Autophagy receptors in developmental clearance of mitochondria.

Abstract

Recent discoveries of autophagy receptors, which specifically recognize different cellular cargo destined for degradation, have opened a new chapter in the autophagy field. Selective cargo recognition by autophagic machinery is important in the context of cellular homeostasis and survival. One of the crucial homeostasis events involving autophagy is the removal of damaged or excessive mitochondria through mitophagy. Future studies on mitochondrial receptors and proteins associated with mitochondrial clearance will help us better understand the role of mitophagy in normal physiological processes as well as in diverse pathological conditions.

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focused on removal of cellular components as well as their turn-
over with a special emphasis on autophagy, a sequestering pro-
cess that involves the formation of double-membrane vesicles.
Controlled and selective removal of damaged or redundant
organelles via autophagy is involved in developmental processes
and is physiologically important in certain disease states.1 Various
types of selective autophagy can be categorized depending on the
substrate, ranging from aggregated proteins, mitochondria or
ribosomes to bacteria and are named accordingly aggrephagy,
mitophagy, ribophagy or xenophagy.2,3 Cargo engulfment mech-
anisms are not well understood, but recent discoveries of numer-
ous autophagic receptors and adaptors that uniquely recognize
cargo species are emerging.2,3
www.landesbioscience.com Autophagy 301
Autophagy 7:3, 301-303; March 2011; © 2011 Landes Bioscience
Autophagy receptors in developmental clearance
of mitochondria
REVIEW
REVIEW
Ivana Novak1,* and Ivan Dikic1,2
1School of Medicine; University of Split; Split, Croatia; 2Frankfurt Institute for Molecular Life Sciences and Institute of Biochemistry II; Goethe University; Germany
Key words: mitophagy, autophagy receptors, mitochondria, Nix, Bnip3, reticulocyte maturation
Introduction
Cellular homeostasis is established through biogenesis and deg-
radation of proteins and organelles. Recently, research has been
The Role of Nix in Mitophagy
Mitochondria are one of the most vital organelles performing
essential functions in the cell, from ATP production to calcium
storage and cell death, and are indispensable for cellular homeo-
stasis.4 Knowledge of the molecular mechanisms of mitochon-
drial recognition and removal by mitophagy has so far been
limited. It is well known that during cellular development low
energy states occur and excess mitochondria are not needed, and
*Correspondence to: Ivana Novak; Email: inovak@mefst.hr
Submitted: 10/04/10; Revised: 12/16/10; Accepted: 12/17/10
DOI: 10.4161/auto.7.3.14509
Recent discoveries of autophagy receptors, which specifically
recognize different cellular cargo destined for degradation,
have opened a new chapter in the autophagy field. Selective
cargo recognition by autophagic machinery is important in
the context of cellular homeostasis and survival. One of the
crucial homeostasis events involving autophagy is the removal
of damaged or excessive mitochondria through mitophagy.
Future studies on mitochondrial receptors and proteins
associated with mitochondrial clearance will help us better
understand the role of mitophagy in normal physiological
processes as well as in diverse pathological conditions.
they may be the source of agents such as reactive oxygen spe-
cies (ROS), cytochrome c and other apoptosis-inducing factors
that when released could cause cell damage and death. In such
a case, mitophagy is activated and occurs following induction or
inhibition of apoptosis.5 Accumulation of damaged mitochon-
dria is associated with neurodegenerative diseases, hypoxia and
cancer, and it is important to comprehend how these abnormal
conditions can be avoided. Moreover, partial or complete mito-
chondrial elimination during development and cell differentia-
tion (e.g., during reticulocyte and lymphocyte maturation, and
spermatogenesis) is likewise essential.
We have recently described Nix (also named Bnip3L) as
mammalian mitophagy receptor important for selective removal
of damaged mitochondria as well as complete removal of mito-
chondria during reticulocyte maturation.6 Nix-deficient mice
develop anemia and severe reticulocytosis caused by inefficient
loss of mitochondria. Although the basic autophagy machinery is
functional in the absence of Nix, mitochonodria engulfment by
autophagosomes is impaired.7,8 Therefore, we were able to demon-
strate that Nix directly interacts with LC3/GABARAP proteins
in an LIR (LC3 Interacting Region)-dependent manner where
a single mutation in the LIR is sufficient to abolish recruitment
of LC3/GABARAP to mitochondria. Analysis of programmed
mitochondrial clearance in reticulocytes of Nix-deficient cells,
where wild-type or LIR mutant Nix was introduced, revealed
that the effect of the LIR mutation on Nix-mediated clear-
ance is partial. This is similar to recent data where the yeast
mitophagy receptor Atg32 was shown to interact with Atg8 in
an LIR-dependent manner but this binding is also partial. For
fully functional mitophagy in yeast, Atg32 needs a second bind-
ing partner, Atg11.9,10 Partial mitochondrial clearance in Nix-
/- reticulocytes also suggests alternative mechanisms leading to
mitophagy (Fig. 1). Further, Nix initiates mitophagy by prepar-
ing mitochondria for recognition by the autophagic machinery.
When mitochondria get depolarized, Nix influences transloca-
tion of the E3 ligase Parkin to mitochondria in order to ubiqui-
tinate mitochondrial proteins and, thus, mark mitochondria for
degradation by autophagy.11,12 The importance of ubiquitin as a
signal in autophagy has been demonstrated by identification of
p62 and NBR1 as autophagy receptors that bind both ubiquiti-
nated proteins, with their UBA domains and LC3/GABARAP,
with LIR domains.13,14 In contrast, physiological localization
of Nix to the mitochondrial outer membrane predisposes it to
deliver mitochondria into autophagosomes without requiring the
intermediate ubiquitin to label the cargo. However, ubiquitin,

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general autophagy and mitophagy in red blood cell differentia-
tion, and have suggested that the basic autophagic machinery is
indispensable for programmed mitochondrial clearance. In con-
trast, mitochondrial mass and number in ex vivo hematopoietic
lineage cells, such as myeloid and dendritic cells, are not affected
by the loss of Atg7. The selective removal of mitochondria by
autophagy during erythropoeisis is essential, and is a necessary
developmental step. Degradation of other organelles in Atg7-/-
erythroid cells, including the nucleus, ER and ribosomes is not
affected.15 However, the Atg7-/- cells do not answer one of the
key questions as to whether the depolarization of mitochondria
is a cause or a consequence of mitochondria recruitment to auto-
phagosomes. Because mitochondria in Nix-/- reticulocytes remain
polarized, it has been hypothesized that Nix targets the mito-
hondria for mitophagy by triggering depolarization.7 However,
the polarization state of mitochondria in Atg7-/- reticulocytes does
not show mitochondrial depolarization, suggesting that Nix is
not the only protein responsible for membrane depolarization.15
Therefore, current results are not giving a clear answer as to
whether all mitochondria need to be depolarized for clearance by
mitophagy. In contrast, it was proven that increased ROS levels
lead to mitochondrial depolarization that signals translocation
of the E3-ligase Parkin to mitochondria where it ubiquitinates
mitochondrial proteins such as VDAC1 and mitofusins, and
consequently obstructs the mitochondrial fission and fusion bal-
ance to finally trigger autophagy.16-18 Moreover, there is emerging
evidence that other autophagy signals might be responsible for
302 Autophagy Volume 7 Issue 3
mitophagy. Another mitophagy receptor candidate is Bnip3, a
Nix homolog previously shown to be involved in autophagic cell
death that occurs independently of apoptosis.19 Hypoxia selec-
tively induces Bnip3-dependent mitophagy through disruption
of the interaction between Beclin 1 and Bcl-2.20 In addition, Nix
was described to be a similar regulator of hypoxia-induced auto-
phagy.21 Involvement of both proteins is important in hypoxia
conditions where knockdown of both Bnip3 and Nix inhibits the
pro-survival activation of hypoxia-induced mitophagy. In that
regard, ectopic expression of both Bnip3 and Nix causes activa-
tion of mitophagy even in normoxia.21
Our preliminary results showed that Bnip3, similar to Nix,
possess an LIR that strongly binds LC3/GABARAP proteins
and its binding is abolished when a single mutation in the LIR
is introduced (Fig. 2). Nix and Bnip3 could, consequently, act
together and participate in mitophagy to finally result in cell
rescue or differentiation. Furthermore, Nix and Bnip3 could be
involved in mitochondrial removal through the same autophagic
mechanism by binding LC3/GABARAP but in different normal
or abnormal conditions. For example, Nix could be involved in
complete removal of mitochondria during cellular differentiation
(in reticulocytes or T-lymphocytes) and Bnip3 in conditions such
as hypoxia. Both could be required when, in normal conditions
and terminally differentiated cells, the population of damaged
mitochondria needs to be removed to protect cells from further
attached to other mitochondrial proteins, might be important for
additional selection of damaged versus healthy mitochondria for
autophagy.
Mitophagy Receptors in Development
Recent studies on Atg7-/- mice revealed the connection between
Figure 1. Mitophagy receptors in yeast and mammals. The yeast
mitophagy receptor Atg32 interacts with the Atg8 and Atg11 proteins
in order to completely remove damaged mitochondria. An interaction
between LC3/GABARAP proteins and the mitophagy receptors, Nix and
Bnip3 is not sufficient for fully functional mitophagy in the mammalian
system; an additional binding partner(s) needs to be defined.
Figure 2. The role of Nix, Bnip3 and ubiquitin in mitophagy. Mitochon-
drion-localized Nix and Bnip3 interact via their N-terminal LIR with LC3/
GABARAP-conjugated to the newly forming autophagosome, thereby
mediating tethering of the nascent phagophore to the target mito-
chondrion. Concomitant ubiquitination of a mitochondrion-localized
substrate protein may, via the Ub-binding autophagy receptors p62
and NBR1, further increase tethering of mitochondrial and phagophore
membranes.

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mitochondrial clearance during reticulocyte matura-
tion. Proc Natl Acad Sci USA 2007; 104:19500-5.
Kanki T, et al. Atg32 is a mitochondrial protein that
confers selectivity during mitophagy. Dev Cell 2009;
17:98-109.
www.landesbioscience.com Autophagy 303
destined for elimination, and what are the upstream signals
that label such mitochondria. What mediates full incorpora-
tion of mitochondria into autophagosomes remains to be further
explored, but it is obvious that this process is tightly connected
with regulation of mitochondrial quality.
Acknowledgements
We are grateful to Janos Terzic and Ivana Marinovic Terzic for
comments and discussions. I.N. is supported by an EMBO Long
term fellowship. Research in the I.D. laboratory is supported by
the Deutsche Forschungsgemeinschaft, the Cluster of Excellence
“Macromolecular Complexes” of the Goethe University
Frankfurt (EXC115) and the European Research Council (ERC)
grant agreement No. [250241-LineUb].
damage or death. Because p62 and NBR1 show redundancy
while clearing aggregated ubiquitinated proteins, it is also possi-
ble that Nix and Bnip3 have overlapping functions as mitophagy
receptors.
Concluding Remarks
The role of Nix as a mitophagy receptor has extensively been
studied in reticulocytes; however, other cellular systems, where
mitochondria are physiologically removed due to developmental
progression, like in spermatocytes or lens cells, needs to be addi-
tionally explored for both Nix and Bnip3. Moreover, future stud-
ies will enable better understanding of the mechanism by which
the mitophagy receptors Nix and Bnip3 recognize mitochondria
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