Download Mitochondrial diseases and the role of the yeast models

MINIREVIEW
Mitochondrial diseases and the role of the yeast models
Teresa Rinaldi1, Cristina Dallabona2, Ileana Ferrero2, Laura Frontali1 & Monique Bolotin-Fukuhara3
1
Department of Cell and Developmental Biology, Pasteur Institute-Cenci Bolognetti Foundation, Sapienza University of Rome, Rome, Italy; 2Department
of Genetics, Biology of Microrganisms, Anthropology, Evolution, University of Parma, Parma, Italy; and 3Institut de Génétique et Microbiologie, CNRSUniversité Paris XI, Orsay, France
Correspondence: Monique BolotinFukuhara, Institut de Génétique et
Microbiologie, Université Paris Sud/XI,
Bâtiment 400, 91405 Orsay Cedex, France.
Tel.: 133 1 69 156 201; fax: 133 1 69 157
296; e-mail: [email protected]
Received 14 June 2010; revised 30 August
2010; accepted 30 August 2010.
Final version published online 14 October 2010.
Abstract
Nowadays, mitochondrial diseases are recognized and studied with much attention
and they cannot be considered anymore as ‘rare diseases’. Yeast has been an
instrumental organism to understand the genetic and molecular aspects of the
many roles of mitochondria within the cells. Thanks to the general conservation of
mitochondrial genes and pathways between human and yeast, it can also be used to
model some diseases. In this review, we focus on the most recent topics,
exemplifying those for which yeast models have been especially valuable.
DOI:10.1111/j.1567-1364.2010.00685.x
Editor: Claude Gaillardin
Keywords
mitochondrial diseases; fusion/fission;
mitochondrial transformation; mitochondrial
tRNAs; mtDNA stability and maintenance.
YEAST RESEARCH
Introduction
In recent years, mitochondrial diseases have been recognized
and studied much more attentively than in previous times.
Attention to the patients and to the necessity of genetic
counselling have considerably improved, but treatments are
still lacking and even mechanisms of the different diseases
are mostly far from being understood. In this article, we will
try to review some latest results and the models that are now
contributing to the understanding of this heterogeneous
group of, mostly, neuromuscular diseases.
Mitochondria are at the heart of cell function. For many
years, emphasis has been placed on their primary function, i.e.
providing the majority of the cellular energy in the form of
ATP. Besides their role in ATP synthesis, mitochondria are also
involved in essential cellular processes: b-oxidation (Bartlett &
Eaton, 2004) and maturation of Fe-S proteins; without the
mitochondrial structure, the cell is not viable (Lill & Kispal,
2000). Mitochondria generate reactive oxygen species (ROS)
during respiration, 1–2% of the oxygen consumed during
respiration being not completely reduced to water. Under
hypoxia, the mitochondrial respiratory chain (RC) also produces nitric oxide, which can generate other reactive nitrogen
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species (RNS). High levels of ROS or RNS produce oxidative
and nitrosative stress and low levels function in cellular
signaling; they also have important implications for several
diseases such as inflammation and cancer (Poyton et al., 2009).
Mitochondria also mediate apoptosis via the mitochondrial
permeability transition pore (review in Cheng et al., 2006;
Pradelli et al., 2010) but probably also via fission/fusion
proteins conserved in evolution (Cheng et al., 2008). The
mitochondria networks associated with the endoplasmic reticulum structure are pivotal to the control of Ca21 signalling
and processes that depend upon them such as apoptosis
(Giorgi et al., 2009). All these pathways are tightly intertwined
and as a consequence mitochondrial dysfunctions are associated with many multifactorial diseases such as diabetes,
cardiac diseases (review in Baines, 2010) or cancer (Diaz-Ruiz
et al., 2009; Mayevsky, 2009; Weinberg & Chandel, 2009). They
are also associated with neurodegenerative diseases such as
Parkinson (reviewed in Lin et al., 2009), Alzheimer (Yan et al.,
2006) and Huntington diseases (Abou-Sleiman et al., 2006;
Quintanilla & Johnson, 2009).
The precise relation between mitochondrial functions
and such multifactorial diseases are not yet elucidated, and
there are still much debates about any causal relationships
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Mitochondrial diseases and the role of the yeast models
(Johannsen & Ravussin, 2009). They are not the topics of
this review and some will be treated in other chapters of this
issue. Here, we will concentrate on diseases due to mutations that directly affect mitochondrial genes or gene
expression in a monogenic manner. Because mitochondrial
biogenesis requires two genetic compartments, the chromosomes and the mitochondrial genome localized within the
mitochondrial compartment, one expects these mutations
to be of nuclear and mitochondrial origins.
Yeast (Saccharomyces cerevisiae) is a simple eukaryotic
organism, with a complete genome sequence and, more
importantly, the best annotated one, thanks to a coordinated
international effort. Many genetic tools that have been
created during these years, including the complete collection
of gene deletions and a considerable number of mechanisms
and pathways existing in higher eukaryotes, have been first
studied and described in yeast. Moreover, about 40% of
human genes whose mutations lead to diseases have an
orthologue in yeast (Bassett et al., 1996) and genomic screens
have been extended to mitochondrial diseases (Steinmetz
et al., 2002). It is the reason for which it has been widely used
to decipher molecular mechanisms underlying diseases in
general. However, the study of mitochondrial functions and
dysfunction is of special interest in yeast because it is in this
organism that mitochondrial genetics and recombination
have been discovered (Bolotin et al., 1971) and that nucleomitochondrial interactions have been studied in depth.
There are also specific reasons for choosing S. cerevisiae for
mitochondrial studies. This organism is petite-positive i.e. it
can lose its mitochondrial genome (assimilated to the rho
factor) provided it is supplied with a fermentative substrate.
Consequently, all mutations of the mitochondrial genome
can be studied without cell lethality. The frequency of
homologous recombination is very high (1% recombination
is considered to correspond to about 100 bp in the mitochondrial genome). It is genetically easy to transfer mitochondria from one nuclear genetic background to another
via karyogamy. Moreover mitochondria can be transformed
making in vitro mutation analysis possible. The richness and
ease of yeast molecular genetics opens big opportunities, and
even the major difference existing between human and yeast
mitochondrial genomes, i.e. the predominant heteroplasmy
of human and the homoplasmy of yeast, can result in the
easier definition of the pathogenic mutations.
To review mitochondrial diseases may be a very difficult
task because the definition might include different kinds of
metabolic disorders or degenerative syndromes. Moreover,
some important aspects have been extensively reviewed and
the reader might refer to very good recent articles by Di
Mauro (2010) for historical aspects, by Wallace et al. (2010)
for bioenergetics, by Spinazzola & Zeviani (2009) for
nucleo–mitochondrial intergenomic cross-talk; a previous
review by Schwimmer et al. (2006) has already given an
FEMS Yeast Res 10 (2010) 1006–1022
important outline of yeast models of mitochondrial diseases. In recent years, it has become clearer how important
the conservation is between human and yeast and hence the
possible use of yeast models. In the present article, we try
therefore to exemplify the newest subjects and those for
which yeast models have been especially valuable.
Yeast models of mitochondrial diseases
due to mutations in nuclear genes
Saccharomyces cerevisiae has played an important role as a
model system to understand the biochemistry and molecular
biology of mammalian cells. The genetic tools available have
also made S. cerevisiae a powerful system to identify gene–
disease relationship. Furthermore, the possibility to duplicate
as haploid or diploid makes this organism a flexible tool for
assessing the dominant or recessive nature of a mutation. Yeast
offers invaluable guidance for approaching human diseasesassociated gene functions particularly concerning mitochondrial ones due to the ability of yeast to survive without a
functional mitochondrial RC, provided a fermentable carbon
source is made available. When the concentration of glucose is
reduced, respiration-deficient yeast mutants grow slowly,
forming small (petite) colonies. Petite, OXPHOS-deficient,
yeast mutants carry mtDNA abnormalities in the form of
multiple rearrangements (rho-minus petites) or as mtDNAless strains (rho-zero petites).
Now, mitochondrial functions are very diverse and so are
the nuclear origins of pathologies. In most cases, the understanding of the defect in yeast was essential to clarify the
human pathology. Mitochondrial diseases caused by mutations in nuclear genes can be grouped into several categories.
Some disorders are due to mutations in RC subunits or in
proteins involved in the corresponding assembly. Others are
due to defects of ATP synthase or of other metabolic enzyme
complexes, such as defects in biosynthetic enzymes for lipids
or cofactors or in enzymes of the Krebs cycle. Several others
are due to deficiencies in mitochondrial protein synthesis or
in the corresponding proofreading apparatus.
As noted in the Introduction, most of these dysfunctions
and the relative correspondence between humans and yeasts
have been reviewed in Schwimmer et al. (2006) and Spinazzola & Zeviani (2009).
In this first part of the present review, we will discuss some
recent research advances where yeast models have made
essential contribution to clarify the relationship between a
nuclear mutation and the corresponding mitochondrial defect.
In the first section, we will discuss mitochondrial diseases
associated with mtDNA instability, in the second section, we
discuss the defects in metabolic assembly factors, while in
the third section we will treat the very recently investigated
cases of nuclear mutations resulting in diseases due to
defects in mitochondrial morphology and interactions.
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1008
Defects associated with mtDNA instability
The maintenance of the mtDNA depends on a variety of
nuclear-encoded proteins. While the most obvious link
between the nuclear DNA and the mtDNA is represented
by the genes coding for the enzymes involved in mtDNA
synthesis, in some other interesting cases, the relationship
between the mutation and the defect is not evident and yeast
models have been very important to enhance knowledge.
The synthesis of mtDNA requires the activity of the
mitochondrial polymerase POLG (Van Goethem et al., 2001)
and helicase TWINKLE (Moraes, 2001; Spelbrink et al., 2001).
In addition, a correct balance of the mitochondrial dNTP pool
is essential for the maintenance of mtDNA copy number. In
fact, mutations can lead to mtDNA depletion such as mutations in the human genes thymidine kinase 2 (TK2) (Saada
et al., 2001) and deoxyguanosine kinase (dGK) (Mandel et al.,
2001), encoding enzymes involved in mitochondrial dNTP
recycling, in p53-regulated ribonucleotide reductase (Bourdon
et al., 2007), involved in the de novo synthesis of dNTPs, and in
thymidine phosphorylase (TP) (Nishino et al., 1999), involved
in the catabolism of thymidine.
Defects associated with mtDNA instability – yeast
models of POLG
The first observation that a disease associated with multiple
deletions of mtDNA is caused by a mutation in a nuclear gene
(Zeviani et al., 1989) dates back to 1989. In 2001, the first
description of POLG mutations associated with mtDNA
deletions (Van Goethem et al., 2001) appeared. So far, 4 150
mutations in POLG have been reported to be associated with
various pathologies (for a complete list, see the database http://
tools.niehs.nih.gov/polg/). Thanks to the conservation between
the human POLG and the yeast orthologue MIP1 (Lecrenier &
Foury, 2000; Foury & Kucej, 2001), it was possible to study in
yeast the effect of POLG mutations found in patients. Yeast has
proved a suitable model to validate the significance of new
pathogenic POLG mutations. Generally speaking, most of
MIP1 mutations equivalent to the POLG mutations led to an
increased mutability of mtDNA, deletion, depletion, or point
mutations. These results may provide relevant information on
the molecular mechanisms of the replication defect underlying
the disease. Most patients are compound heterozygous, or
carry potentially relevant single-nucleotide polymorphisms
(SNPs), which lead to the question as to whether these changes
can determine additive or cooperative effects on the pathomechanisms, ultimately leading to the different clinical phenotypes. Yeast is a useful tool to answer these questions, as the
effects of mutations either singly or in combination, both in cis
and in trans, can be studied (Baruffini et al., 2010).
Some examples of studies in yeast that have contributed to
the understanding of the effects of mutations and of the
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T. Rinaldi et al.
possible molecular mechanisms underlying the disease have
been described here. The introduction of POLG mutations
associated with progressive external ophthalmoplegia (PEO),
L304R, 467T, G923D, R943H, Y955C, A957S, into yeast MIP1
caused increased mtDNA mutability, increased nuclear mutation rates and increased oxidative stress. Mutations in the
polymerase domain caused the most severe phenotype,
whereas the mutation in the exonuclease domain showed a
less severe phenotype (Stuart et al., 2006). Yeast models have
also been constructed of several human mutations alone or in
combination, among which G848S-E1143G and H932YG1051R associated in trans, and A889T-E1143G associated in
cis (Baruffini et al., 2007; Spinazzola et al., 2009; Stricker et al.,
2009). The mutation A889T is slightly dominant in yeast, in
agreement with the hypothesis that the gene penetrance is
influenced by other genetic or environmental factors (Hisama
et al., 2005). Furthermore, in vivo studies in yeast have shown
that the mutation E1143G is not a neutral SNP (Baruffini
et al., 2007), but rather acts as a cis-modulator of Mip1 activity,
in agreement with the observation that in vitro E1143G was
somewhat detrimental to protein stability (Chan et al., 2006).
Experiments in yeast demonstrated that, in contrast to what
was suggested previously (Graziewicz et al., 2004), mtDNA
deletions are not related to accumulation of point mutations
of mtDNA (Baruffini et al., 2006; Stumpf et al., 2010).
In yeast, the genetic and chemical rescue of mutant
phenotype induced by POLG pathological mutations was also
described. It was found that the mtDNA instability caused by
mutations in MIP1 is reduced by increasing the concentration
of the dNTP pool through an increase in RNR1 expression
(Lecrenier & Foury, 1995; Zhao et al., 1998; Chabes et al.,
2003; Baruffini et al., 2006; Stumpf et al., 2010) or deleting
SML1, encoding Rnr1 repressor (Zhao et al., 1998; Baruffini
et al., 2006). Whether the rescue due to an increased pool of
dNTPs occurs preventing the stall of POLG or improving the
efficiency of mtDNA repair, remains to be determined.
Chemical rescue experiments demonstrated that the instability
of mtDNA due to the POLG mutation Y955C depends, at least
in part, on ROS damage (Baruffini et al., 2006). Finally, it is
worth mentioning that there are examples where the effect of
mutation in yeast suggests the existence of another mutation,
in addition to that already identified, as a cause of disease (E.
Baruffini, pers. commun.).
Defects associated with mtDNA instability – yeast
models of ANT1
ANT1 is the gene encoding the muscle-heart-specific isoform of the mitochondrial adenine nucleotide translocator
(Ant). ANT1 mutations are responsible for PEO. The ANT
gene, primarily involved in ATP/ADP exchange across the
inner mitochondrial membrane, is highly conserved in all
eukaryotes, including S. cerevisiae. This allowed to study in
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Mitochondrial diseases and the role of the yeast models
yeast the effect of the mutations identified in patients. An
A114P missense mutation in the human Ant1 protein was
found to be associated with autosomal dominant PEO
(adPEO) (Kaukonen et al., 2000). A128P mutation of the
S. cerevisiae Aac2 protein, equivalent to A114P in human
Ant1, causes a decrease in respiratory growth (Kaukonen
et al., 2000). In a study aimed at evaluating the effect of this
Ant mutant, it was found that it results in depolarization,
structural swelling and disintegration of mitochondria, and
it was hypothesized that the formation of an unregulated
channel, rather than a defect in ATP/ADP exchange, was a
direct pathogenic factor in human adPEO (Chen, 2002).
Four yeast models were constructed, each carrying a
missense mutation identified in the ANT1 gene of adPEO
patients (Fontanesi et al., 2004). All of them displayed a
marked decrease in respiratory growth and a concurrent
reduction of the amount of mitochondrial cytochromes,
cytochrome oxidase activity and cellular respiration (Fontanesi et al., 2004). To evaluate whether the mutations were
dominant in yeast, as in humans, the aac2 mutant alleles were
also inserted in combination with the endogenous wild-type
AAC2 gene. The mutations behaved as dominant for reduction in cytochrome content and increased mtDNA instability
phenotypes indicating that S. cerevisiae is a suitable in vivo
model to study the pathogenicity of the human ANT1
mutations. In yeast models, the efficiency of ATP and ADP
transport was variably affected by the different AAC2 mutations. However, the observation that mutants retain the basic
features of ANT/AAC proteins, together with a significant
level of mitochondrial membrane potential (Fontanesi et al.,
2004; Galassi et al., 2008), strongly argues against the hypothesis that the primary pathogenic role of ANT1 mutations
associated with adPEO is to cause the opening of an unregulated channel followed by structural disintegration of mitochondria (Chen, 2002). It should also be noted that the
uncoupling effects reported by Chen were observed in a strain
overexpressing the aac2A128P mutant allele.
A yeast model carrying the mutation equivalent to the
first (and so far the only) known recessive mutation in the
ANT1 gene (A123D mutation) (Spinazzola & Zeviani,
2009), displayed a complete loss of transport activity, and a
severe OXPHOS phenotype that was largely rescued by
exposure to ROS scavengers, suggesting that increased redox
stress is involved in the pathogenesis of the disease and that
anti-ROS therapy may be beneficial to patients; the mutation was recessive in yeast as in human (Palmieri et al.,
2005). It was reported elsewhere that by increasing the gene
dosage of the A123D mutant allele, the mutation resulted in
a dominant phenotype (Wang et al., 2008). However, this is
neither a natural condition for yeast nor a representative of
the human condition where the gene dosage is a constant.
To overcome the problem posed by a mutation mapping
in domains not conserved between human and yeast, we
FEMS Yeast Res 10 (2010) 1006–1022
took advantage of a yAAC2/hANT1 chimeric construction as
a template to introduce pathogenic hANT1 mutation.
Application to the case of the D104G mutation indicated
that the chimeric construction could be a tool for studying
pathogenic mutations in yeast (Lodi et al., 2006).
The relationship between ANT1 and mtDNA stability is not
obvious. A possible explanation is that, in addition to its
function on ATP/ADP translocation, the Aac2 protein plays a
‘structural’ role that contributes to maintain the integrity of
respiratory complexes in the inner membrane of mitochondria.
mtDNA maintenance (MPV17-SYM1)
Another intriguing example of a gene necessary for the
maintenance of mtDNA is human MPV17, mutation of which
leads to a peculiar form of hepatocerebral mtDNA depletion
syndrome (MDS). Even though Mpv17 mutations are one of
the causes of MDS in humans (Poulton et al., 2009) and the
discovery of this protein has been reported 4 20 years ago, its
function is not yet understood. Originally considered as a
peroxisomal membrane protein (Weiher et al., 1990; Zwacka
et al., 1994), it was later demonstrated that Mpv17 is localized
to the inner mitochondrial membrane (Spinazzola et al.,
2006), as also previously demonstrated for the yeast orthologue Sym1 (Trott & Morano, 2004).
With the aim of clarifying the role of MPV17 in MDS, a
mouse model Mpv17
/
was studied (Viscomi et al.,
2009). This mouse presents, like humans, severe mtDNA
depletion in liver, but, unlike human, only a modest reduction of RC enzyme activities, probably due to an increased
transcription of mtDNA. The yeast orthologue SYM1 was
identified as a heat shock protein with a role in metabolism
and/or tolerance to ethanol (Trott & Morano, 2004).
To validate the significance of pathogenic human mutations, these were introduced into a SYM1-defective yeast
strain (sym1D). The mammalian gene, MPV17, itself can
complement the phenotype of sym1D mutant, thanks to the
high degree of conservation from yeast to human. The
pathological mutations of human MPV17 are deleterious in
yeast: they cause an OXPHOS-negative phenotype and
result in an increase of mtDNA mutability (Spinazzola
et al., 2006). Further studies in yeast have shed some light
on the function of Sym1. The sym1 mutant mitochondria
are morphologically abnormal, with flattened mitochondrial cristae and accumulation of electron-dense particles, as
observed for Mpv17 / mouse mitochondria (Viscomi et al.,
2009), suggesting a role for Mpv17/Sym1 in the structural
preservation of the inner mitochondrial membrane. This
defect is not a consequence of the mtDNA instability
because it has been observed under cultural conditions
where no defect of mtDNA was observed, indicating that
the morphogenetic effects of Sym1 are likely to precede and
possibly determine its effects on mtDNA stability.
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1010
The phenotypes of double mutants (cit1 sym1, cit2 sym1)
and the nature of multicopy suppressors (ODC1, YMC1)
suggest for sym1 null mutant a defect in Krebs cycle confirmed
by an enzymatic analysis that clearly indicates a heavy reduction of succinate dehydrogenase (SDH) activity. Accordingly,
sym1D displays a significant reduction in the amount of
glycogen that is dependent on gluconeogenesis, which is in
turn regulated by the anaplerotic flux of tricarboxylic acid
(TCA) intermediates from mitochondria to the cytosol (Dallabona et al., 2010). Interestingly, patients with Mpv17 mutations suffer from drastic, often fatal, hypoglycaemic crises,
which are likely due to glycogen shortage in the liver (Spinazzola et al., 2006; Parini et al., 2009).
Blue-native gel electrophoresis immunovisualization
clearly demonstrated that Sym1 is part of a high-molecularweight complex (4 650 kDa) (Dallabona et al., 2010). While
further work is necessary to identify the primary role of
Sym1, including the molecular dissection and characterization of the Sym1-containing protein complex, these results
indicate that Sym1 is involved in the structural and functional
stability of the inner mitochondrial membrane, thus controlling crucial mechanisms related to this compartment, including the activity of RC complexes, the morphology of
mitochondria and the maintenance of mtDNA.
Defects in the biosynthesis of SDH assembly
factors
SDH (or complex II or cII) is composed of four subunits
(SDHA-D in humans, SDH1-4 in yeast), all encoded by
nuclear genes. Despite the extensive knowledge on structural
and catalytic properties of the complex, two assembly factors
specific for the SDH have been only recently identified:
SHDAF1 and SDHAF2 (YDR379C-A and SDH5 in yeast).
The identification and characterization of SDH assembly
factors is an example of how yeast can be used as a model
system in ‘two ways’: from yeast to human and back.
The SDHAF1 gene has been identified in humans as
linked to infantile leukoencephalopathy (Ghezzi et al.,
2009). A yeast strain deleted in the SDHAF1 orthologue
YDR379C-A (ydr379c-aD) was OXPHOS incompetent due
to a severe and specific reduction of SDH activity. Transformation with YDR379C-A variants corresponding to the
human mutant alleles did not restore OXPHOS growth of
the ydr379c-aD strain, demonstrating that these mutations
are really the cause of the disease. Experiments performed in
human tissues and in yeast showed a marked reduction of
cII holoenzyme and a Km value for succinate similar in wild
type and in the null mutants, suggesting that defective SDH
activity was caused by a reduced number of enzyme units
rather than by qualitative alterations of complex II (Ghezzi
et al., 2009). Although there are other examples of low cII
content and activity associated with mutations in mitochon2010 Federation of European Microbiological Societies
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T. Rinaldi et al.
drial chaperonins such as yeast Tcm62 (Klanner et al., 2000),
or proteins involved in Fe-S biosynthesis such as human and
yeast frataxin or IscU (Rouault & Tong, 2008), SDHAF1 is
the first protein identified with a specific role in cII
assembly, as other Fe-S-dependent activities were normal in
SDHAF1-defective organisms (Ghezzi et al., 2009).
The function of another assembly factor, SDH5 (EMI5/
YOL071W) was discovered in yeast. The sdh5D mutant is
characterized by defective oxidative growth, impaired respiratory activity, decreased chronological life-span, hypersensitivity
to H2O2, lack of SDH activity and loss of complex II. Sdh5
interacts with Sdh1, but is not a stable component of complex II,
and it is both necessary and sufficient for Sdh1 flavination and
thus for SDH activity (Hao et al., 2009). After the identification
of the yeast Sdh5 and the assignment of the function, it was
demonstrated that mutations in the human orthologue gene
C11 or f79 (renamed hSDH5) are responsible for paraganglioma.
Thus, an uncharacterized mitochondrial protein in yeast was
shown to play a crucial role in the biogenesis and function of
complex II and its mutational inactivation was found to confer
susceptibility to cancer in humans (Hao et al., 2009).
SDH is a peculiar mitochondrial enzyme because its mutation causes both typical mitochondrial disease and cancer.
Mutations in SDHB, SDHC and SDHD are linked with
dominantly inherited paragangliomas and phaechromocytomas (Devlin, 2000; Niemann & Muller, 2000; Astuti et al.,
2001; Baysal et al., 2000; Goffrini et al., 2009), while mutations
in SDHAF1 lead to infantile leukoencephalopathy (Ghezzi
et al., 2009), mutations in SDHAF2 segregate with hereditary
paraganglioma and mutations in SDHA cause Leigh syndrome, an early onset encephalopathy (Bourgeron et al., 1995;
Parfait et al., 2000; Van Coster et al., 2003; Horváth et al.,
2006); SDHA mutations have also been reported in a large
consanguineous family with isolated cardiomyopathy (Levitas
et al., 2010). Only recently, a case of paraganglioma has been
associated with a mutation in SDHA (Burnichon et al., 2010).
SDH is an enzyme of the TCA cycle, and perhaps this is why,
despite being a mitochondrial enzyme, its mutations give rise
to cancer, as was indeed recently observed with other enzymes
of the TCA cycle (Selak et al., 2005; Mithani et al., 2007;
Parsons et al., 2008). The relationship between mitochondrial
dysfunction and cancer, already suggested at his time by
Warburg (1956), and then long forgotten, has received renewed
interest in recent years, as reported by Chen et al. (2009) in
their work ‘in New Perspectives on the Warburg Effect’.
Nuclear mutations affecting the mitochondrial
fusion and fission system
A new class of mitochondrial diseases (which has recently
been the object of considerable attention) is caused by
nuclear mutations affecting mitochondrial morphology
(Detmer & Chan, 2007).
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Mitochondrial diseases and the role of the yeast models
Actually, the refinement of microscopic techniques has
revealed a completely new viewpoint on these organelles:
their shape, their connections, their mobility in the cell and
the corresponding defects.
The mitochondrial network is regulated by two dynamically opposed processes, fusion and fission of the mitochondrial membranes. Most proteins mediating yeast
mitochondrial fusion and fission are conserved in flies,
worms, plants, mice and humans, indicating that the fundamental mechanisms controlling mitochondrial morphology
have been maintained during evolution. This conservation
places S. cerevisiae as a good model for studying molecular
mechanisms of mitochondrial dynamics.
Studies of yeast mutants, based on defects in mitochondrial shape, identify three major morphology pathways:
fusion, fission and tubulation. When fusion is blocked,
mitochondria fragment due to ongoing fission. When
fission is blocked, mitochondria form interconnected nets
due to ongoing fusion. When the tubulation pathway is
disrupted, mitochondria are converted into large spheres.
While the fusion and fission proteins have human homologues, proteins of the tubulation apparatus are not conserved in evolution. Indeed, the first proteins required for
mitochondrial distribution and morphology (McConnell
et al., 1990), and tubulation, (Sogo & Yaffe, 1994) were
identified in the early 1990s by genetic screens in S.
cerevisiae.
For many years, the fission/fusion equilibrium has been
investigated mainly in yeast, but it was soon evident that the
proteins involved in this key aspect of mitochondrial function are highly conserved in evolution. Accordingly, mammalian cells exhibit the same fission/fusion events observed
in yeast and several human, mainly neurodegenerative,
pathologies derive from alteration in this equilibrium.
Mitochondrial fusion requires the evolutionarily conserved GTPase called Fzo1 (fuzzy onions) identified in 1998
in budding yeast (Hermann et al., 1998; Rapaport et al.,
1998) and Mfn (mitofusin) in mammals (Santel & Fuller,
2001; Eura et al., 2003). Mgm1 (Shepard & Yaffe, 1999;
Wong et al., 2000) is a second GTPase essential for mitochondrial fusion; The human orthologue of Mgm1 is OPA1
(Alexander et al., 2000; Delettre et al., 2000). Mgm1 is
present in two forms: one integrated into the inner membrane and a second in the intermembrane space, which is
produced by proteolytic processing. The third protein of the
fusion complex is Ugo1, present only in fungi (Sesaki &
Jensen, 2001). Ugo1p is an outer membrane protein with its
N terminus exposed to the cytoplasm and its C terminus in
the intermembrane space.
Mitochondrial fission requires a family of conserved,
dynamin-related GTPases, called Dnm1 in yeast (Bleazard
et al., 1999) and Drp1 in humans (Smirnova et al., 2001).
Dnm1 is a cytoplasmic protein and it can assemble into
punctate structures on mitochondria in the sites of future
mitochondrial fission. The evolutionarily conserved integral
membrane proteins, Fis1 in yeast (Mozdy et al., 2000) and
hFis1 in humans (James et al., 2003; Yoon et al., 2003), play
an essential role in mitochondrial fission. Fis1 is a tailanchored outer mitochondrial membrane protein with its
N-terminal domain exposed to the cytoplasm. Fis1 recruits
the Dnm1 to promote mitochondrial fission.
Studies in yeast revealed two additional proteins required
for mitochondrial fission: Mdv1 (Fekkes et al., 2000; Tieu &
Nunnari, 2000; Cerveny et al., 2001) and Caf4 (Griffin et al.,
2005). They collaborate to recruit Dnm1 into punctate
structures on mitochondria. No human homologues of
Mdv1 and Caf4 have been identified.
Figure 1 shows a cartoon of inner and outer mitochondrial membranes with localized yeast proteins involved in
fusion and fission apparatuses.
Loss of fusion results in mitochondrial fragmentation due
to ongoing fission events. Fragmented mitochondria
Fission defect
Fusion defect
Fission
apparatus
Fusion
apparatus
Dnm1/DRP1
Fzo1/MFN1,2
Ugo1
Mgm1/OPA1
Caf4
Fis1/hFIS1
Mdv1
Mdm33
mim
mom
Fig. 1. Cartoon of inner and outer mitochondrial membranes with localized yeast proteins involved in fusion and fission apparatuses. Fusion and fission
defects as they appear in yeast are also shown. Red colour indicates yeast proteins having high homology with human proteins involved in mitochondrial
morphology-based diseases. Photographs were adapted from Okamoto & Shaw (2005).
FEMS Yeast Res 10 (2010) 1006–1022
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T. Rinaldi et al.
Table 1. Yeast genes involved in mitochondrial morphology and their diseases-associated human orthologues
Yeast gene Function
Human gene
Disease
Human genes mutated in human diseases with homologous genes in yeast
FZO1
Fusion
MNF2
CMT2A
(Charcot-Marie-Tooth 2A)
MGM1
Fusion
OPA1
ADOA
DNM1
–
Fission
Fission
DRP1
GDAP1
(ganglioside-induced
differentiationassociated protein)
–
CMT4A (Charcot-MarieTooth 4A) and CMT2K
(a dominant form)
Mutations identified
in human diseases
Description
% of identity
Autosomal dominant
peripheral neuropathy
Autosomal dominant
optic atrophy
Neonatal lethality
Autosomal recessive
peripheral neuropathy
Low homology only in 757 aa, 50 mutations
the GTPase domain
33% in the GTPase
960 aa, 117 mutations
domain
51%
699 aa, 1 mutation
358 aa, 29 mutations
For a complete list of human peripheral neuropathic mutations, see http://www.molgen.ua.ac.be/CMTMutations/
eventually lose mtDNA by unknown mechanisms. Yeast cells
defective in fusion cannot, therefore, grow on nonfermentable media, which require respiration for energy production (Chen et al., 2010). Mutations in the nuclear-encoded
proteins involved in the above apparatuses result in important alterations in mitochondrial morphology and this has a
relevant influence on the mitochondrial movements.
Fusion is important to protect mitochondrial function by
enabling protein complementation, mtDNA repair and
equal distribution of metabolites. Fission instead helps to
isolate damaged mitochondria and promotes autophagy
(Chen & Chan, 2009).
Mitochondrial mobility has an important function in
yeast: suffice it to say that the presence of mitochondrial
structures is necessary in the bud to ensure viability (Garcı́aRodrı́guez et al., 2009; T. Rinaldi, pers. commun.).
On the other hand, in human neurons, the mitochondrial
movement for long distances towards the synapses is essential for neural functions and, if defective, leads to neurodegeneration (Knott et al., 2008).
In Table 1, we report a list of human genes, with
orthologues in yeast, involved in mitochondrial morphology, which produce, when mutated, neurodegenerative
diseases. In a situation in which mitochondrial movements
are essential for neuron physiology, mitochondria having an
altered shape should be eliminated; therefore, a complex
mechanism exists connecting mitochondrial dynamics,
fusion/fission equilibrium, movement and mitophagy.
Several neurodegenerative diseases caused by mutations
in the fission–fusion genes have been described (Liesa et al.,
2009). Mutations in MFN2 gene cause Charcot–Marie–
Tooth (CMT) subtype 2A, a peripheral neuropathy that is
characterized by muscle weakness and axonal degradation of
sensory and motor neurons (Kijima et al., 2005; Züchner
et al., 2004). Mutations in OPA1 cause the most common
form of optic atrophy: the autosomal dominant optic
atrophy (ADOA). Patients with ADOA exhibit progressive
loss of vision and degeneration of the optic nerve and retinal
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ganglion cells (Carelli et al., 2004). In addition, some
mutations in the OPA1 GTPase domain cause ‘ADOA-plus’
phenotypes that are also characterized by deafness, sensorymotor neuropathy and muscle-movement disorders (Amati-Bonneau et al., 2008; Hudson et al., 2008).
Mutations in fission apparatus seem to be more severe.
Recently, a dominant-negative mutation was reported in the
human DRP1 gene (Waterham et al., 2007). The patient
exhibited elongated mitochondria around the nucleus, a
feature that is characteristic of impaired mitochondrial
fission. Unfortunately, the patient died 37 days after birth.
The neurological features of this patient consisted of severe
neonatal hypotonia, abnormal brain development and
abnormal gyral pattern. The early onset suggests that mutations in fission proteins are much more severe than those of
the fusion mutations. Mutations in the human GDAP1 gene,
with no homologue in yeast, cause CMT4A, another subtype
of CMT syndrome (Baxter et al., 2002; Cuesta et al., 2002).
The homozygous GDAP1 mutation causes early onset and
more severe progression. GDAP1 protein localizes in the
outer membrane and seems to participate in DRP1-dependent mitochondrial fission (Niemann et al., 2005).
To the list of diseases certainly deriving from mutations in
proteins involved, at the level of mitochondrial membranes,
in the fusion/fission equilibrium, we should probably add, at
present, the Huntington disease, which heavily involves
mitochondria, and is monogenic and not polygenic like
Parkinson and Alzheimer diseases. This fatal autosomic
dominant disease is related to the presence in the gene of
an increased number of CAG triplets (n 4 35–40, which is
the normal range). In these cases, the mutated Huntingtin,
mtHtt, produces a fatal neuropathy and dementia in adulthood. The molecular mechanism of this disease is debated,
but severe mitochondrial damage is present. If it is confirmed that Htt has a function in regulating the relationships
among cell organelles, mitochondrial defects might be
generated by malfunctioning of the fusion/fission equilibrium (Reddy et al., 2009).
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Mitochondrial diseases and the role of the yeast models
This group of mitochondrial defects is only now starting
to be studied and clarified in detail, but it is already clear
that both inner and outer mitochondrial membranes are
very dynamic compartments in which some proteins are
stably localized, but others participate only when needed to
determine the necessary structure. These phenomena are
certainly present in all eukaryotic cells, but S. cerevisiae has
been the model in which this complex dynamics has been
studied (Khurana & Lindquist, 2010).
Mitochondrial-encoded mutations
Human mitochondrial diseases due to mutations
in the mitochondrial genome: a short overview
The human mitochondrial genome codes for 13 proteins of
the RC: seven for complex I (ND1, ND2, ND3, NND4, ND4L,
ND5 and ND6), cyt b from complex III, three for complex IV
(CO1, COII, COIIII) and two (ATP6 and ATP8) for complex
V. In addition, the human mitochondrial genome also
encodes 22 tRNAs and two rRNAs, which are different from
the cytoplasmic ones. Apart for complex I which is absent,
all OXPHOS proteins as well as tRNAs and rRNAs have their
equivalent in S. cerevisiae. Mutation rate in mitochondria
is high (around 3 10 5 to 3 10 6 as compared with
2.5 10 8 for the nuclear genome; Nachman et al., 1996;
Schriner et al., 2000). In theory, one would expect pathologic
mutations to be randomly distributed in all the coding
sequences, but this is not so. The list of potential (reported)
and confirmed mutations is regularly updated on the MITOMAP website (http://www.mitomap.org). To this day, 27
confirmed tRNA mutations have been described, while 25
mutations in total have been identified in the other coding
regions. This mutational bias in favour of tRNA genes, which
represent only about 1/10th of the genome sequence, is also
observed among all reported cases, and among tRNAs, a
second bias is observed in favour of tRNALeu UUR. A possible
explanation for this nonrandom distribution may be the more
or less deleterious effects of some mutations that could be
selected against in the germline because this phenomenon has
been observed in mice in vivo (Fan et al., 2008; Stewart et al.,
2008), but this is probably not the only explanation.
Very poorly diagnosed 20 years ago and largely underestimated, the presence of potentially pathogenic mtDNA
mutations is now estimated to be 1 : 200 (Elliott et al., 2008)
but result in mitochondrial diseases in only 1 : 5000 cases
(Schaefer et al., 2008). This does not allow these diseases to
be considered as rare anymore.
A large variety of syndromes is associated with these
mutations (deletions, rearrangements and point mutations).
They will not be detailed here but more information can be
found on the MITOMAP website and in two very recent
reviews (Wallace & Fan, 2009; Tuppen et al., 2010).
FEMS Yeast Res 10 (2010) 1006–1022
The specificities of mitochondrially encoded
mutations
As compared with diseases derived from nuclear-encoded
mutations, mitochondrial-encoded ones have their own
specificities.
All mtDNA deletions and many mtDNA point mutations
are heteroplasmic. Because a cell contains many mtDNA
molecules, most cells contain in fact a mixture of mtDNA
wild-type allele and mtDNA mutated allele. This proportion
may vary in the progeny and in the different tissues. It is now
clear that the severity of the disease is often associated with
the relative proportion of mutated molecules, introducing
the notion of ‘threshold’. It is also logical to expect more
deleterious effects of the mutations in tissues that are
strongly high-energy dependent, such as the heart, the brain
or the muscles (see Tuppen et al., 2010 for a discussion of
these points). However, these two basic facts do not account
for all the observations that have been made. For example,
many different diseases are associated with mt tRNA mutations, which cannot be related only to the molecular effect of
the mutation: the same mutation in the same tRNA can be
sometimes associated with a large variety of syndromes from
mild to severe, and different tRNAs when mutated confer
the same syndromes (reviewed in MITOMAP, 2009; Scaglia
& Wong, 2008). It is probable that the nuclear genetic
background of each individual plays a large role in the
syndrome outcome. How could it be explained that a
mother carrying a homoplasmic tRNAVal mutation
(C1624T) is asymptomatic while her seven children from
different partners were all suffering from extremely severe
pathogenesis? A profound RC deficiency was observed in
both the mother and her children (Mc Farland et al., 2002).
The role of such ‘nuclear modifier gene’, necessary for the
tRNA mutation to express the disease phenotype, has been
reported in a tRNA Ile mutation (Davidson et al., 2009).
The complexity of the system where oogenesis and
differentiation probably play an important role is far from
being understood. The cellular programme and in particular
the transcriptional regulation that presides to these critical
steps as well as the mtDNA bottleneck that takes place
during the oogenesis (reviewed in Tuppen et al., 2010) are
certainly key factors to explain the variable penetrance and
the phenotypic variability that are observed.
The role of the yeast model
Considering the complexity of mitochondrial diseases in
general, animal models of such diseases are urgently needed.
With the possibility to make transgenic mice at will, they
have been largely developed for nuclear- encoded mutations.
This is far from being the case for diseases due to mutations
in the mitochondrial genome. Nucleic acids are naturally
not imported into yeast mitochondria with the exception of
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1014
a cytoplasmic tRNA Lys (Tarassov et al., 1995) and mammalian mitochondria cannot be transformed with an in vitro
modified mitochondrial gene. Several factors could explain
it: the very high number of mtDNA copy, the low efficiency
of recombination and the absence of a powerful selective
system. Even the incorporation of exogenous RNA into
isolated mammalian mitochondria by electroporation has
been unsuccessful because the RNA is not expressed
(McGregor et al., 2001). Alternative strategies have been set
up, based on somatic genetics, with the goal to construct
genetically modified ES cells that can be later reintroduced
into the mouse female germline. A few mice models carrying
specific mutations have been produced (see reviews in
Tyynismaa & Suomalainen, 2009; Wallace & Fan, 2009).
Manipulation of the mitochondrial genome in the germline
based on the selection of mutations resistant to mitochondrially targeted restriction enzyme have been elegantly
applied to Drosophila (Xu et al., 2008) and this method
could certainly also be applied to mice. However, these
constructions are laborious and there are arguments (Fan
et al., 2008) suggesting that a mouse model carrying a highly
pathogenic mutation may not be possible. To circumvent
these difficulties, researchers have tried to indirectly engineer the mtDNA by acting upon nuclear genes involved in
mtDNA replication and maintenance. Particularly interesting are the ‘Mutator mice’ (Trifunovic et al., 2004). The
in-depth analysis made by Foury et al. (2004) on the yeast
mtDNA polymerase (coded by the MIP1gene) has shown
that its proofreading domain can be mutated, leading in
some cases to a strong mutator activity. Transposed to
mouse, appropriate mutations also lead to a strong
enhancement of mitochondrial mutations among which
one can be lucky and find the ‘pathogenic mutation’ that is
searched. This study is once more a very good example of
how yeast can help in expertly guiding what could be done
in mammals. For the moment construction of appropriate
mice models is still very laborious and specific mutations
very difficult to obtain. Despite these limitations, the various
mice mutants that have been developed have been instrumental to mimic some syndromes and to show that mtDNA
mutations can be the primary cause of neurodegenerative
diseases. They also permitted to understand tissue specificity
and the segregation of mtDNA molecules as well as to
investigate the role of mtDNA in ageing and other degenerative phenotypes (Kujoth et al., 2005).
In contrast to the difficulties encountered to establish
mitochondrially mutated mice, the yeast system offers some
facilities to create at will the precise desired mutations.
While nobody pretends that the information obtained
will provide knowledge on how mitochondrial diseases
appear and are transmitted in a sophisticated differentiated
organism, yeast ‘models’ of human mitochondrial diseases
offer information on genetic and molecular aspects; they
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T. Rinaldi et al.
also allow to distinguish between the significant number of
neutral mtDNA variants and pathogenic mutations, a question that in humans is difficult to resolve because of the high
mutational rate of the mitochondrial genome and the
presence of population-specific polymorphism (Tuppen
et al., 2010). In addition, the power of yeast genetics allows
to easily screen for compensatory mutations (see Exploiting
these model yeast strains).
The key point, if only one, which amply justifies the
utilization of yeast, is the possibility to transform mitochondria. Initially shown by Johnston et al. (1988) and Fox et al.
(1988), the biolistic transformation (shooting at high velocity microprojectiles layered with manipulated DNA) has
been improved enough to be used as a routine technique
(Bonnefoy & Fox, 2007). Second, and opposite to what
is observed in mice where many interesting mutations
are eliminated after transmission to the female germline
(see discussion in the previous paragraph), all mutations
whatever their consequences can be kept in yeast. Yeast
(S. cerevisiae) can survive on fermentative carbon substrates
with completely nonfunctional mitochondria. One could
add that it is very easy to change the nuclear background for
the same mitochondrial genome, based on a karyogamy
mutant (kar1-1; Conde & Fink, 1976). The kar 1-1 mutation
considerably delays the karyogamy and allows to reassociate
a given mitochondrial genotype with a new nuclear one.
Finally, the fact that yeast becomes very rapidly homoplasmic when two mitochondrial populations are mixed could
also be an advantage. This property does not allow to study
the threshold effect (which can however be mimicked by the
different nuclear background), but it leads to the simplification of a very complex system, which is a welcome first step
to start any analysis.
Some examples of ‘humanized’ yeasts
Saccharomyces cerevisiae mitochondrial transformation is
mostly based on two important characteristics (1) the
possibility for yeast mitochondria to efficiently replicate
many sequences – and in particular plasmids – which do
not have the true mitochondrial replication origins (Fox
et al., 1988) and (2) the high efficiency of homologous
recombination. The fact that mitochondrial genetics has
been developed for years in this organism offers a third
essential tool: appropriate mutations and markers. Biolistic
transformation is ‘inefficient’ in the sense that most cells are
killed by the process. Selecting survivors and appropriate
recombinants is based on a positive selection, supported by
appropriate genetic markers. A detailed description of this
process and associated protocols can be found in Bonnefoy
& Fox (2007). Finally, we should mention the essential fact
that the products of mitochondrial genes (proteins or RNA)
are largely conserved between human and yeast.
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Mitochondrial diseases and the role of the yeast models
Cyt b is certainly the protein that presents the largest
spectrum of mutations; some of them were isolated by
random mutagenesis many years ago, but the panel was
largely extended via biolistic transformation (Meunier,
2001; Bratton et al., 2003; Fisher et al., 2004a, b; Blakely
et al., 2005; Wenz et al., 2006). The positive screening is
based on the ability to grow on respiratory medium
(see Wenz et al., 2006 for details). Site-directed mutagenesis
was also used to model selected regions of the mammalian
Qo site in yeast cyt b in order to further understand the
differential efficacy of these Qo-site inhibitor in the mammalian and pathogen bc1 complexes (Kessl et al., 2005). In
these cases, selection was based on inhibitor resistance
(Fisher & Meunier, 2005). These mutations were informative to define the biochemical defects of mutations and to
explain their effects in relation with the structural threedimensional (3D) scaffold. Suppressors were isolated (phenotypic reversion to the respiratory competent phenotype)
and shown to be intragenic. They explain or confirm the 3D
interpretation and its effect on the phenotype (see some
examples in the review by Fisher & Meunier, 2001). A new
screening method, based on the ARG8m mutation, has
been devised to allow selection of any cyt b mutations
whatever their phenotype (functional or nonfunctional).
ARG8m is an allotopically expressed nuclear gene now
expressed in the mitochondrial genome (Steele et al., 1996)
and the screen is based on arginine auxotrophy/prototrophy
(Ding et al., 2008).
The latter method has also been used to create mutations
of ATP6. One of its advantages is also to eliminate deletions of
mtDNA (rho /rho1), which accumulate as a consequence of
the primary mutation because arginine prototrophy requires
a functional translation absent in rho cells. Four mutations
that mimic human pathological ones are described (Rak et al.,
2007; Kucharczyk et al., 2009a, b, 2010). A thorough biochemical investigation has shown that these mutations have similar
impact on the ATPase of human and yeast origin. They
sometimes can clarify contradictory results or offer new
informations not yet obtained in humans. Consequently, they
open new directions for investigations. The last example of
‘humanized’ yeast strains is the case of tRNA mutations.
Human mt tRNA mutations are over-represented and exhibit
a very large diversity, structural (any domain of the cloverleaf
structure), biochemical (processing, aminoacylation, interaction with ribosome, stability, etc.) and phenotypic (from severe
syndrome such as MERFF or MELAS to milder ones such as
diabetes or CPEO). A precise list and description of the human
pathogenic mutations can be found in Scaglia & Wong (2008).
It is also in a tRNA that the first ‘dominant’ mitochondrial
mutation has been identified (C5545T in tRNATrpS, Sacconi
et al., 2008), in contradiction with the ‘threshold rule’.
In yeast, the first mutations introduced into tRNALeu UUR
gene were MELAS pathogenic mutations. Because this is a
FEMS Yeast Res 10 (2010) 1006–1022
severe syndrome, one could expect that yeast cells bearing
the same mutations would exhibit a strong phenotype.
Based on this hypothesis and because it was impossible to
screen with the ARG8m gene, which requires an active
mitochondrial protein synthesis absent in this type of
mutant, the authors relied on sequencing the tRNALeu UUR
gene of only about 30 ‘petite’ colonies. Indeed the mutation
was identified in two colonies, confirming the expected
strong yeast phenotype. Such cells were not able to grow on
respiratory substrates and moreover produced a high
percentage of rho cells. (Feuermann et al., 2003). Mutations for which the phenotype could be very weak or barely
detectable would require one to sequence the gene in
thousand or more colonies, considering the recombination
frequency. To circumvent these difficulties, the authors later
on turned to the use of an artificially created restriction site
(ACRS)-PCR technique – an analysis that does not preclude
any resulting phenotype – and introduced other mutations
either in the tRNALeu UUR gene or in other tRNA genes.
Molecular and structural consequences of those were analysed in detail (Montanari et al., 2008; De Luca et al., 2009)
based on semi-denaturing Northern blots in which the
amount, the size and the aminoacylation of the tRNAs could
be examined. Two conclusions could be drawn from such
studies: (1) a correlation between the severity of the in vivo
phenotypes of yeast tRNA mutants and those obtained by in
vitro studies of human tRNA mutants supports the view that
yeast is a suitable model to study the cellular and molecular
effects of tRNA mutations involved in human pathologies.
This correlation was also observed for molecular effects. For
example, the aminoacylation defect of the A3243G mutation
(Sohm et al., 2003) is also observed in yeast (Montanari
et al., 2008). The same similarity holds true for the thermosensitive aminoacylation defect of the yeast T3250C equivalent mutation, which parallels the moderate severity of the
in vitro aminoacylation defect of the human mutation. (2)
The phenotypic effects of the mutations is considerably
dependent on the nuclear background of the wild-type
strain in which the mutations have been introduced, but
the relative strength of the phenotypes within one background is conserved in the others.
Exploiting these model yeast strains
As shown previously, once yeast mutations similar to
human pathogenic mutations have been created, the first
objective is to genetically and biochemically characterize
them, a task that is made easier by the facilities to grow yeast
cells (in terms of rapidity, yield and simplicity of manipulation) and the homoplasmic state. Moreover, one irreplaceable quality of yeast is the power of its genetics. Once a
mutation is obtained, there is no limit to search for
compensatory mutations (suppressors), which will alleviate
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1016
the defective phenotype. Conversely, it is also possible to
search for synthetic lethal mutations that, when associated
with a mutation with a nondetectable or weak phenotype,
will worsen it. Suppressors can be selected either as secondary mutation or as wild-type gene dosage modification
(multicopy suppressors). These possibilities have indeed
been exploited with the mutations described above.
The search for suppressors has already been described (see
Some examples of ‘humanized’ yeasts) in the case of cyt b
mutations; all of them turned out to be intragenic second-site
mutations, and, when placed within the 3D structural model,
they provided insights into the molecular interactions within
the structure. Such knowledge had important consequences,
in particular for drug selection (Fisher & Meunier, 2008). A
different strategy was applied to identify suppressors for
ATP6 and tRNA genes. The phenotypic correction was looked
for with multicopy suppressor, i.e. a change in wild-type gene
amount. The gene ODC1, a member of the mitochondrial
carrier family that exchanges intermediates of the TCA cycle
across the inner membrane is able, when overexpressed, to
increase ATP production, thus correcting mutations with an
impaired ATP synthesis (Schwimmer et al., 2005). It seems
that under such conditions the ODC1 gene can improve the
respiratory capacity of the yeast NARP T8993G mutation
(cited in Kucharczyk et al., 2009c). In this case, the gene
overexpression allows a metabolic correction, by producing
alternative ATP synthesis.
Overexpression of the TUF1 gene, which encodes the
mitochondrial translation elongation factor mtEF-Tu was
initially identified in a screen to correct the defective
phenotype of several MELAS mutations of the yeast mt
tRNALeu UUR (Feuermann et al., 2003). Interestingly, it
appeared later that the same gene can in fact correct all
types of tRNA mutations. Cognate tRNA synthetases can
also compensate the mutations (De Luca et al., 2006, 2009).
The effect of tRNA synthetase could be understood because
overexpression can overcome weaker affinity for the substrate, but the fact that mutated leucyl-tRNA synthetase with
highly reduced catalytic activity maintains full suppressing
effect rather suggests a chaperone-like and/or stabilizing
function and this is probably also the case for the general
suppression effect of mtEF-Tu (De Luca et al., 2009).
Future developments
Saccharomyces cerevisiae has proven to be a performing
organism to mimic human pathological mutations of mitochondrial origin but it has its own limitations. For example,
complex I is absent and replaced by a nuclear-encoded NADH
dehydrogenase. However, biolistic transformation is being
developed in other yeasts. Recently, mitochondria of Candida
glabrata were transformed with appearance of heteroplasmy.
Deepening this observation may provide interesting informa2010 Federation of European Microbiological Societies
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T. Rinaldi et al.
tion to control it (Zhou et al., 2010). Because Yarrowia
lipolytica possesses the mitochondrial-encoded subunits of
complex I, it may be used in the future to model complex I
human mitochondrial mutations. At present, only Chlamydomonas reinhardii for which mitochondria can also be transformed are used to study this complex (Remacle et al., 2006).
The work in S. cerevisiae is only possible if the human and
yeast gene products are highly similar, which means that all
tRNA mutations cannot be analysed. To overcome this
difficulty, efforts are made to have a complete human tRNA
gene introduced into the yeast mitochondrial genome.
Recently, Y. Zhou and M. Bolotin-Fukuhara (pers. commun.) have been able to show that the human tRNALeu UUR
can be transcribed and properly matured, including the
terminal CCA, into yeast mitochondria. It remains to be
seen if it can be correctly aminoacylated and functional.
Based on exploiting the natural RNA import system,
Kolesnikova et al. (2010) have developed a set of small RNA
molecules opening the possibility of creating a new mitochondrial vector system able to target therapeutic oligoribonucleotides into deficient human mitochondria.
Alternatively, gene therapy based on mitochondrial gene
versions expressed in the nucleus and artificially reimported
into mitochondria or nuclear suppressors able to correct the
defective mitochondrial gene can be foreseen in the future.
Multicopy suppression is especially valuable for such
approaches. The case of Ef-Tu, which is a general suppressor
of all mutated tRNAs described up to now, is of special
interest and all the more because it has been recently shown
that it is also efficient to correct defective patient cell lines
(Sasarman et al., 2008). Finally, one should not overlook the
pharmaceutical approach. Yeast cells are easy to manipulate,
fast to grow and such process can be robotized at low costs.
They constitute therefore a method of choice for a rapid first
screening of pharmaceutical products active in human
diseases. Yeasts have been successfully used to find drugs
active against mammalian prions (Bach et al., 2003;
Tribouillard et al., 2006), and promising molecules active
on yeast models of ATP synthase deficiencies have already
been isolated (cited in Kucharczyk et al., 2009c).
Concluding remarks
In this review, we have discussed different aspects of mitochondrial diseases. Because, as noted in the Introduction,
several good reviews already exist, some of them focused
on yeast, we have chosen to focus on the more recently
developed aspects of mitochondrial defects, in particular
those concerning mtDNA maintenance, the importance of
mitochondrial morphology and the mitochondrial transformation.
The first important remark to be made is concerned with
the increasing complexity of the mitochondrial world that
FEMS Yeast Res 10 (2010) 1006–1022
1017
Mitochondrial diseases and the role of the yeast models
pathologies are disclosing to us. Starting from the second
part of the last century, we first were fascinated by the
presence of two genomes evolving in the same eukaryotic
cell, but now we see in detail the dynamic relationship
between the two genomes and the different organelles. The
importance of mitochondria in complex pathologies and in
the effects of ageing certainly adds to this complexity.
Here, we come to a central point: in all the described
pathologies, the possibility to establish a yeast model has
been an invaluable tool to control and verify basic effects.
The possibility to control yeast ploidy, to shift mitochondria
to different nuclear contexts and to immediately understand
the effect of an mtDNA mutation in the homoplasmic yeast
mitochondria have largely demonstrated the value of this
model. The new knowledge on the fine-tuning of mitochondrial functions by proteins localized in inner and outer
mitochondrial membranes, but also contacting inner and
outer plasm, opens a new field in which the yeast model is
indispensable and in which a new aspect, namely mitochondrial motility, becomes essential.
Finally, the successful use of yeast mitochondrial transformation can be an essential step towards a mitochondrial
pharmacology.
Moreover, we would like to stress the very high conservation of these complex functions between humans and
yeasts and hence the general applicability of the yeast models
even when pathological characteristics should be considered.
Acknowledgements
M.B-F has been continuously supported by the ‘Association
Française Contre les Myopathies (AFM) for the work on
yeast mitochondrial tRNA models. L.F. has been supported
by Telethon project GGP07164.
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