 2% glucose, while cells consumed approximately
half of the glucose in 4% glucose medium (Figure 3). These
data indicate that there was no correlation between the
concentration of glucose remaining in the medium and the
progression of mitochondrial fragmentation.
nor respiratory rate, and the rate of glucose consumption
6Oxidative Medicine and Cellular Longevity
correlated with mitochondrial fragmentation observed in
high glucose concentrations. We then further investigate the
cause of early mitochondrial fragmentation in cells grown at
high glucose concentrations (2% and 4%) by analyzing the
mitochondrial morphology of cells lacking genes involved in
maintaining mitochondrial morphology.
3.2. Progression of Mitochondrial Fragmentation in High Glu-
cose Is Independent of Mitochondrial Fission. Mitochondrial
morphology is modulated by a delicate balance between
replicative lifespan . We therefore determined whether
the mitochondrial fragmentation observed in cells grown
in a high level of glucose was regulated by factors affecting
mitochondrial fission by examining mutant strains (dnm1Δ
a mutation in the WHI2 gene which rescues the mitochon-
drial respiratory defect caused by FIS1 deficiency, which also
causes a failure to suppress cell growth during amino acid
deprivation . The mutant cells were transformed with
the ACO1-GFP construct and grown in 2% or 4% glucose
medium under the same condition described above.
The dnm1Δ mutant defective in mitochondrial fission
displayed fragmentation comparable to that of the wild type
(Table 1, see also Supplementary Figure S1 available online
at http://dx.doi.org/10.1155/2013/636287). A slight reduction
was expected to show a reduced level of mitochondrial
fragmentation ; however, when grown in 4% glucose, it
in the percentage of dnm1Δ cells that harbored fragmented
in 2% glucose. However, similarly to dnm1Δ, mitochondrial
fragmented mitochondria. These results indicated that mito-
chondrial fragmentation was unavoidable when cells were
fission. Hence, mitochondrial fragmentation observed in
Cells deleted for the mitochondrial fusion gene FZO1
lack mitochondrial DNA and had severely deformed mito-
chondria in both glucose concentrations examined, and it
was therefore difficult to determine whether there was any
involvement of mitochondrial fusion in the fragmentation of
mitochondria using this mutant.
cose. Nevertheless, mitochondrial fragmentation progressed
in the dnm1Δ strain under the high glucose conditions. Cells
lacking FIS1 also showed a reduction in the percentage of
cells containing mitochondrial fragmentation when grown
fragmentation was observed when fis1Δ cells were grown in
4% glucose, resulting in 63% of cells containing completely
3.3. Inhibition of TOR Signaling Pathway Reduces Mitochon-
drial Fragmentation. Since nutrient availability might play a
greater role than mitochondrial fission processes in modu-
lating mitochondrial fragmentation when cells were grown
at high glucose concentration, we examined mutant strains
lacking genes involved in glucose sensing (SNF3, RGT2),
glucose metabolism (HXK2, GPA2, PDE1, and PDE2), and
general nutrient sensing (TOR1). Mutant cells transformed
with the ACO1-GFP construct were grown in 2% or 4%
glucose medium as described above.
Mutants with a deletion affecting glucose sensing or
glucose metabolism showed 50% to 84% of cells with totally
fragmented mitochondria morphology after 72 hours of
growth in either 2% or 4% glucose (Table 1). Among the
mutants screened, only cells lacking the TOR1 gene showed
a substantial reduction in the percentage of cells with totally
fragmented mitochondria when grown in 2% or 4% glucose
(Table 1; Supplementary Figure S2). Mitochondrial fragmen-
of the TOR pathway, the wild-type cells were treated with
10nM rapamycin to inhibit both TOR1 and TOR2 gene
products. Cells treated with rapamycin showed an even
greater reduction in total mitochondrial fragmentation than
grown in 4% glucose and 2% glucose, respectively, after 72
hours. As an alternative approach to genetic manipulation
in the tor1Δ strain, with only 12% to 15% of the cells showing
for 72 hours in 2% or 4% glucose media containing 10nM
rapamycin, respectively (Table 1; Supplementary Figure S3).
drial fragmentation while inhibition of the TOR pathway by
rapamycin, which also inhibits TOR2, further repressed the
extent of mitochondrial fragmentation during cell growth in
high glucose levels.
totally fragmented mitochondria when the cells were grown
drial fragmentation occurred after cells had grown in media,
we tested whether cells grown in different concentrations
of glucose excreted metabolites are capable of stimulating
conditioned medium (in which cells had been grown in 0.5%
or 4% glucose for either 24 hours or 48 hours) was collected
and then used to replace the growth medium of cells grown
that caused mitochondrial fragmentation in exponential
phase cells, regardless of the glucose concentration of the
medium in which the cells were pregrown (Figure 4). Mito-
chondrial fragmentation was observed as early as 2h after
transfer into this medium. Fragmentation also occurred for
stationary phase cells pregrown in medium containing 2%
and 4% glucose. In contrast, stationary phase cells pregrown
in 0.5% glucose were resistant to mitochondrial fragmen-
tation induced by the same medium. It was hypothesized
that mitochondrial fragmentation was prevented in these
cells because nutrients became depleted, and autophagy was
activated earlier than in the other growth regimes.
In order to investigate the involvement of autophagy in
resistance to conditioned medium-induced mitochondrial
fragmentation, the autophagy mutant strains Δuth1, Δatg1,
ditioned medium (4% glucose, 48 hours). The mitochondria
and Δatg5 were grown to stationary phase in medium
containing 0.5% glucose and then transferred into the con-
Oxidative Medicine and Cellular Longevity7
Figure 4: Conditioned medium from S. cerevisiae grown in 4% glucose triggered mitochondrial fragmentation, which was delayed by
autophagy. (a) The wild-type cells pregrown to exponential phase for 6 hours in 0.5%, 2%, and 4% glucose to exponential phase were
(b) The wild-type cells grown to stationary phase for 48 hours in 0.5%, 2%, and 4% glucose, then transferred to the 4% conditioned medium
shown are representative of the populations. Note: the micrograph for the Δuth1 mutant transferred into 4% 48h represents the morphology
(4% 48h) or 0.5% conditioned medium (0.5% 48h), and mitochondrial morphology was observed. (c) Mutants affected in autophagy (Δatg1
of half the population of the cells, and the inset represents that of the other half of the population.
and Δuth1) were grown to stationary phase as under (b), and mitochondrial morphology was observed. Scale bar: 5휇m. The micrographs
of the Δatg1 and Δatg5 mutants became fragmented, but not
resistance to the metabolites that stimulated mitochondrial
fragmentation and that starvation may be able to delay mito-
chondrial fragmentation. Indeed, delayed fragmentation was
observed in the cells growing in 10-fold diluted SC medium
containing 2% glucose compared to the cells growing in
normal SC medium with 2% glucose (data not shown).
Since conditioned medium (4% glucose, 48 hours)
appeared to contain a substance that stimulated fragmen-
tation of mitochondria, it was analysed further. Treatment
with diluted spent medium did not cause mitochondrial
fragmentation in S. cerevisiae pregrown in any of the glucose
concentrations (Figure 5(a)), indicating that the effect was
probably not due to the physical disturbance of changing
the medium but due to the concentration of the glucose
metabolites present. These cells maintained tubular mito-
exchanged. In addition, vacuum evaporation of the condi-
fragmentation (Figure 5(a)) indicating that the stimulatory
substance/s were volatile. Interestingly, mitochondrial frag-
mentation stimulated by the conditioned medium was found
to be reversible once the medium was removed (Figure 5(b)).
Oxidative Medicine and Cellular Longevity
those of the mitophagy mutant Δuth1. These results indi-
cated that general autophagy was important for conferring
3.5. The Observed Mitochondrial Fragmentation Was Not due
to Intracellular Acidification. Since the metabolite(s) respon-
sible for mitochondrial fragmentation was(were) volatile,
we analysed all of the 48 hours conditioned media (0.5%
mass spectrometry. Three volatile substances with higher
concentrations in the 4% glucose-conditioned medium were
detected: acetic acid, ethanol, and 2,3-butanediol (Supple-
mentary Table S1). Of the three compounds, acetic acid was
the only one that resulted in mitochondrial fragmentation
when separately added to the cells (Supplementary Figure
Mitochondrial fragmentation triggered by acetic acid
release of protons or to accumulation of acetate. In addition
to acetic acid, benzoic acid and 2,4-dinitrophenol (2,4-DNP)
also triggered mitochondrial fragmentation (Figure S1). One
feature that is common to these three compounds is their
ability to lead to acidification within the cells, and therefore
we analyzed the intracellular concentration of acetate and
type cells growing in 2% and 4% glucose than those growing
in 0.5% (See Supplementary Table S2). The intracellular pH
of the cells grown in different concentrations of glucose was
measured using the pH-sensitive GPF probe pHluorin. No
significant correlation between intracellular pH and mito-
chondrial fragmentation was found (Supplementary Figure
S5). Although mitochondrial fragmentation was already
results indicated that intracellular acidification was unlikely
to be responsible for triggering mitochondrial fragmentation
and that acetate or some metabolite derived from it is more
likely to be responsible.
tation. One of the many cellular processes regulated by the
and organelles and makes amino acids and other essential
metabolites to the cell  available. To determine whether
autophagy plays a role in the reduction of mitochondrial
defective for initiation of autophagy (atg1Δ) was transformed
Cells deleted for ATG1 displayed higher percentages
(approximately 75% after 72 hours incubation) of mito-
chondrial fragmentation than the wild type, indicating
that autophagy acts to reduce the onset of mitochondrial
fragmentation in 2% and 4% glucose-grown cells. Since
phology under these conditions, cells lacking genes affecting
Surprisingly, deletion of UTH1 or ATG32 did not affect mito-
chondrial fragmentation compared to that in the wild-type
cells, indicating that mitochondrial-specific autophagy alone
did not substantially suppress mitochondrial fragmentation.
However, general autophagy, involving ATG1 appears to play
a vitally important role for reducing mitochondrial fragmen-
elevated the fragmentation of mitochondria seen in cells
grown on higher glucose levels.
Subsequently, we checked whether the TOR pathway
regulated the function of autophagy in reducing mitochon-
drial fragmentation. The autophagy mutants were treated
with rapamycin, and total mitochondrial fragmentation was
examined. A reduction of mitochondrial fragmentation in
with ACO1-GFP construct to examine mitochondrial frag-
mentation (Table 1).
rapamycin-treated uth1Δ was observed (Table 1), which was
suppression effect of rapamycin observed was independent
consistent with the finding that deletion of UTH1 did not
have an impact on mitochondrial fragmentation and that the
of UTH1. In the atg1Δ mutant, although treatment with
fragmentation remained much higher than in rapamycin-
treated wild-type cells. Hence, rapamycin inhibition of the
TOR pathway led to suppression of mitochondrial fragmen-
tation, but this was largely dependent on the presence of
a functional autophagy pathway. Therefore, it is likely that
autophagy functions downstream of the TOR pathway in
maintaining mitochondria in a nonfragmented state.
rapamycin reduced mitochondrial fragmentation compared
to the untreated mutant cells, the level of mitochondrial
3.7. Role of Autophagy in Mitochondrial Fragmentation
Induced by Glucose Metabolites. Having identified a cellular
what triggered mitochondrial fragmentation in these cells.
Oxidative Medicine and Cellular Longevity9
Figure 5: Reversibility of conditioned medium-induced mitochondrial fragmentation. (a) The mitochondria of the wild-type cells grown in
2% glucose did not fragment when they were transferred into diluted (4% 48h dilu) and evaporated (4% 48h evap) conditioned media (4%
glucose, 48 hours). Mitochondria were observed at 2 hours, 20 hours, and 62 hours after media exchange. Only the micrographs taken at
20 hours after medium exchange are shown. (b) Mitochondrial fragmentation of the wild-type cells grown in 2% glucose was triggered by
the 4% conditioned medium (+4% 48h). Cells were grown in the 4% conditioned medium for 24 hours before transferring into the 0.5%
conditioned medium (+0.5% 48h). The morphology of mitochondria was observed 2 hours after transfer. Scale bar: 5휇m. The micrographs
shown are representative of the populations.
Mitochondria are the major site of reactive oxygen species
(ROS) production, and an elevation of ROS could be one
of the causes of mitochondrial fragmentation. We examined
the levels of superoxide anion by DHE staining of cells
growing in 0.5%, 2%, and 4% glucose over a 72h time course
and flow cytometry analysis to determine whether elevation
in superoxide levels was correlated with the occurrence of
Cellular superoxide levels increased over time regardless
of the concentration of glucose, as shown in Figure 6. Cells
grown in 0.5% glucose had the highest superoxide level
after 24 hours growth, which is consistent with the fact that
respiratory activity was the highest for these cells at that time
had the highest level of superoxide followed by those grown
in 2% glucose and then those grown in 0.5% glucose. It is
therefore unlikely that an increase in ROS level triggered
mitochondrial fragmentation during cell growth, since the
onset of elevated levels of ROS in 0.5% glucose-grown cells
did not lead to mitochondrial fragmentation.
Since fragmentation occurred 24 hours after inoculation
in media, we analyzed whether the glucose metabolites
accumulated in the medium during growth stimulated mito-
medium (in which cells were grown for either 24 hours
or 48 hours) originating from 4% glucose or 0.5% glucose
medium was collected and then was used to replace the
or stationary phase (48 hours).
The 48 hours conditioned medium that was initially
supplemented with 4% glucose (4% conditioned medium)
contained substances that caused mitochondrial fragmenta-
tion in wild-type cells in exponential phase, regardless of
the glucose concentration of the medium in which cells
were pregrown (Figure 4(a)). For instance, mitochondrial
fragmentation was observed as soon as two hours after
transferring cells into the 4% conditioned medium. This
fragmentation was also found for stationary phase wild-type
cells pregrown in medium containing 2% or 4% glucose
in 0.5% glucose did not display fragmented mitochondria
after transfer into the 4% conditioned medium (Figure 4(b)).
We hypothesized that the early nutrient depletion in 0.5%
to induction of mitochondrial fragmentation.
In order to investigate the involvement of autophagy
in this mitochondrial fragmentation process, the atg1Δ and
tioned media (Figure 4(c)). Mitochondrial fragmentation
conditioned medium. On the other hand, uth1Δ cells pre-
uth1Δ autophagy mutants were grown for 48 hours in 0.5%,
2%, and 4% glucose and then transferred into the condi-
was observed in the atg1Δ mutant cells, including those
grown in 0.5% glucose mediumwere partiallyresistant to 4%
pregrown in 0.5% glucose, after transfer into the 4% glucose
10Oxidative Medicine and Cellular Longevity
Cells with elevated ROS (%)
Figure 6: ROS levels in S. cerevisiae at 24h, 48h, and 72h of growth. Wild-type cells grown in 0.5%, 2%, and 4% glucose were collected at
the indicated times and stained with 5휇g/mL DHE to detect superoxide radicals. Fluorescence intensities were analyzed by flow cytometry.
rectangles). Data are the averages from two independent experiments; bars indicate the range of data obtained.
(a) The clear and filled histograms represent the cells without and with DHE, respectively. (b) Percentage cells showing elevated ROS levels
at each incubation time for cells grown in 0.5% glucose (clear rectangles), 2% glucose (lighter grey rectangles), and 4% glucose (darker grey
conditioned medium-induced mitochondrial fragmentation
(approximately 50% of the total population displayed tubular
mitochondria). These results indicated that activation of
general autophagy during starvation played an important
role in conferring resistance to those metabolites present
in the conditioned medium that stimulated mitochondrial
Conversely, wild-type cells transferred into 0.5% condi-
were pregrown (Figure 4(c)). The fragmented mitochondria
in the atg1Δ, and uth1Δ mutants also returned to a tubular
The effects seen using 4% conditioned medium to stimu-
ical disturbanceof changing themedium.When conditioned
medium was removed and fresh medium was supplemented
to cells, there was no fragmentation in cells pregrown in
any of the glucose concentrations used. Interestingly, mito-
chondrial fragmentation stimulated by the 4% conditioned
medium was found to be reversible once the medium was
replaced by the 0.5% conditioned medium (Figure 5). The
reversible nature of the process indicated that the cells were
not yet committed to any deleterious effects that may result
from mitochondrial fragmentation.
structure after cells were transferred into 0.5% conditioned
medium, although these cells required a longer time for
3.8. Mitochondrial Fragmentation and Chronological Lifes-
pan. The above results demonstrated that S. cerevisiae cells
grown in high glucose concentrations not only possessed
fragmented mitochondria but also showed higher levels
of oxidative stress than those grown in calorie-restricted
conditions. It is well known that S. cerevisiae cells that are
restricted in their calorie intake have longer chronological
and replicative lifespans [36, 37], that maintenance of the
morphology of mitochondria is important for cell survival
since the mutants that preserve tubular mitochondrial struc-
ture (such as Δdnm1) live longer than the wild-type cells
of cells grown in 2% and 4% glucose would extend their
Since mitochondrial fragmentation in 4% or 2% glucose-
grown cells could be reversed in 0.5% conditioned medium
(Figure 5(b)) and vice versa (Figure 5(a)), we determined
whether chronological lifespan (CLS) could also be reversed
for 48 hours and then transferred into conditioned media as
shown in Figure 6, and their CLS were assessed.
CLS compared to those grown in higher glucose concen-
trations (Figure 7). Interestingly, their lifespan was short-
ened when these cells were transferred into 4% conditioned
medium. On the other hand, the lifespan of cells grown in
4% glucose medium was extended following their transfer
into 0.5% conditioned medium. Burtner et al.  have also
shown that the CLS was reversible by substituting spent
growth medium in a similar way. Here, we show that the CLS
of S. cerevisiae varied depending on the type of medium into
which cells were exchanged and that this correlated with the
reversible changes in mitochondrial morphology.
, and the mutants that progress early to mitochondrial
fragmentation have shorter lifespan . This led us to
Oxidative Medicine and Cellular Longevity 11
No changeNo changeNo change
0.5% 48h0.5% 48h
manipulation. Wild-type cells were grown in media containing 0.5%, 2%, and 4% glucose for 48 hours and exchanged into the conditioned
media originally supplemented with 0.5% (0.5% 48h) or 4% (4% 48h) glucose. Cell viability was assessed by spotting diluted cultures onto
YPD plates at indicated times and compared with that of the cells without any media exchange (no change).
Mitochondrial morphology is dynamic and responds to
ogy is modulated by the balance between fusion and fission
processes [5, 40, 41]. However, factors such as apoptotic
signals or oxidative stress cause an imbalance between these
two processes resulting in fragmentation of mitochondria
to a punctate morphology [6, 26, 42, 43], and this altered
morphology occurs on induction of cell death and during
ageing of cells . Here, we have shown that mitochondrial
morphology of S. cerevisiae changes depending on the con-
centration of glucose in the medium and that high glucose
availability triggers mitochondrial fragmentation in yeast.
This process is largely independent of mitochondrial fission
ent sensing mechanisms involving the TOR pathway via the
general autophagy process that it modulates. Early onset of
respiratory activity correlated with an increased level of ROS
in cells grown in low glucose levels as expected, but it is
unlikely that mitochondrial damage caused by an increasing
level of ROS is a trigger for mitochondrial fragmentation
since cells grown in low glucose were respiring, produc-
ing relatively high levels of ROS (superoxide anion) yet
maintained their mitochondrial structure during prolonged
ing the mitochondrial structure, a set of deletion mutants
was examined for the phenotype of reduced mitochondrial
fragmentation in the presence of higher concentrations of
that repressed mitochondrial fragmentation under higher
glucose levels (2% and 4%). Wild-type cells treated with
rapamycin also displayed nonfragmented mitochondria for
a prolonged period under these conditions. Together, these
results demonstrated that regulation of the TOR pathway
ture. Both caloric restriction and inhibition of TOR delayed
fragmentation in an autophagy-dependent manner since
deletion of the ATG1 gene led to an increased fragmentation
under both conditions. Interestingly, selective elimination of
damaged mitochondria by mitophagy in response to mito-
chondrial dysfunction  by mutating the UTH1 or ATG32
genes did not affect the fragmentation of mitochondria
effect of autophagy is most likely by modulation of nutrient
than mitochondria, since they are degraded only by selective
autophagy . It is possible that the early low level of
ROS observed in respiring cells in 0.5% glucose condition
may have activated autophagy to maintain mitochondrial
structure under this condition .
The importance of autophagy in cellular lifespan exten-
sion is highlighted by the demonstration of its importance in
C. elegans during dietary restriction  and that autophagy
and amino acid homeostasis are required for extended CLS
in S. cerevisiae . Autophagy is necessary for rapamycin-
induced lifespan extension . Induction of autophagy by
cells . Based on our data, the prevention or delay of the
an important role in lifespan extension.
It is interesting that both the fragmentation of mitochon-
dria and the shortening of CLS of cells grown in high glucose
condition are reversible. Fragmented mitochondria in cells
were able to return to a tubular morphology, and the cellular
lifespan was extended when high glucose medium was
replaced by the conditioned medium originating from cells
grown on low glucose. The reversibility with respect to CLS
has also been shown . For mitochondrial morphology
this change occurred within 2 hours of replacement. This
reversibility of mitochondrialfragmentationis dependent on
general autophagic processes. Further study on the composi-
tion of the conditioned media and comparison between the
one that shortens lifespan and the one that lengthens may
reveal which factor(s) present in the medium cause early cell
death. These results further point to the correlation between
mitochondrial morphology and chronological lifespan in S.
caloric restriction on cell aging.
This work was supported by grants from the Australian
Research Council. Yuen T. Lam was supported by the
Australian Postgraduate Award. The authors are grateful to
Professor Trevor Lithgow for his kind donation of pUC35-
CIT1-Dsred plasmid and thank Gabriel Perrone for the help
with flow cytometry analysis.
12Oxidative Medicine and Cellular Longevity
 G. A. Cortopassi and A. Wong, “Mitochondria in organismal
aging and degeneration,” Biochimica et Biophysica Acta, vol.
1410, no. 2, pp. 183–193, 1999.
 D. R. Green and J. C. Reed, “Mitochondria and apoptosis,”
Science, vol. 281, no. 5381, pp. 1309–1312, 1998.
in Cell Biology, vol. 17, no. 1, pp. 6–12, 2007.
 C. Q. Scheckhuber, N. Erjavec, A. Tinazli, A. Hamann, T.
results in increased life span and fitness of two fungal ageing
models,” Nature Cell Biology, vol. 9, no. 1, pp. 99–105, 2007.
 G. J. Hermann and J. M. Shaw, “Mitochondrial dynamics in
yeast,” Annual Review of Cell and Developmental Biology, vol.
14, pp. 265–303, 1998.
sectioning and computer graphics reconstruction,” Biologie
Cellulaire, vol. 28, pp. 37–56, 1977.
 J. Bereiter-Hahn and M. Voth, “Dynamics of mitochondria in
living cells: shape changes, dislocations, fusion, and fission of
mitochondria,” Microscopy Research and Technique, vol. 27, no.
3, pp. 198–219, 1994.
 H. Plattner and G. Schatz, “Promitochondria of anaerobically
grown yeast. III. Morphology,” Biochemistry, vol. 8, no. 1, pp.
 H. P. Hoffmann and C. J. Avers, “Mitochondrion of yeast:
ultrastructural evidence for one giant, branched organelle per
cell,” Science, vol. 181, no. 4101, pp. 749–751, 1973.
 W. Visser, E. A. van Spronsen, N. Nanninga, J. T. Pronk, J. G.
Kuenen, and J. P. Van Dijken, “Effects of growth conditions
on mitochondrial morphology in Saccharomyces cerevisiae,”
Antonie van Leeuwenhoek, vol. 67, no. 3, pp. 243–253, 1995.
 T. Yu, S. S. Sheu, J. L. Robotham, and Y. Yoon, “Mitochondrial
fission mediates high glucose-induced cell death through ele-
vated production of reactive oxygen species,” Cardiovascular
Research, vol. 79, no. 2, pp. 341–351, 2008.
 E. J. Masoro, “Overview of caloric restriction and ageing,”
Mechanisms of Ageing and Development, vol. 126, no. 9, pp. 913–
 J. C. Jiang, E. Jaruga, M. V. Repnevskaya, and S. M. Jazwinski,
“An intervention resembling caloric restriction prolongs life
span and retards aging in yeast,” The FASEB Journal, vol. 14, no.
14, pp. 2135–2137, 2000.
 M. Kaeberlein, R. W. Powers III, K. K. Steffen et al., “Cell
biology: regulation of yeast replicative life span by TOR and
Sch9 response to nutrients,” Science, vol. 310, no. 5751, pp. 1193–
 R. W. Powers III, M. Kaeberlein, S. D. Caldwell, B. K. Kennedy,
and S. Fields, “Extension of chronological life span in yeast by
decreased TOR pathway signaling,” Genes and Development,
vol. 20, no. 2, pp. 174–184, 2006.
 J. R. Rohde, R. Bastidas, R. Puria, and M. E. Cardenas, “Nutri-
tional control via Tor signaling in Saccharomyces cerevisiae,”
 F. Reggiori and D. J. Klionsky, “Autophagy in the eukaryotic
cell,” Eukaryotic Cell, vol. 1, no. 1, pp. 11–21, 2002.
 C. W. Wang and D. J. Klionsky, “The molecular mechanism of
 W. Droge, “Autophagy and aging-importance of amino acid
levels,” Mechanisms of Ageing and Development, vol. 125, no. 3,
pp. 161–168, 2004.
 W. A. Dunn Jr., J. M. Cregg, J. A. K. W. Kiel et al., “Pexophagy:
the selective autophagy of peroxisomes,” Autophagy, vol. 1, no.
2, pp. 75–83, 2005.
 J. J. Lemasters, “Selective mitochondrial autophagy, or
mitophagy, as a targeted defense against oxidative stress,
mitochondrial dysfunction, and aging,” Rejuvenation Research,
vol. 8, no. 1, pp. 3–5, 2005.
 G. Juhasz, “Atg7-dependent autophagy promotes neuronal
health, stress tolerance, and longevity but is dispensable for
metamorphosis in Drosophila,” Genes & Development, vol. 21,
pp. 3061–3066, 2007.
 M. L. T´ oth, T. Sigmond,´E. Borsos et al., “Longevity path-
ways converge on autophagy genes to regulate life span in
Caenorhabditis elegans,” Autophagy, vol. 4, no. 3, pp. 330–338,
 A. L. Alvers, L. K. Fishwick, M. S. Wood et al., “Autophagy
and amino acid homeostasis are required for chronological
longevity in Saccharomyces cerevisiae,” Aging Cell, vol. 8, no. 4,
pp. 353–369, 2009.
 K. Jia and B. Levine, “Autophagy is required for dietary
restriction-mediated life span extension in C. elegans,”
Autophagy, vol. 3, no. 6, pp. 597–599, 2007.
 N. Alic, V. J. Higgins, and I. W. Dawes, “Identification of a
Saccharomyces cerevisiae gene that is required for G1 arrest in
response to the lipid oxidation product linoleic acid hydroper-
 C. L. Brett, D. N. Tukaye, S. Mukherjee, and R. Rao, “The yeast
endosomal Na+(K+)/H+ exchanger Nhx1 regulates cellular pH
to control vesicle trafficking,” Molecular Biology of the Cell, vol.
16, no. 3, pp. 1396–1405, 2005.
 T. Drakulic, M. D. Temple, R. Guido et al., “Involvement
of oxidative stress response genes in redox homeostasis, the
level of reactive oxygen species, and ageing in Saccharomyces
cerevisiae,” FEMS Yeast Research, vol. 5, no. 12, pp. 1215–1228,
 H. Klinger, M. Rinnerthaler, Y. T. Lam et al., “Quantitation
of (a)symmetric inheritance of functional and of oxidatively
damaged mitochondrial aconitase in the cell division of old
yeast mother cells,” Experimental Gerontology, vol. 45, no. 7-8,
pp. 533–542, 2010.
and J. M. Hardwick, “Fis1 deficiency selects for compensatory
Cell Death and Differentiation, vol. 15, no. 12, pp. 1838–1846,
 T. Kanki, K. Wang, Y. Cao, M. Baba, and D. J. Klionsky,
mitophagy,” Developmental Cell, vol. 17, no. 1, pp. 98–109, 2009.
“Mitochondria-anchored receptor Atg32 mediates degradation
of mitochondria via selective autophagy,” Developmental Cell,
vol. 17, no. 1, pp. 87–97, 2009.
 I. Kiˇ sˇ sov´ a, M. Deffieu, S. Manon, and N. Camougrand, “Uth1p
is involved in the autophagic degradation of mitochondria,”
Oxidative Medicine and Cellular Longevity13
Journal of Biological Chemistry, vol. 279, no. 37, pp. 39068–
 N. M. Camougrand, M. Mouassite, G. M. Velours, and M.
G. Guerin, “The ‘SUN’ family: UTH1, an ageing gene, is also
involved in the regulation of mitochondria biogenesis in Sac-
charomyces cerevisiae,” Archives of Biochemistry and Biophysics,
vol. 375, no. 1, pp. 154–160, 2000.
 J. C. Jiang, E. Jaruga, M. V. Repnevskaya, and S. M. Jazwinski,
“An intervention resembling caloric restriction prolongs life
span and retards aging in yeast,” The FASEB Journal, vol. 14, no.
14, pp. 2135–2137, 2000.
 D. L. Smith Jr., J. M. McClure, M. Matecic, and J. S. Smith,
“Calorie restriction extends the chronological lifespan of Sac-
vol. 6, no. 5, pp. 649–662, 2007.
 R. Sugioka, S. Shimizu, and Y. Tsujimoto, “Fzo1, a protein
Biological Chemistry, vol. 279, no. 50, pp. 52726–52734, 2004.
 C. R. Burtner, C. J. Murakami, B. K. Kennedy, and M. Kaeber-
lein, “A molecular mechanism of chronological aging in yeast,”
Cell Cycle, vol. 8, no. 8, pp. 1256–1270, 2009.
 J. M. Shaw and J. Nunnari, “Mitochondrial dynamics and
division in budding yeast,” Trends in Cell Biology, vol. 12, no.
4, pp. 178–184, 2002.
 B. Westermann, “Mitochondrial fusion and fission in cell life
and death,” Nature Reviews Molecular Cell Biology, vol. 11, pp.
and M. Cˆ orte-Real, “Cytochrome c release and mitochondria
involvement in programmed cell death induced by acetic acid
in Saccharomyces cerevisiae,” Molecular Biology of the Cell, vol.
13, no. 8, pp. 2598–2606, 2002.
 S. Matsuyama and J. C. Reed, “Mitochondria-dependent apop-
vol. 7, no. 12, pp. 1155–1165, 2000.
 R. J. Braun and B. Westermann, “Mitochondrial dynamics in
yeast cell death and aging,” Biochemical Society Transactions,
vol. 39, no. 5, pp. 1520–1526, 2011.
no. 47, pp. 32386–32393, 2008.
 R. Scherz-Shouval, E. Shvets, E. Fass, H. Shorer, L. Gil, and Z.
specifically regulate the activity of Atg4,” The EMBO Journal,
vol. 26, no. 7, pp. 1749–1760, 2007.
 A. L. Alvers, M. S. Wood, D. Hu, A. C. Kaywell, W. A. Dunn
Jr., and J. P. Aris, “Autophagy is required for extension of yeast
chronological life span by rapamycin,” Autophagy, vol. 5, no. 6,
pp. 847–849, 2009.
 T. Eisenberg, H. Knauer, A. Schauer et al., “Induction of auto-
phagy by spermidine promotes longevity,” Nature Cell Biology,
vol. 11, no. 11, pp. 1305–1314, 2009.