MINIREVIEW
Mitochondria in ageing: there is metabolism beyond the ROS
Michael Breitenbach1, Mark Rinnerthaler1, Johannes Hartl2, Anna Stincone2, Jakob Vowinckel2,
Hannelore Breitenbach-Koller1 & Markus Ralser2,3
1
Department of Cell Biology, University of Salzburg, Salzburg, Austria; 2Department of Biochemistry and Cambridge Systems Biology Centre,
University of Cambridge, Cambridge, UK; and 3Division of Physiology and Metabolism, MRC National Institute for Medical Research, London, UK
Correspondence: Markus Ralser, Division of
Physiology and Metabolism, MRC National
Institute for Medical Research, the Ridgeway,
Mill Hill, London NW7 1AA, UK.
Tel.: +44 1223 761346;
fax: +44 1223 766002;
e-mail: [email protected]
Received 6 September 2013; revised 19
December 2013; accepted 21 December
2013. Final version published online 16
January 2014.
DOI: 10.1111/1567-1364.12134
Editor: Austen Ganley
Keywords
iron sulfur cluster; mitophagy; TCA cycle;
chronological ageing; replicative ageing;
hibernating ageing.
Abstract
Mitochondria are responsible for a series of metabolic functions. Superoxide
leakage from the respiratory chain and the resulting cascade of reactive oxygen
species-induced damage, as well as mitochondrial metabolism in programmed
cell death, have been intensively studied during ageing in single-cellular and
higher organisms. Changes in mitochondrial physiology and metabolism resulting in ROS are thus considered to be hallmarks of ageing. In this review, we
address ‘other’ metabolic activities of mitochondria, carbon metabolism (the
TCA cycle and related underground metabolism), the synthesis of Fe/S clusters
and the metabolic consequences of mitophagy. These important mitochondrial
activities are hitherto less well-studied in the context of cellular and organismic
ageing. In budding yeast, they strongly influence replicative, chronological and
hibernating lifespan, connecting the diverse ageing phenotypes studied in this
single-cellular model organism. Moreover, there is evidence that similar processes equally contribute to ageing of higher organisms as well. In this scenario,
increasing loss of metabolic integrity would be one driving force that contributes to the ageing process. Understanding mitochondrial metabolism may thus
be required for achieving a unifying theory of eukaryotic ageing.
YEAST RESEARCH
Introduction
It has been speculated since the 1950s that mitochondria
execute a central role in the ageing process, yet only now
is this field developing rapidly with many important
recent discoveries. The ‘prehistory’ of ‘the mitochondrial
theory of ageing’ (here called mTOA for shortness) has
been pioneered by the ‘oxygen free radical TOA’ and the
‘somatic mutation TOA’ and therefore, indirectly, by
Medawar (Medawar, 1952), Gerschmann (Gerschman
et al., 1954), Harman (Harman, 1956), Szilard (Szilard,
1959), and Orgel (Orgel, 1963). However, mitochondria
as a source of both reactive oxygen species (ROS; Harman, 1972) and antioxidant processes, exemplified by the
biological role of superoxide dismutase, (McCord &
Fridovich, 1969) were recognized much later. These
canonical functions of mitochondria in ageing have been
the subject of comprehensive timely reviews (Barros et al.,
2010; Ugidos et al., 2010; Pan, 2011; Schleit et al., 2013)
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Published by John Wiley & Sons Ltd. All rights reserved
including two book chapters in a volume dedicated to the
yeast ageing processes (Breitenbach et al., 2012; Longo &
Fabrizio, 2012). Some of the seemingly disparate thoughts
about the causes of ageing can now be integrated under a
still preliminary but increasingly unifying hypothesis, in
which mitochondrial physiology and the ageing-related
metabolic changes are correlated with gene mutations that
target proteins that are active in stress response and
confer lifespan extension.
The last two decades have seen radical changes in the
field of ageing and stress response. These include the elucidation of a large number of biochemical pathways for
oxidative stress defence, which are in many cases specific
for certain kinds of damage and particular subcellular
compartments (Thorpe et al., 2004; Aung-Htut et al.,
2012), and the role of ROS (superoxide and hydrogen peroxide) as signalling substances for growth and cell differentiation in situations where no particular oxidative stress
occurred in the cells. Further, the role of mitochondria in
FEMS Yeast Res 14 (2014) 198–212
Mitochondrial metabolism in ageing cells
programmed cell death and the ability of mitochondria to
adjust to different physiological situations have been
explored with respect to morphology, gene expression,
protein traffic and cross talk with the nucleo/cytoplasmic
system. Moreover, mitochondrial segregation during cell
division, including rejuvenation, fission, fusion and mitophagy has been studied and found to change with ageing
(Barros et al., 2010; Ugidos et al., 2010; Pan, 2011; Breitenbach et al., 2012; Mao & Klionsky, 2013).
However, mitochondria have, from a physiological perspective, major additional roles in cellular metabolism.
They host the respiratory chain, the TCA cycle and the
compartment of iron/sulphur cluster assembly in nonplant eukaryotes. Furthermore, mitochondria play a crucial role in the synthesis of lipids and membrane
compounds such as ceramides (Aerts et al., 2008; Salminen et al., 2012). Ageing is a consequence of metabolic
activities. During the last few years, a remarkable renaissance of metabolism research has been initiated, based on
powerful new analytical and mathematical tools. It was
demonstrated that metabolic adaptation – and this also
applies to ageing – is faster and more dynamic at the
enzyme and small metabolite level compared with the
well-known transcriptional level, as rapid responses are
necessary for survival when environmental situations
change (Ralser et al., 2009; Gruning et al., 2010; Buescher
et al., 2012). Furthermore, it has become clear that metabolic fluxes are to a large extent regulated on a posttranslational and metabolic level, and hence, metabolic
activities can only be extracted from transcriptome and
proteome studies if combined with metabolite quantification and/or flux analysis (Bakker et al., 1999; Buescher
et al., 2012).
Mitochondrial metabolism during yeast
ageing
Two ageing models are intensively studied in the yeast,
S. cerevisiae: chronological and replicative ageing. The
study of both have led to the discovery of ageing factors
(i.e. TOR, SCH9/AMPK, SIR2) that are now of prime
importance in plants, insects and mammals (Blagosklonny, 2008; Steinberg & Kemp, 2009; Ralser et al.,
2012). However, these ageing measures are physiologically
different processes, especially with regards to metabolism.
Chronological ageing is another term for survival of the
stationary phase after nutrients, or space, become limiting
and cells no longer divide (Fabrizio & Longo, 2003; Longo & Fabrizio, 2012). The metabolic requirements necessary for this ‘silent’ metabolism are very different from
those of growing cells. Survival of the stationary phase
has been called a model for the survival of postmitotic
cells in the human body, like for instance, neurons in the
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199
central nervous system. However, at least a percentage of
human postmitotic cells are metabolically very active and
are in that respect not similar to stationary yeast cells. In
addition, a stationary culture is not 100% silent. Occasionally, cells divide, and there are phases when subpopulations show proliferation, known as adaptive re-growth
(Longo & Fabrizio, 2012).
A special case of chronological lifespan represents longtime survival under conditions with very low metabolic
activity, as found at low temperature. Mutant colonies of
the laboratory yeast strain BY4741 (an S288c descendant)
that show a prolonged hibernating lifespan can survive
several years at 4 °C. Again, the nature of the gene deletions identified indicates that their contribution to prolonged survival is facilitated by altered metabolism,
including mitochondrial metabolism and respiration
(Postma et al., 2009).
In contrast, eukaryotic and prokaryotic cells can even
in the continuous presence of a nutrient supply undergo
only a limited number of cell cycles. In yeast, this phenomenon is used as a measure for lifespan, termed
mother cell-specific or replicative ageing (for review: Breitenbach et al., 2004). The old mother cells start to display
irregular cell cycles after about 25 cell generations (median value for many laboratory strains). Then, they lose
checkpoint control and start cell cycles before the previous cell cycle has been completed (Nestelbacher et al.,
1999), leading to genomic instability (Veatch et al.,
2009). Replicatively aged mother cells are very large
(compared with their newborn daughters) and finally
undergo apoptosis (Laun et al., 2001). Comparing about
500 yeast deletion mutations, it turns out that there is
relatively little concordance between the replicative, chronological and hibernating lifespans (Laun et al., 2006;
Postma et al., 2009), indicating that they represent
distinct facets of the ageing process.
Evidence for a decline in mitochondrial
integrity during ageing
In the search for a common feature of the different
ageing processes, it was found that apoptosis, internal
oxidative stress and characteristic changes of mitochondrial morphology are common to old mother cells and
chronologically aged cells. In particular, the familiar
mitochondrial network of growing yeast cells transforms
to many small roundish mitochondria that are stained
intensively with dihydrorhodamine or dihydroethidium
(Breitenbach et al., 2012), indicating the presence of
ROS. This phenotype resembles small (or fragmented),
globose (roundish) mitochondria of cells challenged by
H2O2. The typical mitochondrial network of a normal
yeast cell, a cell exposed to hydrogen peroxide, and one
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M. Breitenbach et al.
(a)
(b)
Fig. 1. The mitochondrial network disassembles during ageing and oxidative stress. (a) Mitochondrial morphology in a young, aged and H2O2exposed yeast cell as obtained by super-resolution fluorescence microscopy. Wild-type yeast (YSBN11) was transformed with an mtGFP
expression plasmid (Westermann & Neupert, 2000). Cells were harvested, fixed with para-formaldehyde and mounted in Vectashield on glass
cover slips. Images were acquired using an OMX microscope (General Electric), equipped with a 488 nm laser and a 6091.4 NA oil objective in
structured illumination mode with z-sections of 125 nm spacing. Images were processed using SoftWorx (General Electric) and 3D images were
reconstructed using Volocity (Perkin Elmer). Upper panel: Exponentially growing (young) cells in G2 in synthetic complete media with 2% glucose
as carbon source. Note the elaborate mitochondrial network. Middle panel: Exponentially growing (young) cells were treated with 0.5 mM H2O2
in water for 45 min. Mitochondria are now small and roundish. Lower panel: A representative aged cell from a culture grown to stationary
phase is depicted. Note the fragmentation of the mitochondrial network. (b) Aconitase localization in a young and replicatively old yeast cell.
Wild-type yeast was transformed with an inducible expression construct encoding aconitase (ACO1-GFP) and analysed by fluorescence
microscopy (Zeiss Axioscope) and DIC (adapted from Klinger et al., 2010). Upper panel: Exponentially growing (young) yeast cells in G2 phase,
upon growth in synthetic complete media with 2% glucose as carbon source. Note the elaborate mitochondrial network. Length bar: 5 lm,
Lower panel: An old mother cell isolated as fraction V by elutriation centrifugation (Jarolim et al., 2004). Note that the old mother cell is larger
than a young cell and contains fragmented roundish mitochondria. Length bar: 5 lm [pictures in Fig. 1b are reproduced with permission from
the authors (Klinger et al., 2010)].
representative aged cell is illustrated, picturing a mitochondria-localized GFP (Westermann & Neupert, 2000)
by super-resolution microscopy (Fig. 1). The mitochondrial network of these cells is compared with the localisation of aconitase-GFP fusion protein (Aco1-GFP) within
a representative cell of a young and a replicatively aged
yeast population, as separated by elutriation centrifugation [reproduced from (Klinger et al., 2010) Fig. 1b]. The
mitochondrial ageing phenotypes observed in yeast display a strong similarity to aged mitochondria of higher
cells, where mitochondria change their physiology, separate into single globose units and show an overwhelming
signature of oxidative stress (Shigenaga et al., 1994).
Fragmentation of the mitochondrial network by treatment
of yeast cells with 0.5 mM H2O2 is reversible. The yeast
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Published by John Wiley & Sons Ltd. All rights reserved
cells reform the normal network after the end of oxidative stress and continue growth. This is in contrast to the
overall globular pattern of fragmented mitochondria in a
terminally old cell, where the globular pattern is locked
in, as these cells are unable to dynamically restore the
mitochondrial network (Fig. 1a and b).
During ageing in vivo and in vitro, physiological
changes resulting from oxidative insults are found in the
mitochondria of higher cells as well. Mitochondrial mass
in ageing rat brain and liver is severely decreased, as is
the activity of mitochondrial enzymes, including respiratory complexes (Navarro & Boveris, 2004). In addition, a
deficiency of mitophagy has been described (Hubbard
et al., 2012), contributing to overall defects in the decomposition of damaged macromolecules or organelles.
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201
Mitochondrial metabolism in ageing cells
Correlated with these impairments of mitochondrial
structure and function, mutations in the mitochondrial
DNA clearly increase in old age. This has been studied
extensively in vivo in a mouse model that expresses a
mitochondrial DNA polymerase with no active proofreading function (Polg mutant mice; Trifunovic et al., 2004;
Kujoth et al., 2005), with new results recently summarized by (Bratic & Larsson, 2013). Polg mutant mice show
increased mutation frequency in ageing, and the homozygous animals display a very impressive spectrum of premature ageing phenotypes (Trifunovic et al., 2004; Kujoth
et al., 2005). However, the nature of the main cause of
these phenotypes is questioned by the analysis of heterozygous animals which display a mutation frequency that
is orders of magnitude higher than the mitochondrial
mutation frequency in aged wild-type mice, yet they do
not show signs of premature ageing (Khrapko & Vijg,
2007). Logic thus dictates that the accumulation of mitochondrial mutations during life can be excluded as a
main cause of the ageing process of this mammal. Moreover, the second assumption that gene loss caused by
deletions in mitochondrial DNA are causative for ageing
(Vermulst et al., 2008) remains doubtful as well, as the
‘deleter’ mouse expressing a human dominant mutant
form of the mitochondrial DNA helicase twinkle acquires
mitochondrial DNA deletions similar in number to the
ones found in very old wild-type mice. The mutant mice
show a progressive defect in mitochondrial respiration
like the human patients from which the mutant form of
twinkle was isolated. However, they show no features of a
premature ageing syndrome (Tyynismaa et al., 2005; Park
& Larsson, 2011). Although other, yet unproven interpretations have been put forward (Ahlqvist et al., 2012; Bratic
& Larsson, 2013), this controversy is still unresolved.
The debate about the correct interpretation of this animal model exemplifies a common problem in contemporary ageing research. It is relatively simple to find
correlations of certain biochemical markers with age and
lifespan, but extremely difficult to obtain proof for causal
relationships. One candidate for the causal relationships
of mutations inducing mitochondrial dysfunction and
ageing are uncharacterized enzymatic functions that in
their normal form serve to buffer cellular stress.
Additional support for this ‘buffering’ hypothesis
comes from studies of mitochondrial and nuclear mutations that occur in aged wild-type yeast cells. The main
information available on this comes from Gottschling and
colleagues (McMurray & Gottschling, 2003; Veatch et al.,
2009; Lindstrom et al., 2011). Their results reveal that
mothers throw off respiratory deficient (‘petite’) mutant
daughters. The reason why this effect is seen only at an
advanced age is the heteroplasmic (the presence of a
mixture of more than one type of an organellar genome)
FEMS Yeast Res 14 (2014) 198–212
nature of the mitochondrial mutations, meaning that one
cell harbours both wild-type and mutant mitochondrial
genomes. These must become homoplasmic (when all
mitochondrial genomes in a cell contain the same allele)
through mitotic segregation. In the homoplasmic cells,
only one type of mitochondrial (in this case mutant) genome is present, leading to a phenotype characteristic for a
recessive mitochondrial mutation. At the replicative age
where this becomes relevant, one can assume that the
quality control process of mitophagy is already compromised. Furthermore, mother cells possess an increased
frequency of gene conversion at the MET15 locus which
is heterozygous in the genetic system BY4743 used to test
this hypothesis (McMurray & Gottschling, 2003).
Unspecific biochemical reactivity and
metabolite repair
Besides damage to proteins and nucleic acids, a third
cause of the loss in mitochondrial integrity could be the
accumulation of toxic metabolic intermediates. Several
metabolites are prone to oxidation, and other unwanted
chemical entities emerge from the side reactions of ‘promiscuous’ enzymes or by spontaneous chemical reactions
(Linster et al., 2013). For frequent metabolic errors, evolution invented specific repair strategies. For instance, the
accumulation of D-2-hydroxyglutarate, a side product of
isocitrate dehydrogenase in the reaction to a-ketoglutarate, is prevented by an enzyme termed D-2-hydroxyglutarate dehydrogenase (Struys et al., 2005). A second
example is cis-aconitate, which spontaneously interconverts into the more stable trans-aconitate and inhibits
aconitase in the mitochondrial TCA cycle. The inhibition
of aconitase, however, is prevented by a trans-aconitate
methyltransferase, Tmt1 that converts trans-aconitate to a
monomethylester (Cai & Clarke, 1999; Cai et al., 2001). It
is less understood how cells deal with rare chemical modifications (Linster et al., 2013; Van Schaftingen et al.,
2013). Intuitively, these metabolites lacking a specific
clearance mechanism would accumulate over time and
interfere with normal metabolic reactions. The contribution of this underground metabolism to the ageing process
is without question very interesting, but only barely
understood at present.
Biosynthesis, degradation and function
of Fe/S proteins as determinants of
ageing
In nonplant eukaryotes, mitochondria are the only compartment where iron/sulphur clusters are assembled. These
clusters retain iron in its reduced form, and function
as cofactors in enzymatic redox reactions. Iron/sulphur
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202
proteins are of central importance for many biological
processes; most prominent are the redox reactions of the
mitochondrial electron transport chain. Because of the
toxicity of free ferrous ions as well as sulphide, the synthesis of Fe2-S2 as well as Fe4-S4 clusters is highly regulated.
In bacteria, three different and partially overlapping biosynthetic pathways for Fe/S clusters have been found
[reviewed in (Fontecave & Ollagnier-de-Choudens, 2008)].
The ‘nitrogen fixation pathway’ occurs in bacteria and Archaea; the ‘sulphur mobilization pathway’ in bacteria and
the chloroplasts of green plants, and the third pathway,
‘ISC (iron sulphur cluster) pathway’ occurs in bacteria
and mitochondria (Rawat & Stemmler, 2011), and is best
described in S. cerevisiae.
Iron/sulphur clusters prevent the presence of soluble,
reduced iron within the cell, necessitating a complex
transport pathway. In a first step, Fe2+ is transported
across the inner mitochondrial membrane, and then, the
ferrous iron is bound and oxidized by the iron chaperone, frataxin [Yfh1; reviewed in (Lill, 2009; Philpott,
2012)]. Before the iron/sulphur cluster is assembled at the
scaffold proteins Isu1/Isu2, these proteins have to be sulphurylated. The cysteine desulfurase Nfs1 and its activator
Isd11 form a persulphide at Nfs1 by abstracting sulphur
from the amino acid cysteine that is converted to an alanine residue and afterwards this –SSH group is transferred to Isu1. The Fe/S cluster assembly at Isu1 is strictly
dependent on reduction of the persulphide sulphur to
sulphide and the electron is transferred from either
NADH or NADPH to Isu via the ferredoxin reductase
Arh1 and the ferredoxin Yah1 (Lill, 2009; Philpott, 2012).
The resulting previously so-called ‘inorganic’ sulphide
ions of the clusters (four in the Fe4-S4 and two in the
Fe2-S2 clusters) can be liberated as toxic H2S molecules
when the clusters are destroyed. For instance, the Fe4-S4
cluster of aconitase is extremely sensitive and is easily
destroyed by molecular oxygen (Gardner, 2002).
Replicative ageing is accompanied with a gradual
decline of the inner mitochondrial membrane potential,
finally leading to dysfunctional mitochondria (Lai et al.,
2002). As mitochondrial iron uptake via Mrs3/Mrs4 is
strictly dependent on the mitochondrial membrane potential (Muhlenhoff et al., 2003), Fe/S cluster synthesis will
be deeply affected in old yeast cells. In fact, it was
demonstrated that loss or damage of mitochondrial DNA
is associated with a reduced membrane potential and a
reduced Fe/S cluster biosynthesis. This in turn has a direct
effect on the nuclear genome by promoting its instability
and loss of heterozygosity (Veatch et al., 2009), due to a
lack of iron sulphur cluster proteins, including DNA polymerase subunits, that are required for DNA replication.
The increase in mutations such as base substitutions,
deletions, insertions and chromosomal rearrangements
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M. Breitenbach et al.
observed in chronologically aged yeast cells is thus indirectly related to a decline in mitochondrial membrane
potential (Wei et al., 2011).
This effect may be amplified by the Fe/S cluster
dependency of several repair enzymes such as the DNA
N-glycosylase (Ntg2) that is involved in the base excision
repair (Meadows et al., 2003); Rad3, a Fe/S cluster containing 5′ to 3′ DNA helicase, that is participating in the
nucleotide excision repair as well as in the repair of double-strand breaks, needs for the fulfilment of its function
among others two Fe/S cluster containing proteins: a
DNA primase Pri2 (Sung et al., 1987; Holmes & Haber,
1999) and Pol3, the catalytic subunit of the DNA polymerase delta (Chanet & Heude, 2003). Consistently, nonlethal single gene deletions of the aforementioned nuclear
Fe/S proteins leads to dramatic decreases in chronological
lifespan (Powers et al., 2006; Fabrizio et al., 2010). The
Fe/S cluster containing helicase/nuclease Dna2 belongs to
the family of RecQ helicases (Hoopes et al., 2002) that
includes the yeast Sgs1 helicase (Gangloff et al., 1994). A
well-known representative of this family is the human
helicase WRN, responsible for the autosomal recessive
premature ageing disease, Werner’s syndrome (Gray
et al., 1997). Similar to this observation, nonlethal dna2
mutations lead to enlarged and fragmented nucleoli and a
dramatic reduction (by 86%) in replicative lifespan
(Hoopes et al., 2002).
Fe/S clusters and reactive oxygen species
Besides their important biochemical role, Fe/S clusters are
– in concert with the mitochondrial electron transport
chain – a source of reactive oxygen species. Superoxide
produced by the respiratory chain can, if accumulated
above normal cellular levels, damage redox sensitive proteins, such as the TCA enzyme aconitase, Aco1, by oxidizing its Fe4-S4 cluster, with the consequence that Fe2+ is
released (Gardner, 2002). This release of Fe2+ is an additional source of ROS by producing hydroxyl radicals via
the Fenton reaction (Liochev & Fridovich, 1994; Longo
et al., 1999; Cantu et al., 2009). Eukaryotic cells have
developed a sophisticated but not fully understood mechanism to cope with this problem. Part of the solution
appears to be through asymmetric segregation. For
instance, the active enzyme Aco1 is primarily passed on
to yeast daughter cells, whereas the damaged, inactive
protein is retained in the mother cells (Klinger et al.,
2010).
In contrast, an increase in mutation frequency, or
mutation fixation, during chronological ageing cannot, by
definition, be a dominant mechanism, as replicative DNA
synthesis does not take place in nondividing cells. However, cells are not fully silent, possess basic metabolism
FEMS Yeast Res 14 (2014) 198–212
Mitochondrial metabolism in ageing cells
and repair synthesis of DNA does take place (Longo &
Fabrizio, 2012). Consequently, mutations have been studied in postmitotic nondividing cells. These include mutations conferring canavanine resistance, reversion of
missense and frameshift mutations, gross chromosomal
rearrangements, and mutagenesis induced by homologous
recombination (Wei et al., 2011).
Mitochondria-centric metabolic
processes important for the ageing
process
Fe/S cluster biosynthesis is important, but not the only
mitochondrial metabolic process associated with ageing.
Another obvious mitochondrial metabolic connection to
ageing is the TCA cycle, a central metabolic pathway
located to this organelle. Evidence that TCA activity
changes during ageing originated in the 1960s from plant
studies (Laties, 1964). During yeast chronological ageing,
succinate dehydrogenase (SDH), enzymes of the glyoxylate cycle (GCL) (isocitrate lyase (ICL) and malate synthase (MLS)), and enzymes of ethanol oxidation (alcohol
dehydrogenase (ADH) and acetaldehyde dehydrogenase
(ACDH)) are activated, while the classic TCA enzymes
citrate synthase (CS), a-ketoglutarate dehydrogenase
(KGDH) and malate dehydrogenase (MDH) decrease in
activity (Samokhvalov et al., 2004), indicating dynamic
changes of the TCA cycle during ageing. These observations are supported by the observation that metabolic
enzymes are subject to oxidative modifications while cells
age chronologically (Shenton & Grant, 2003; Klinger
et al., 2010; Brandes et al., 2013).
A causal connection of mitochondrial carbon metabolism with ageing is indicated by experiments that demonstrate altered ageing phenotypes when TCA enzymes are
deleted or overexpressed. To illustrate this connectivity,
we obtained phenotypic information from the yeast genome database (SGD; Cherry et al., 1998) and created a
mitochondria-centric interaction network, taking into
account genetic and physical interactions as annotated in
the BioGrid database. This network illustrates that despite
replicative, chronological and hibernating lifespan being
physiologically distinct phenotypes, the genes coding for
mitochondrial proteins that are causative for these phenotypes, are tightly interconnected (Fig. 2). As a caveat, we
want to mention that the high-throughput genome data
used for the connectivity network in many cases still need
to be confirmed by precisely aimed experiments.
Examples of these interactions (Fig. 2) involve several
metabolic enzymes. The deletion of citrate synthase CIT1,
for instance, was identified to prolong hibernating lifespan (Postma et al., 2009). This enzyme catalyses the
condensation of acetyl coenzyme A and oxaloacetate to
FEMS Yeast Res 14 (2014) 198–212
203
form citrate and is thus responsible for the most rate limiting step of the TCA cycle (Suissa et al., 1984). CIT1 in
turn connects directly to three enzymes that are important for chronological lifespan, DIA4, LPD1 and ACH1.
The first of these genes, DIA4, is not a metabolic enzyme
in the classic sense but is a (yet putative) mitochondrial
seryl-tRNA synthethase. Aminoacyl-tRNA synthethases
attach amino acids specifically to cognate tRNAs and are
therefore directly or indirectly implicated in the regulation of amino acid homoeostasis and translation (Laxman
et al., 2013).
The second enzyme, LPD1, has a central function in
connecting glycolysis and the TCA cycle in the biosynthesis of glycine. It encodes for dihydrolipoamide dehydrogenase, located in the mitochondrial matrix as a component
of the glycine decarboxylase complex (GDC), 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase (PDH)
complexes. Mutations in LPD1 abolish activity of these
complexes (Sinclair & Dawes, 1995; Pronk et al., 1996;
Dickinson et al., 1997; Zaman et al., 1999). Evidence for
the importance of LPD1 in ageing comes from studies of
LAT1, coding for dihydrolipoamide acetyltransferase, the
(E2) component of the PDH complex. Deleting the LAT1
gene abolished a chronological lifespan extension induced
by caloric restriction, while overexpression of this protein
prolonged chronological lifespan, which was nonadditive
with caloric restriction (Easlon et al., 2007).
The third gene, ACH1 possesses a CoA-transferase
activity and catalyses the CoASH transfer from succinylCoA to acetate generating acetyl-CoA (Lee et al., 1990).
Cells lacking this gene accumulate high amounts of extracellular acetic acid, and during chronological ageing accumulate reactive oxygen species, obtain mitochondrial
damage and exhibit an early onset of apoptosis (Orlandi
et al., 2012). Interestingly, this gene is connected to LSC1,
coding for the alpha subunit of succinyl-CoA ligase, the
enzyme that catalyses the nucleotide-dependent conversion of succinyl-CoA to succinate. Although the chronological lifespan of the LSC1 deletion mutant is very short
(Powers et al., 2006; Fabrizio et al., 2010), deletion of this
gene extends the hibernating lifespan phenotype (Postma
et al., 2009). This discrepancy may be explained by the
physiological difference between chronological and hibernating lifespan. These examples illustrate the close, yet
often opposing, connection between chronological and
hibernating lifespan phenotypes.
In sum, the three enzymes discussed in exemplary form
here represent in paradigmatic form the importance of
metabolic flux in the ageing process. They are either constituents of the TCA cycle, or feed into or withdraw from
it, and each one is controlled by oxidative stress.
Regarding mitochondrial metabolism, replicative lifespan appears to be metabolically more distinct from the
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204
M. Breitenbach et al.
Fig. 2. Mitochondrial metabolic genes link yeast lifespan phenotypes. Computational interaction network among mitochondrial metabolic genes
that influence chronological, replicative and hibernating lifespan. Genes with both metabolic function and altered replicative (gene symbol in
pink), chronological (green) or hibernating (blue) lifespan phenotypes, were extracted according to their annotation in the Saccharomyces
Genome Database (Cherry et al., 1998) as of August 2013. The genes were annotated according to their primary localization (‘Compartments’)
using OrganelleDB (Wiwatwattana et al., 2007). The physical and genetic interaction network was obtained employing BioGRID(Stark et al.,
2006), and visualized with Cytoscape (Smoot et al., 2011). References for the individual interactions are given in the Supporting Information
Table S1.
chronological and hibernating lifespans, yet connected
through peroxiredoxin and thioredoxin systems, which
operate under conditions of more continuous oxidative
stress. ROS and free-radical theory of ageing are not the
focus of this review; however, metabolic redox reactions
are among the first processes affected by an excess of oxidizing over reducing molecules. Special attention has to
be given to the mitochondrial peroxiredoxin Prx1, whose
activity is strongly associated with control of replicative
lifespan (Unlu & Koc, 2007). In a similar vein, it was
found that a gain of function allele of the cytoplasmic
peroxiredoxin, Tsa1, causes premature replicative ageing
(Timmermann et al., 2010). On the other hand, the presence of Tsa1 has been shown to be required for the
extension of replicative lifespan by caloric restriction
(Molin et al., 2011).
In summary, there is convincing, yet mostly correlative,
evidence that the TCA cycle plays an important role during ageing. Remarkably, the TCA cycle seems to connect
between the three yeast ageing phenotypes, although
chronological and hibernating lifespan appear more
extensively integrated than replicative ageing. However,
the TCA cycle has been less studied in ageing than other
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Published by John Wiley & Sons Ltd. All rights reserved
metabolic processes, and much work remains to be done
to clarify its relationship to lifespan.
Involvement of mitophagy in the yeast
ageing processes
Mitophagy, the specific degradation of parts of the mitochondrial network through the process of autophagy,
serves a number of distinct physiological needs: (1) adaptation to nutrients and carbon source such as the shift
from glycerol to glucose that results in the degradation of
many mitochondria, (2) adaptation to starvation in stationary phase, which includes the degradation of mitochondria to gain an extra supply of nutrients for survival
of the stationary phase (Tal et al., 2007; Bhatia-Kissova &
Camougrand, 2010) and (3) finally, as a form of mitochondrial quality control resulting in the removal of toxic
waste and damaged macromolecules.
Autophagy, in general, and mitophagy, in particular,
are needed for the rejuvenation process of daughter cells
in cell divisions of replicatively aged mother cells (discussed below). The same mechanism that guarantees rejuvenation of the daughter cells and therefore survival of
FEMS Yeast Res 14 (2014) 198–212
Mitochondrial metabolism in ageing cells
the species leads to deposition of damaged molecules in
the ageing mother cells. Surprisingly, little is presently
known about the exact chemical nature of the damaged
macromolecules and organelles that exist in aged cells.
Protein carbonyls (Aguilaniu et al., 2003) are one form of
oxidation products accessible for study, as an easy
method for their detection is available (Goto et al., 1999;
Beal, 2002; Levine, 2002). Other products of cellular
decomposition, however, maybe of equal importance.
This includes already discussed substances that originate
as side reactions from metabolism(D’Ari & Casadesus,
1998; Linster et al., 2013).
The mechanism and physiological significance of mitophagy has been elucidated during recent years in considerable detail [reviewed in (Bhatia-Kissova & Camougrand,
2010; Hirota et al., 2012; Novak, 2012; Palikaras &
Tavernarakis, 2012)]. The pathway of mitophagy consists
of a mitochondrial specific part (to be described below)
that leads to engulfment of mitochondria in the phagosome. This part of the pathway is dependent on only a
few specific proteins, while the ‘unspecific’ part of the
pathway is to a large degree identical with macroautophagy, or other known specialized forms of autophagy
(Bhatia-Kissova & Camougrand, 2010).
Yeast has been a leading model organism in deciphering autophagy and mitophagy. Original nomenclature
named the involved genes either aut- (Thumm et al.,
1994), apg- (Tsukada & Ohsumi, 1993), or cvt-mutants
(Harding et al., 1995), but the gene nomenclature has
eventually been unified to ‘ATG’ and involves 33 genes so
far (Klionsky et al., 2003).
Mitophagy commences with recruitment of a special
surface marker, Atg32, whose translocation is most probably induced upon a critical loss of mitochondrial membrane potential (Priault et al., 2005; Kondo-Okamoto
et al., 2012). A decline of this potential in ageing is
observed, as well as damage of membrane compounds
(Dmitriev & Titov, 2010; Paradies et al., 2011). In parallel, loss of mitochondrial metabolic capacity could also be
triggered by opening of the mitochondrial permeability
transition pore (Rodriguez-Enriquez et al., 2004), thus
creating a link between mitophagy and apoptosis, which
occurs in replicatively as well as in chronologically aged
yeast cells and is their main cause of death [reviewed in
(Breitenbach et al., 2012; Laun et al., 2012)]. It follows
that mitophagy has pro-survival as well as a pro-death
functions (Abeliovich, 2007; Tal et al., 2007) depending
on the physiological situation. Here, we focus on the
potential survival-promoting function of yeast mitophagy,
which appears weakened in old mother cells.
Atg32 possesses close homologues among fungi but is
weakly conserved in other eukaryotes (Kondo-Okamoto
et al., 2012). However, Atg32 is not the only
FEMS Yeast Res 14 (2014) 198–212
205
mitochondrial protein involved in leading mitochondria
into mitophagy. This process also involves Atg33, Aup1,
Fzo1 and Por1 (a synonym of yeast Vdac1), and potentially
Uth1 (Kissova et al., 2004). These proteins possess a partially redundant function. Deletion of any one of them still
allows mitophagy to occur (Kissova et al., 2004; BhatiaKissova & Camougrand, 2010). The situation is complicated by the fact that these ‘entry’ proteins are needed to
different degrees depending on the subtype of mitophagy.
The transition of mitochondria in yeast cells during
stationary phase and mother cell-specific ageing into a
large number of small roundish, separated, mitochondria
(Klinger et al., 2010), suggests that these small mitochondria may be more accessible for the phagosome (Mao &
Klionsky, 2013). Indeed, in other situations where mitophagy is needed, the mitochondrial network disassembles
in a similar fashion, and the fragments of the network are
subsequently enclosed in the phagosomes (Twig & Shirihai, 2011). The disassembly procedure depends on the
mitochondrial fission machinery. Staining with redoxsensitive dyes and with mitochondria-specific fluorescent
protein markers (ro-GFP) indicates that parts of the
mitochondrial network are slightly depolarized without
compromising the rest of the network. The depolarized
regions are separated by the fission process and incorporated into phagosomes (Twig & Shirihai, 2011; Mao &
Klionsky, 2013).
In contrast to yeast, mammalian cells create the mitophagic ‘eat me’ signal (Vernon & Tang, 2013) in a different way. In response to mitochondrial damage, the E3
ubiquitin ligase parkin is recruited to the mitochondrial
surface and phosphorylated by the mitochondrial outer
membrane protein PINK1. Activated Parkin then ubiquitinates mitochondrial outer membrane proteins such as
the mitofusins Mfn1/2 and VDAC1 (Rodriguez-Enriquez
et al., 2004; Novak, 2012). As a consequence, mitofusins
are degraded and the isolated mitochondrion cannot fuse
again with the mitochondrial network, entering a one
way street to mitophagy. Here, the ubiquitinated
hVDAC1 serves as an anchor for the p62 module which
firmly links the mitochondrion to the phagosome via LC3
(the human homologue of Atg8; Rodriguez-Enriquez
et al., 2004; Novak, 2012). Several other mitochondrial
outer membrane proteins are also ubiquitinated at this
stage (Tom70, 40 and 20; Miro 1 and 2; Yoshii et al.,
2011). Remarkably, the involvement of an ubiquitinating
enzyme in mitophagy provides testimony for the crosstalk
between the proteasome and the autophagic pathway in
the decomposition of cellular compounds.
In human cells, mitophagy can be induced by ironchelation with drugs (deferiprone) by a mechanism independent of PINK1 and Parkin, again emphasizing the
connection between iron metabolism and mitophagy
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
206
(Allen et al., 2013). Mutations in both PINK1 and Parkin
can lead to the devastating neurodegenerative Parkinson’s
disease, a typical disease of old age, demonstrating once
more the close relationship of mitophagy with ageing
(Hauser & Hastings, 2013).
Once mitochondria are channelled to the autophagosome the mitochondria-specific part of mitophagy concludes. The continuing ‘macro autophagy’ pathway
ultimately leads to enclosure of the mitochondrion in the
lytic compartment (the yeast vacuole) where not only
proteins but also lipids and other biomolecules are
degraded.
Mitophagy throughout the stationary phase was studied
in detail and serves the purpose of supplying cells with
the nutrients necessary for restructuring their architecture
(for instance, the structure of the cell wall) and metabolically adapting to survival in times of starvation (Kissova
et al., 2004; Tal et al., 2007). The ability to do so was
crucial during evolution, as microbial cells in the wild are
frequently confronted with a scarcity of nutrients. As
nutrients are limited during chronological ageing, it is
suspected that mutations compromising mitophagy in
stationary phase should shorten the chronological lifespan. This prediction is supported by existing whole genome high-throughput data (Powers et al., 2006; Fabrizio
et al., 2010). In both databases, deletions of all but one
(UTH1) of the proteins that have been identified so far as
being directly or indirectly specific for mitophagy (Atg32,
Atg33, Uth1, Aup1, Fzo1, Por1), are short-lived in stationary phase. The chronological lifespan of the corresponding deletion mutants was significantly shorter than
wild type, although these mutants are respiration-active.
In addition, the two interactors Atg11 and Atg8 (Palikaras
& Tavernarakis, 2012) of Atg32 in mitophagy, together
with more than 30 ATG genes (Nakatogawa et al., 2009)
are generally needed for macroautophagy. They are
expected to show a strong negative effect on chronological lifespan when deleted, which is confirmed by
published data (Powers et al., 2006; Fabrizio et al., 2010).
The reason for this strong effect seems to be the necessity
to also degrade nonmitochondrial cellular components by
autophagy to survive starvation.
Interestingly, the known pathways of regulating mitophagy are closely interconnected with metabolic regulation. The increase in both the chronological and
replicative lifespan which is observed when treating yeast
cells with low doses of rapamycin (Bjedov & Partridge,
2011) is dependent on rapamycin-mediated removal of
the inhibitory action of yeast Tor1 on autophagy and
mitophagy (Rubinsztein et al., 2011). Likewise, activity of
the RAS/PKA system of yeast inhibits mitophagy (Budovskaya et al., 2004). If the up-regulation of mitophagy
observed in wild-type cells upon reaching diauxie and the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
M. Breitenbach et al.
stationary phase is prevented by genetically activating
RAS2, or inactivating its interacting genes such as WHI2,
survival of the stationary phase and chronological lifespan
are compromised leading to cell death (Mendl et al.,
2011). Hence, the metabolic activity and the ability to
adapt to nutrient starvation seem to be a major link in
connecting mitophagy with ageing.
Role of UTH1 in the two ageing
processes
The uth1D deletion mutant was shown to be chronologically long-lived (Kissova et al., 2004). The tentative explanation given is that Uth1, a mitochondrial outer
membrane protein, has no influence on autophagy and
only a partial influence on mitophagy.
UTH1 was studied in detail with respect to replicative
ageing as well. Like the chronological lifespan (Kissova
et al., 2004), the replicative lifespan of uth1D yeast is
increased significantly (Kennedy et al., 1995). The role of
Uth1 in the ageing processes is still not fully understood
and somewhat controversial. The reason for this may be the
unusually large number of biochemical functions that has
been attributed to this SUN-family mitochondrial protein
(Camougrand et al., 2004).
Role of polyamine metabolites
spermine and spermidine
Another, presumably critical, but yet to be fully elucidated role in the process of autophagy is attributed to the
polyamine metabolites spermine and spermidine. These
metabolites are considered to be free-radical scavengers
and are highly concentrated within cells (Lovaas & Carlin,
1991). During ageing, they decline in concentration as
observed in various organisms (Minois et al., 2011). Vice
versa, spermine/spermidine supplementation extends lifespan in laboratory models, including yeast replicative ageing (Eisenberg et al., 2009; Morselli et al., 2009), and
restores age-induced memory impairment in drosophila
(Gupta et al., 2013). Both, lifespan extension and memory improvement correlate with the activation of autophagy (Eisenberg et al., 2009; Gupta et al., 2013), and as
spermine and spermidine are time-keepers of the stress
response (Kruger et al., 2013), their function appears
centre stage for age-related autophagy.
Apparently, the multiple (but closely related) forms of
autophagy, including mitophagy, cannot completely
remove the unwanted and detrimental components of
aged yeast mother cells. Therefore, another ingenious
invention serves the purpose of quality control: asymmetric segregation of damaged material, including
mitochondria (Aguilaniu et al., 2003; Klinger et al., 2010).
FEMS Yeast Res 14 (2014) 198–212
Mitochondrial metabolism in ageing cells
207
Fig. 3. Schematic overview of carbon metabolism, Fe/S cluster biosynthesis and mitophagy in ageing. A sufficient Dw across the inner
mitochondrial membrane is necessary for protein import into mitochondria and for the biosynthesis of Fe/S clusters (ISC) and is essential for
survival of the cell. Only one of the TCA cycle enzymes, aconitase, contains a structural (non-redox active) Fe/S cluster rendering it sensitive to
oxidants. ATG32 is located on the surface of damaged mitochondria and interacts with ATG8 on the preautophagosome. ATG8 and ATG32,
yeast autophagy genes; ISC, iron sulfur cluster; TCA, tricarboxylic acid cycle; Dw, electrochemical potential gradient across the inner
mitochondrial membrane.
Accordingly, the aged mother cell retains damaged substances upon cell division, which protects the young
daughter cell from age-dependent damage. This asymmetric segregation is in our view a general mechanism of
living cells, allowing a cellular ‘rejuvenation’ process
occurring at least once per life cycle, that is essential for
survival of a species.
Conclusions
Mitochondria are intensively studied in regard to ageing,
but most research has focused on mitochondrial ATP
production, ROS leakage from the respiratory chain, and
age-dependent induction of apoptosis. Here, we discussed
three central functions of mitochondrial metabolism that
may be equally important: (1) The biosynthesis of Fe/S
clusters which is an essential task of mitochondria in
FEMS Yeast Res 14 (2014) 198–212
eukaryotes, (2) mitochondrial carbon metabolism (TCA
cycle), a central source of reducing equivalents and biosynthethic intermediates for the cell and (3) the process
of mitophagy, which disassembles the mitochondrial network as result of stress and ageing and helps to maintain
the energy state during starvation and to decompose
damaged molecules in old mother cells or stationary cell
cultures (Fig. 3).
Importantly, all three processes are highly dynamic
metabolic processes and their interconnectivity may buffer adverse cellular conditions, either caused by mutation
in one of their components or caused by intra- or extracellular toxic challenge. Evidence suggests that toxic waste
load increases with time, and by interfering with normal
cellular activities, it inhibits function of metabolic pathways. Once the dynamics of complete metabolic pathways
are compromised, the ageing process may be irreversible.
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
208
Importantly, mitochondrial metabolic processes influence different facets of ageing and form connections
between replicative, chronological, and hibernating lifespan in yeast as well as to ageing in mammals. Partially
overlapping mechanisms that arise from their biochemical
function, and the regulation of basic cellular metabolic
activity, potentially explain these ageing phenotypes. A
unifying concept to understand the role of metabolism in
ageing thus requires considering the metabolic integrity
of mitochondria, adding additional complexity to the
immensely important role of oxidative stress and
programmed cell death in the ageing process.
Acknowledgements
We are grateful for the support of N. Lawrence of the
Gurdon Institute Imaging Facility (University of Cambridge) in super-resolution microscopy, and to Benedikt
Westermann, University of Bayreuth, for providing the
mtGFP expression plasmid. We are grateful to FWF (Austria) for grant S9302-B05 (to MB) and to the EC (Brussels, Europe) for project MIMAGE (contract 512020, to
MB), the Wellcome Trust (RG 093735/Z/10/Z) (to MR)
and the ERC (Starting grant 260809) (to MR). M.R. is a
Wellcome Trust Research Career Development and
Wellcome-Beit prize fellow.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. References and interaction details for the molecular network of mitochondria-specific ageing factors in yeast.
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