© The Authors Journal compilation © 2013 Biochemical Society

Essays Biochem. (2013) 55, 93–104: doi: 10.1042/BSE0550093

8
Mitophagy
Thomas MacVicar1
School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol BS8 1TD, U.K.

Abstract
Mitophagy describes the selective targeting and degradation of mitochondria by
the autophagy pathway. In this process, defective mitochondria are first purged
from the mitochondrial network then delivered to the lysosome by the autophagy
machinery. Mitophagy has emerged as a key facet of mitochondrial quality control
and has been implicated in a variety of human diseases. Disturbances in the cellu-
lar control of mitophagy can result in a dysfunctional mitochondrial network with
grave implications for high energy demanding tissue. The present chapter reviews
the recent advancements in the study of mitophagy mechanisms and regulation.

Keywords:
Atg32, erythropoiesis, mitochondrial dynamics, mitochondrial dysfunction, Nix, parkin,
Parkinson’s disease, PINK1.

Introduction
For decades, mitochondria have been recognized as the powerhouses of eukaryotic cells and
we now appreciate that they function in other key cellular processes such as calcium signal-
ling and apoptosis. The mitochondrial network is an amazingly dynamic and adaptable orga-
nelle system that must remain healthy in order to meet changing demands for ATP. Indeed,
disturbances in mitochondrial homoeostasis result in an increasingly damaged and dysfunc-
tional mitochondrial network, leading to a range of human pathologies including ischaemia,
diabetes and neurodegeneration [1]. Dysfunctional mitochondria have the potential to generate
vast quantities of ROS (reactive oxygen species) and can become ‘leaky’, releasing pro-apoptotic
proteins into the cytoplasm, often with catastrophic consequences. If unchecked, deleteri-
ous mtDNA (mitochondrial DNA) mutations are also likely to manifest within the network

1
email tom.macvicar@bristol.ac.uk

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and further compromise mitochondrial activity [2]. Cells must therefore efficiently remove
and  replace damaged mitochondria with healthy ones in order to maintain their energy
supply.
Damaged mitochondria are targeted for degradation in the lysosomes via a specific
autophagic pathway called mitophagy [3]. When mitophagy was first described, the distinction
between it and the non-selective autophagy pathway was hazy. However, following the discov-
ery of proteins that function solely in the autophagic degradation of mitochondria [4], it soon
became clear that mitophagy has evolved as a unique quality control mechanism. By sequester-
ing dysfunctional (or sometimes redundant) mitochondria into double membrane autophago-
somes and trafficking them to lysosomes, cells are able to neutralize any threat posed by the
faulty powerhouses. Over the last 10 years, developments in techniques such as live-cell imag-
ing have allowed us to make exciting progress in the study of mitophagy dynamics and
regulation.

Mitophagy mechanisms
Mitophagy has been studied in a number of different systems both in vitro and in vivo and it
has emerged that mitochondrial degradation can be initiated by different environmental and
developmental cues. It is now clear that mitophagy is not solely a defence mechanism triggered
by mitochondrial damage. By exploring the molecular mechanisms of mitophagy in more
detail, we can begin to understand how it has evolved as an essential feature of both cellular
quality control and adaptation.

Mitophagy in yeast
Mitophagy in yeast depends on the delivery of mitochondria to the acidic lysosome-like vacu-
ole for degradation. Yeast initiate mitophagy in response to a number of conditions including
nitrogen starvation and rapamycin treatment [5]. They have proven to be important in demon-
strating that mitophagy can proceed via tightly regulated mechanisms distinct from the com-
mon autophagy pathway. Proteins such as the OMM (outer mitochondrial membrane) protein
Uth1p were first demonstrated to regulate mitophagy independently of autophagy under cer-
tain conditions [4]. Subsequent genetic screens identified another OMM protein, Atg32, as
being essential for mitophagy [6,7]. In order to guide ill-fated mitochondria to the autophago-
some, Atg32 binds directly with the resident autophagosome protein Atg8 and also interacts
with the previously identified selective autophagy adaptor protein Atg11 [8] (Figure 1).
Positive regulation of the Atg32–Atg11 interaction requires MAPK (mitogen-activated protein
kinase)-dependent phosphorylation of Atg32 at Ser114 [9]. This phosphorylation event is criti-
cal to mitophagy as mutation of the serine residue results in abolishment of the mitophagy
response to nitrogen starvation [9]. Although Atg32 has no known higher eukaryote homo-
logue, these seminal studies have certainly removed any doubts surrounding the selective
nature of mitophagy.
Work in yeast has also advanced our understanding of how mitophagy is regulated during
shifts in metabolism. For example, activation of mitophagy following an exchange of respira-
tory to fermentative conditions appears to signify a housekeeping role for mitophagy as redun-
dant mitochondria are selected for degradation [5].

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T. MacVicar 95

Figure 1. Mitophagy in yeast

Yeast mitochondria are targeted to the isolation membrane by the OMM protein Atg32. Atg32
can directly bind the autophagosome protein Atg8 or via interaction with an adapter protein
Atg11. Atg11 may provide extra control of autophagosome assembly around mitochondria by
interacting with both Atg32 and Atg8. Following the initial targeting, autophagosomes seal
around mitochondria and eventually fuse with the acidic vacuole for degradation of the
entrapped organelle [6–8].

Mitophagy during erythropoiesis

A striking example of mitophagy being employed to remove healthy yet redundant mitochon-
dria occurs during mammalian red blood cell development (erythropoiesis). Mature red blood
cells are devoid of mitochondria following the selective elimination of their entire mitochon-
drial population by a distinct mitophagy pathway [10]. During normal erythroid terminal dif-
ferentiation, expression of the OMM BNIP3-like protein Nix is augmented [11] and this is
necessary for complete mitochondrial depletion [12,13]. Importantly, an absence of Nix causes
defective erythroid maturation and anaemia in mice [12].
Similar to Atg32 in yeast, Nix confers the selectivity for mitochondrial engulfment by the
autophagosome. As discussed in Chapter 7 of this volume, Nix directly interacts with
autophagosomal marker proteins LC3 (light-chain 3) and GABARAP (γ-aminobutyric acid
receptor-associated protein) via its LIR (LC3-interacting region) motif [14] allowing the
recruitment and formation of autophagosomes around the mitochondria (Figure 2).
Interestingly, however, rescue of mitophagy in Nix−/− maturing blood cells is only partially sup-
pressed by mutation of the LIR motif and recently a newly characterized 16-amino-acid cyto-
plasmic region of Nix called the MER (minimal essential region) has been described as critical
for Nix activity [15]. Although LC3 does not interact with the MER [15], identification of other
possible mitophagy-related interaction partners will improve our mechanistic understanding
of mitochondrial clearance in developing red blood cells. Besides interacting directly with
autophagosome components, Nix can also regulate mitophagy by facilitating mitochondrial
membrane depolarization and ROS generation [16]. A Nix-regulated increase in ROS produc-
tion may provide a key step in mitophagy by enhancing autophagosome biogenesis via mTOR
[mammalian (also known as mechanistic) target of rapamycin] inhibition [16].
Nix-orchestrated mitophagy during erythropoiesis is arguably an example of pre-pro-
grammed mitochondrial clearance. How then do mammalian cells react to unexpected

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Figure 2. Nix regulated mitophagy during red blood cell development

Maturing red blood cells increase Nix expression on the OMM. Nix binds to LC3, the
mammalian homologue of Atg8, through its cytoplasmic LIR motif. This mediates the
engulfment of mitochondria by forming autophagosomes [11,12,15].

mitochondrial damage in order to preserve a healthy mitochondrial population? This question

appears to be particularly significant in respiring differentiated cells such as neurons and
hepatocytes. Given the pathological consequences of mitochondrial dysfunction in such tissue,
extensive research has begun to elucidate the mechanisms that exist to selectively target and
eradicate damaged mitochondria.

Parkin-mediated mitophagy and beyond

Mutations in the E3-ubiquitin ligase parkin [17] and PINK1 [PTEN (phosphatase and tensin
homologue deleted on chromosome 10)-induced putative kinase 1] [18] were found to cause
autosomal recessive forms of PD (Parkinson’s disease); however, it was not initially clear how
these two proteins contributed to the severe pathogenesis. The spotlight was cast on a possible
role in mitochondrial quality control when in vivo studies demonstrated that these mutations
manifest as mitochondrial dysfunction. Drosophila harbouring PINK1/parkin mutations suffer
dopaminergic neuron loss and locomotion defects, associated with abnormal mitochondria
[19,20]. Subsequently, elegant Drosophila genetics were employed to demonstrate that parkin
functions downstream from PINK1 in the same pathway [20,21].
The question remained of how mutations in PINK1 and parkin might result in the accu-
mulation of dysfunctional mitochondria. An answer was provided when Narendra et al. [22]
discovered that parkin normally functions to target damaged mitochondria for degradation by
mitophagy. Live-cell imaging of cultured cells expressing fluorescently tagged parkin revealed
the remarkably dynamic recruitment of cytosolic parkin to damaged mitochondria. These par-
kin-decorated mitochondria are then sequestered into autophagosomes and delivered to lys-
osomes for degradation. Astoundingly, many parkin-overexpressing cells can clear their entire
mitochondrial population after the induction of global mitochondrial damage by treatment
with mitochondrial depolarizing drugs.

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T. MacVicar 97

Questions remain of how physiologically relevant such wholesale examples of mitophagy

are, not least because more bioenergetically active cells have been demonstrated to resist parkin-
mediated mitophagy in response to depolarizing drugs [23]. In that study, cells with a greater
dependence on oxidative phosphorylation were found to inhibit parkin translocation to depo-
larized mitochondria [23]. Important discrepancies also exist between the functions of endoge-
nous and overexpressed parkin [24]. Further work examining the role of endogenous parkin,
particularly in disease-vulnerable neurons, is essential. Surprisingly, parkin-knockout mice do
not display hallmark PD phenotypes such as reduced motor ability and decreased neurological
function [25]. It remains unclear why parkin-deficient fly and mouse models vary so much in
the severity of Parkinsonism symptoms. Nevertheless, both in vitro and in vivo experiments
continue to be essential for further dissection of the parkin/PINK1 mitophagy pathway.
In a healthy mitochondrion, PINK1 is rapidly turned over by constitutive proteolytic pro-
cessing. However, following insult and dissipation of the inner membrane potential (mtΔΨ),
which is required to drive ATP production and mitochondrial protein import, PINK1 becomes
stabilized on the OMM permitting it to recruit its binding partner parkin [26,27]. PINK1 inter-
action with parkin is necessary for parkin self-association and activation of parkin’s ubiquitin
ligase activity [28]. Once activated, parkin ubiquitinates targets on the OMM allowing the clus-
tering of damaged mitochondria and their engulfment by autophagosomes (Figure 3).
Parkin is not the only ubiquitin ligase shown to direct mitophagy. Increased activity of the
ubiquitin ligase Mul1 results in enhanced mitophagy during skeletal muscle wasting.
Suppression of Mul1 maintains mitochondrial number and partially prevents muscle wasting
in mice exposed to muscle-wasting stimuli [29]. Finally, gp78 (glycoprotein 78) is another E3
ubiquitin ligase recently identified to drive depolarization-induced mitophagy in a parkin-
independent manner [30]. Interestingly, gp78 is ER (endoplasmic reticulum)-associated and,

Figure 3. Parkin-mediated mitophagy

In a healthy mitochondrion with an active membrane potential (↑mtΔΨ), PINK1 is imported to
the IMM and constitutively turned over by proteolysis. However, following a compromise of
mitochondrial activity and loss of membrane potential (↓mtΔΨ), PINK1 stabilizes on the OMM
allowing recruitment of the E3-ubiquitin ligase parkin. Parkin activation results in the
ubiquitination of OMM proteins that act as an ‘eat-me’ signal recognized by a nascent
autophagosome. LC3 can bind ubiquitin directly or may interact indirectly via adapter protein
p62 [22,26].

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Figure 4. Mitochondrial dynamics and mitophagy

Mitochondrial fission is used to purge unwanted mitochondria from the network. The GTPase
Drp1 drives dynamin-like scission of mitochondria. Parkin and other ubiquitin ligases can also
facilitate the fission of depolarized mitochondria by targeting the mitofusins for degradation.
Once divided from the network, exiled mitochondria can be engulfed by autophagosomes or,
alternatively, if they remain healthy with an intact membrane potential, they may be permitted
to re-fuse with the network. Mitochondrial fusion is coordinated by the OMM GTPases Mfn1
and Mfn2 and the IMM OPA1 (not shown). Enhanced fusion can protect mitochondria from
autophagy degradation [45,46,49].

upon mitochondrial depolarization, its ubiquitination activity recruits LC3 to engulf mito-
chondria in close proximity to the ER [30].
Exactly how autophagosomes are recruited to parkin-decorated mitochondria remains an
intriguing question. The adaptor protein p62 can bind both ubiquitin and LC3 and is found to
accumulate on mitochondria that have been polyubiquitinated by parkin. Despite this,
although a role for p62 in mitochondrial clustering looks likely [31], conflicting reports exist as
to whether p62 is actually essential for the recruitment of autophagosomes to mitochondria
(or vice versa) [31,32]. As we have seen with Atg32 in yeast and Nix in mammalian red blood
cells, mitophagy does not always depend on cytosolic adapter proteins for recruitment of the
autophagosome. Indeed, hypoxia-induced mitophagy also requires the direct interaction
between an integral OMM protein FUNDC1 and autophagosomal LC3. FUNDC1 binds LC3
via its LIR motif and this interaction is enhanced on FUNDC1 dephosphorylation during
hypoxic conditions [33]. Hypoxia-induced mitophagy therefore offers another mechanism
whereby mitochondria are targeted for mitophagy by regulated and direct interaction between
OMM and autophagosome proteins.
Regardless of targeting, further work is certainly required to elucidate the actual origins
of the ‘mitophagosome’. As discussed in Chapter 3 of this volume, several organelles have so far
been implicated in autophagosome biogenesis including the mitochondria themselves [34]. It
has also been suggested recently that the autophagic isolation membrane can form de novo on
depolarized mitochondria as opposed to being recruited from elsewhere in the cytoplasm [35].
There is much left to learn about the role played by mitochondria in autophagosome biogene-
sis, however, it is now apparent that mitochondria can also regulate their own degradation by
strict control of their morphology. The remainder of the present chapter focuses on the excit-
ing progress being made in the understanding of how, and why, mitophagy is regulated by
mitochondrial dynamics.

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T. MacVicar 99

Mitophagy regulation by mitochondrial

dynamics
It is impossible to discuss mitophagy without exploring the regulatory role played by mito-
chondrial dynamics. Mitochondrial fusion and fission have been implicated in many of the
classical mitochondrial-associated cellular pathways including calcium signalling, apoptosis
and the cell cycle. To explore how mitochondrial dynamics regulates mitophagy we must first
introduce the fission and fusion machinery.

Mitochondrial dynamics: a balance

between fusion and fission
Mitochondrial fission is driven by the dynamin-related protein Drp1 [36], and OMM proteins
Fis1 [37] and Mff [38]. Unlike Fis1 and Mff, the GTPase Drp1 is a cytoplasmic protein that
localizes to sites of mitochondrial fission in a rigorously regulated manner. Drp1 is the target
of several key post-translational modifications including sumoylation, ubiquitination and
phosphorylation [39]. These modifications either enhance or disrupt Drp1 activity in a num-
ber of ways such as by regulating Drp1 translocation to the mitochondria or by mediating
Drp1 assembly into spiral structures at the future sites of mitochondrial division.
The mitochondrial fusion machinery also consists of three key proteins: the OMM GTPases
Mfn1 (mitofusin 1) and Mfn2 [40] and the IMM (inner mitochondrial membrane) protein
OPA1 (optic atrophy 1) [41]. Although structurally similar, Mfn1 and Mfn2 have been associated
with separate functions. Of note, Mfn2 tethers mitochondria to the ER at contact sites that have
been shown recently to play an important role in autophagosome biogenesis [42]. On the IMM,
OPA1 is regulated by constitutive and inducible proteolytic cleavage and besides driving IMM
fusion, OPA1 also serves to remodel cristae; key for ATP production and apoptosis [43].

Linking fission with mitophagy

It is generally accepted that mitochondrial fission is a pre-requisite for mitophagy in many
mammalian cell types. As others have pointed out, it is logical for an elongated mitochondrion
of over 5 μm in length to be chopped up before engulfment by 0.5-μm-diameter autophago-
somes [44]. But beyond this, it is clear that the fission and fusion machinery offer invaluable
quality control and can help determine whether a mitochondrion is destined for degradation.
Twig et al. [45] first demonstrated elegantly that fission can generate fragmented mitochondria
with a low membrane potential and reduced levels of OPA1. These mitochondria therefore have
an inherent inability to re-fuse with the healthy mitochondrial network. Following their expul-
sion from the mitochondrial network, the mitochondria are finally removed by mitophagy.
In parkin-mediated mitophagy, parkin translocation to depolarized mitochondria triggers
the ubiquitination and proteasome-dependent degradation of the mitofusins [46], thus further
isolating dysfunctional mitochondria from the rest of the network. In other pathways, Mfn2 is
targeted by the muscle-wasting mitophagy ubiquitin ligase Mul1 [29] and both Mfn1 and Mfn2
are ubiquitinated by the mitophagy regulator gp78 [30]. Therefore obstructing mitochondrial
fusion by mitofusin degradation appears to be a common step in mitophagy initiation.

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Mitochondrial parkin recruitment also causes degradation of the kinesin motor adaptor
protein Miro [47]. This detaches kinesin from the mitochondrial surface leading to an arrest of
mitochondrial motility that may facilitate mitophagy. Such a mechanism is particularly signifi-
cant in neurons where mitochondrial transport is key for the distribution of functional mito-
chondria along axons to synapses. So by preventing the transport of damaged and potentially
toxic mitochondria to these vital domains, PINK1 and parkin may be limiting the risk posed
by dysfunctional mitochondria. In addition, Miro localizes to ER–mitochondrial contact sites
and it remains to be seen whether disruption of ER–mitochondrial tethering is a key step in
initiating mitophagy [48].

Fusion protects mitochondria from

mitophagy
Given the importance of mitochondrial fission in assisting mitophagy, is it conceivable that
mitochondria can resist mitophagy under certain conditions by inhibiting fission and promot-
ing fusion? Owing to metabolic and bioenergetic demands, it sometimes becomes the case that
mitophagy must be kept in check in order to maintain a respiring mitochondrial population.
For example, mitophagy is blocked in yeast that are fed on a carbon source making mitochon-
dria essential for metabolism [8]. An exciting possibility is that the metabolic status of a cell
can feed into the mitochondrial dynamics machinery. Evidence for this comes from recent
reports that describe how mitochondria undergo fusion and are spared from mitophagy dur-
ing starvation and thus are able to maintain ATP levels. Cells can therefore protect their pre-
cious mitochondria during starvation-induced macroautophagy by maintaining an elongated
mitochondrial network [49,50]. In both studies it was found that starvation-induced activation
of PKA (protein kinase A) leads to inhibitory phosphorylation of fission factor Drp1 at Ser637
and dephosphorylation at Ser616. This blocks the translocation of Drp1 to the mitochondria
and hence inhibits mitochondrial fission.
Despite recent advances, there is a long way to go if we are to truly understand the cross-
talk between mitochondrial dynamics and mitophagy and how this affects disease pathology.
Mitochondrial elongation depends on the type of nutrient starvation indicating that tight con-
trol of mitochondrial dynamics depends on intricate signalling pathways yet to be properly
defined [51]. It is also important to bear in mind that mitochondrial networks differ from cell
type to cell type. In yeast, for example, mitophagy occurs independently of mitochondrial fis-
sion [52]. Interestingly, on simulation of fasting by nutrient deprivation in the presence of
glucagon, mitophagy is actually enhanced in GFP–LC3-expressing primary mouse hepatocytes
[53]. Morphologically, the mitochondria do not appear fused in these hepatocytes and
autophagosomes are even found to form around mitochondria that have an intact membrane
potential [53]. This provides strong evidence that mitochondrial depolarization is not always a
pre-requisite for the selective removal of mitochondria.

Conclusion
Mitophagy is fundamental to mitochondrial quality control and homoeostasis and its impor-
tance is demonstrated by the pathological consequences of its mis-regulation. Since its initial

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T. MacVicar 101

recognition as a bona fide example of a specific autophagy pathway, the sheer diversity of
mitophagy regulatory mechanisms continues to surprise. Cells regulate mitochondrial degra-
dation not only through control of the autophagy machinery, but also via delicate tuning of
mitochondrial fusion and fission. It remains to be seen whether other cellular processes linked
with mitochondria also have a role to play in mitophagy regulation. Calcium, for instance, has
emerged as a key modulator of autophagy [54]. Given the central role played by mitochondria
in calcium signalling [55], it too could have important functions in mitophagy control.
Better understanding of the mechanisms involved in mitochondrial quality control will
hopefully hold serious therapeutic potential for the number of devastating human diseases
associated with mitochondrial dysfunction.

Summary
• Mitophagy is the selective degradation of dysfunctional or redundant mito-
chondria by the autophagy machinery.
• Disturbances in mitochondrial quality control have been implicated in a range
of human pathologies including neurodegeneration.
• Mitophagy-specific proteins exist that target mitochondria for degradation
by directly or indirectly interacting with autophagosomes, these include
Atg32 in yeast and Nix in mammalian red blood cells.
• The PINK1/parkin pathway selectively targets damaged mitochondria and
its disruption has been implicated in early-onset forms of PD.
• Mitochondrial dynamics play a key role in regulating mitophagy; fission iso-
lates damaged mitochondria from the network, whereas fusion protects
healthy mitochondria from engulfment by autophagosomes.

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