Download Hungry for Power: Elimination of Mitochondria by Mitophagy

SHOWCASE ON RESEARCH
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Hungry for Power:
Elimination of Mitochondria by Mitophagy
Alexander May and Mark Prescott*
Department of Biochemistry and Molecular Biology, and ARC Centre of Excellence for Structural and
Functional Microbial Genomics, Monash University, Clayton, Victoria 3800
*Corresponding author: [email protected]
Introduction
It is often said that nothing in life is free, and the role
played by the mitochondrion in eukaryotic cells is
no exception. Through oxidative phosphorylation,
mitochondria provide large amounts of energy to the cell,
which has made possible an energy-intensive way of life
and, ultimately, the evolution of increasingly complex
organisms. The cost, however, is that the production
of energy by this unique organelle produces many
potentially dangerous by-products, most notably reactive
oxygen species (ROS), which can damage a cell’s internal
structures, especially as the population of mitochondria
accumulate damage and age. The cell’s relationship
with its mitochondria must therefore be one of balance,
harnessing energy while mitochondrial ATP production
is efficient and removing mitochondria once they present
a hazard to the eukaryotic cell.
The challenges of removing the bulk of an entire
organelle, which is beyond the capacity of protein
degradation systems such as the proteasome, are overcome
by utilising the autophagic machinery of the cell. In broad
terms, this involves the isolation of cargo (in this case
mitochondria) into a double-membrane structure known as
the autophagosome and its delivery, via membrane fusion,
to the lysosome (in mammalian cells) or the vacuole (in yeast)
for degradation (Fig. 1, reviewed in (1)). This autophagy of
mitochondria, termed mitophagy (2), is a controlled process
that achieves selective removal of the organelle in response
to intracellular cues and extracellular stimuli. In addition
to the underlying quality control function, mitophagy is
employed to adjust mitochondrial mass during changes
in the availability of nutrients and during stationary phase
as yeast cells adapt to their environment, whereas the
process is observed during development and immune cell
Fig. 1. A simplified illustration of how autophagy,
specifically macroautophagy, occurs in yeast. Initiation
of autophagy is followed by a membrane expansion
event that ultimately encloses components destined for
degradation in the autophagosome. The autophagosome
then traffics to and fuses with the vacuole or lysosome,
releasing its contents into the lumen for degradation by
hydrolases. Finally, degraded components are returned
to the cytoplasm for reuse. Microautophagy (not shown)
is also an important autophagic route in yeast.
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Fig. 2. Mitophagy in yeast. According to one current model, mitochondrial stress (!) is sensed by Atg33p, which relays
the signal on to Atg32p, and in turn recruits core Atg proteins. Atg11p has been implicated in all types of selective
autophagy in yeast and most likely plays a scaffolding role in the interaction between Atg32p and Atg8p. Stressed
mitochondria can then be enclosed in an autophagosome and delivered to the vacuole for removal and recycling.
maturation in mammalian cells. Although conceptually
simple, the various roles of mitophagy suggest mechanistic
and regulatory complexity, offering a fascinating insight
into the cell’s Faustian pact with the mitochondrion.
Mitophagy in Yeast
Much of our knowledge of mitophagy has been obtained
through experiments examining the budding yeast
Saccharomyces cerevisiae, the key model organism of the
field. In addition to numerous ‘core’ ATG (autophagy)
genes, which are involved in all autophagic processes,
mitophagy in yeast relies on a number of additional genes,
the functions of many of which are yet to be elucidated.
Genetic screens by two separate groups have identified
two genes, ATG32 and ATG33, which are specific to
mitophagy (3,4). The precise function of their encoded
proteins is not fully understood; the outer membrane
(OM) protein Atg32p is thought to play a central role in
bringing a mitochondrion selected for degradation into
contact with the core autophagic machinery, whereas
Atg33p, also on the OM, is implicated in the sensing of
stress that triggers mitochondrial degradation (Fig. 2).
Once mitophagy is triggered, the selected mitochondrion
is delivered to the core autophagic machinery through an
interaction of Atg32p with Atg8p, the protein behind the
biogenesis and expansion of the autophagosome and the
homolog of mammalian LC3. The interaction between
Atg32p and Atg8p is facilitated by an Atg8 interacting
Fig. 3. Mitophagy in mammalian cells.
A. In the NIX-dependent pathway, associated with reticulocyte maturation, LC3 is recruited following the recognition
of mitochondrial stress (!), which induces autophagosome formation.
B. Mitochondrial stress can also be recognised by PINK1, which recruits Parkin. Parkin then ubiquitinates yet unknown
factors (?) that possibly initiate mitophagy. Both pathways result in delivery to the lysosome and degradation.
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motif (AIM), which is crucial to efficient mitophagy. The
adaptor protein Atg11p, which is essential for all selective
forms of autophagy identified to date, is also indispensible
in this interaction.
Additionally, a number of proteins originally identified in
targeted studies by their function in other processes have
also been implicated in mitophagy. These include Uth1p
(a SUN family protein involved in ageing and cell wall
biogenesis (5)), Aup1p (involved in the retrograde signalling
pathway between the mitochondrion and nucleus (6))
and Mdm38p (a mitochondrial inner membrane protein
associated with K+/H+ exchange (7)). The diversity of
proteins involved in mitophagy reinforces the point that the
degradation of mitochondria is an important and complex
cellular activity, enmeshed as a basic process in the cellular
milieu. However, our inability to describe this process in
any detail beyond a basic model also suggests that many
proteins involved in mitophagy are yet to be uncovered.
Clearly, much work remains to be undertaken in the field.
Mitophagy in Mammals
The results of research on mammalian cells have
proven to be equally intriguing, and further research has
been spurred on mainly by the association of perturbed
mitochondrial turnover with several neurodegenerative
disorders, including Parkinson’s disease and Alzheimer’s
disease (8). At the cellular level, mitochondrial integrity
appears to be maintained through mitochondrial dynamics,
with poorly performing mitochondria – as measured by
a loss of membrane potential – being isolated by fission
and prepared for degradation (9). Although similarities
in general autophagic mechanisms are apparent when
considering mammalian and yeast cells, the mechanism
behind mitophagy at the level of the organelle appears
to differ in mammalian cells from that of yeast, and
mammalian homologues to Atg32p and Atg33p are yet to
be described. However, proteins specific to mitophagy in
mammalian cells have been identified and offer an insight
into how mitochondria are selected for removal.
In mammalian cells, mitophagy is implicated in a range of
cellular functions in addition to that of the quality control of
mitochondria. An example is the maturation of reticulocytes,
a process requiring the removal of the cell’s entire population
of mitochondria as the cells develop into erythrocytes. This
process has been found to be mediated by a mitochondrial
OM protein, NIP3-like protein X (NIX) (10) (Fig. 3A). Like
the yeast protein Atg32p, the Bcl-2 family protein NIX
contains an AIM, which is responsible for interaction with
the mammalian homologue of Atg8, LC3. This interaction
brings mitochondria into contact with the autophagic
machinery, allowing mitophagy to occur. In reticulocytes,
NIX expression is upregulated immediately before the
elimination of the cell’s mitochondria, and deletion of the
gene is observed to block mitochondrial elimination. At the
same time, overexpression of another Bcl-2 family protein,
Bnip3, has also been shown to result in increased mitophagy
(11). While Bcl-2 family proteins are implicated in apoptosis,
it seems to be their ability to alter mitochondrial membrane
potential – a crucial factor in initiating mitophagy – that is
behind autophagic degradation.
Fig. 4. Schematic diagram of Rosella. The biosensor
incorporates a red fluorescent protein (DsRed T3) and
pH-sensitive green fluorescent protein (SEP) joined by a
short linker region (grey region). A targeting sequence
occupies the N-terminus. Excitation (Ex) and emission
(Em) wavelengths are indicated above.
Fig. 5. Output
of the Rosella
assay. The pHsensitive green
fluorescent protein
of mitochondriatargeted Rosella
fluoresces only
at cytosolic pH,
whereas the
red-emitting
fluorescent protein
fluoresces at both
cytosolic and
vacuolar pH. In
wild-type cells
(shown here)
cultured in nutrient-rich conditions, red and green emission is absent from the vacuole (A). However, upon removal
of nitrogen from the medium (starvation) vacuolar delivery of mitochondria (mitophagy), a characteristic red
vacuolar fluorescence is observed (B, white arrows).
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In addition to the NIX- and Bnip3-mediated induction
of mitophagy, the literature suggests the involvement
of other proteins in the priming of mitochondria for
elimination (Fig. 3B). Two proteins, PINK1 and PARKIN,
have received particular attention due to their association
with neurodegenerative disorders. In particular, PARKIN
loss-of-function mutations are the most prevalent genetic
mutation associated with early-onset Parkinson’s disease
(12). From available data, it is hypothesised that the
mitochondrial OM-localised Ser/Thr kinase PINK1 plays a
role in the detection of mitochondrial stress by an unknown
mechanism. PINK1 then communicates this stress by
binding PARKIN, a cytosolic ubiquitin ligase, which
subsequently ubiquitinates target proteins, bringing about
mitophagy (13). The targets of ubiquitination are not clearly
understood, but it is likely that they promote the fission of
stressed organelles from the greater mitochondrial network
for degradation, as perturbation of either of these genes
results in swollen mitochondrial morphology in cells.
Future Research
While the cooperation of such sensing and induction
mechanisms offers an attractive model of how mitophagy
occurs in mammalian cells, more work is essential to
describe the process at a level of detail useful for clinically
oriented research. For example, very little is known about
the regulation of mitophagy in both yeast and mammalian
cells. As with many autophagic processes, it is reasonable
to assume that, due to the fundamental role mitophagy
plays in cellular homeostasis, much more remains to be
learnt about the mechanisms of this process and its role
in the maintenance of homeostasis in cells. With the rapid
pace of research in the field, the coming years will see the
significance of this process reflected in important advances
in understanding of the unique relationship between
eukaryotic cells and the mitochondrion.
At the Monash laboratory, we investigate the mechanisms
and regulation of mitophagy by interrogating a number of
complementary yeast strain collections using a fluorescence
assay based on a novel biosensor known as Rosella (Fig. 4).
Rosella incorporates two fluorescent proteins, one red (pHstable) and the other green (pH-sensitive), which are joined
by a linker region (14,15). In our studies of mitophagy,
Rosella is targeted to the mitochondrial matrix. Green
fluorescence emission is strong at pH > 7.0, but is quenched
at vacuolar pH (< 6.0), while red fluorescence emission
remains strong in the pH range 4 to 11. Accordingly,
delivery of Rosella-tagged mitochondria to the acidic lumen
of the vacuole is accompanied by the quenching of green
but not red fluorescence, as demonstrated by red vacuolar
fluorescence in the merged image (Fig. 5B). By scoring
the proportion of cells undergoing mitophagy at selected
time points under different conditions, and in different
strains, an indication of the contribution and importance of
individual genes in mitophagy can be obtained. Utilising
this approach, we have uncovered the involvement of a
number of genes including a previously uncharacterised
open reading frame, OTP1, which appears to take part in an
early phase of mitophagy, possibly in concert with UTH1,
as well as a variety of other genes that appear to play a role
in the process. With further research, we hope to establish
how OTP1 together with other mitophagy genes contribute
to the simultaneously complex and elegant means of
mitochondrial homeostasis that is mitophagy.
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