FORUM REVIEW ARTICLE
Mitochondria Autophagy in Yeast
Tomotake Kanki,1Daniel J. Klionsky,2and Koji Okamoto3
The mitochondrion is an organelle that carries out a number of important metabolic processes such as fatty acid
oxidation, the citric acid cycle, and oxidative phosphorylation. However, this multitasking organelle also gen-
erates reactive oxygen species (ROS), which can cause oxidative stress resulting in self-damage. This type of
mitochondrial damage can lead to the further production of ROS and a resulting downward spiral with regard
to mitochondrial capability. This is extremely problematic because the accumulation of dysfunctional mito-
chondria is related to aging, cancer, and neurodegenerative diseases. Accordingly, appropriate quality control of
this organelle is important to maintain proper cellular homeostasis. It has been thought that selective mito-
chondria autophagy (mitophagy) contributes to the maintenance of mitochondrial quality by eliminating
damaged or excess mitochondria, although little is known about the mechanism. Recent studies in yeast iden-
tified several mitophagy-related proteins, which have been characterized with regard to their function and
regulation. In this article, we review recent advances in the physiology and molecular mechanism of mitophagy
and discuss the similarities and differences of this degradation process between yeast and mammalian cells.
Antioxid. Redox Signal. 14, 1989–2001.
emerge andsequester cytoplasmiccomponentsas cargoes,and
then the vesicles deliver those cargoes into the lysosome/vac-
uole. The delivered components are degraded by the resident
hydrolytic enzymes into small molecules, which are reused by
the cell for survival. This catalytic process is highly conserved
among eukaryotes and is called ‘‘macroautophagy.’’ In addi-
tion to its function as a cellular stress response that is observed
in most eukaryotes, autophagy plays diverse roles in cellular
development, immune response, aging, and tumor suppres-
sion and in the prevention of many diseases such as cancer,
infection, diabetes, neurodegenerative diseases, gastrointesti-
nal disorders, and cardiomyopathy in mammalian cells (51).
In addition to macroautophagy, there are two morpho-
logically different types of autophagy: microautophagy and
chaperone-mediated autophagy (CMA). Microautophagy
sequesters cytoplasmic components by direct invagination or
In contrast, CMA is a chaperone-dependent process relying
on both cytosolic and lysosomal hsc70to move substrates into
the lysosome lumen and on hsp90 to stabilize the membrane-
bound LAMP-2A receptor (41). The substrate proteins that
n response to various types of cellular stress, such as
nutrient starvation, cytosolic double-membrane vesicles
have the consensus amino acid sequence KFERQ or a similar
motif are unfolded and directly translocated across the lyso-
somal membrane. CMA has been identified in higher eu-
karyotes, but not in yeast.
All of the cytoplasmic components, including proteins,
nucleic acid, and organelles, can be sequestered by macro-
autophagy as a cargo. Actually, the presence of organelles,
such as mitochondria, in the lysosome or vacuole was fre-
quently found in both mammalian cells and yeast (8, 84).
However, it has long been unclear whether these organelles
are nonselectively, preferentially, or selectively degraded by
autophagy. Recent studies, particularly in yeast, revealed that
some organelles or proteins are selectively degraded by an
autophagic process. Now it is known that mitochondria,
peroxisomes, ribosomes, endoplasmic reticulum, protein ag-
gregates, and invasive microbes are selectively degraded by
autophagy, and these selective autophagic processes are
called mitophagy, pexophagy, ribophagy, reticulophagy, ag-
grephagy, and xenophagy, respectively (4, 39, 42, 45, 79, 89).
In addition to these specific types of autophagy, yeast cells
have adapted the autophagic process for a biosynthetic pur-
pose in the form of the cytoplasm-to-vacuole targeting (Cvt)
pathway (40). This pathway selectively delivers at least
two resident hydrolases, aminopeptidase I (Ape1) and a-
mannosidase (Ams1), to the vacuole using most of the same
1Department of Clinical Chemistry and Laboratory Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.
2Life Sciences Institute, University of Michigan, Ann Arbor, Michigan.
3Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan.
ANTIOXIDANTS & REDOX SIGNALING
Volume 14, Number 10, 2011
ª Mary Ann Liebert, Inc.
autophagic machinery that is used for nonselective autop-
hagy. Yeast leucine aminopeptidase III (Lap3) has been re-
cently reported to be selectively degraded through a Cvt
pathway-related process, but only under starvation condi-
tions when the protein was overexpressed (24). Although
macropexophagy and the Cvt pathway are morphologically
is conventionally used to represent bulk (i.e., nonspecific)
autophagy essentially as an antonym to selective autophagy.
Hereafter, we use ‘‘macroautophagy’’ when referring to bulk
The mitochondrion is an organelle that is integrally in-
volved in cellular energetics, carrying out various catabolic
processes. In particular, mitochondrial oxidative phosphory-
lation supplies a large amount of energy that contributes to a
range of cellular activities. However, this process also gener-
ates reactive oxygen species (ROS) that can damage the or-
ganelle. Damaged proteins and DNA in mitochondria cause
further production of ROS, and the accumulation of mito-
chondrial damage is related to aging, cancer, and neurode-
generative diseases (92). Accordingly, the cell has devised
specific mechanisms to ensure proper quality control of this
organelle. Mitochondria have their own quality control sys-
peroxidase (1). Further, recent evidence suggests that mito-
phagy eliminates mitochondria that contain excess damage
beyond the capacity of the above quality control systems to
effect repair. In mammals, mitophagy is closely related to
cellular physiology, whereas mitochondrial dysfunction is
associated with certain diseases. For example, during ery-
throid cell maturation, the mitochondrial outer-membrane
of-function mutations of the PARK2 and PARK6 genes, which
encode Parkin and PTEN-induced putative kinase 1 (PINK1),
respectively, cause Parkinson disease; PINK1 can stably lo-
calize on the outer membrane of impaired mitochondria and
recruit Parkin from the cytosol to the mitochondrial mem-
brane, thus promoting mitophagic degradation (14, 48, 54, 55,
91). However, the further mechanism for engulfing mito-
chondria has not been elucidated in mammalian cells. As with
the autophagy-related (Atg) proteins, which were first char-
acterized in yeast (38, 53), studies in this unicellular eukaryote
recently uncovered some of the first mitophagy-related fac-
to the molecular mechanism of mitophagy in yeast.
Evidence for Selective Mitochondria Autophagy
The presence of mitochondria within the yeast vacuole was
first reported in 1992 (84). Initially, this phenomenon was
thought to be the result of nonselective engulfment of mito-
chondria by macroautophagy. However, recently, several
lines of evidence suggest that impaired mitochondria are se-
lectively degraded by autophagy in yeast. In the fmc1 null
mutant, the FoF1ATPase subunits aggregate, and the mito-
chondrial membrane potential is impaired at 37?C. Under
anaerobic conditions at 37?C, this mutant strain induces au-
tophagy and preferentially removes the dysfunctional mito-
chondria. Importantly, the fmc1 mutant has an increase in
cellular ATP, confirming that autophagy in this setting is not
induced by ATP depletion. This case is, to our knowledge, the
first report demonstrating the elimination of impaired mito-
chondria by autophagy in yeast (71).
There are two more studies suggesting that dysfunctional
mitochondria are eliminated by autophagy. The first concerns
Mdm38, a mitochondrial inner membrane protein with K+/
H+exchange activity. Depletion of Mdm38 causes loss of the
inner membrane potential, mitochondrial swelling, and
fragmentation; eventually, these abnormal mitochondria are
eliminated by autophagy (64, 65). In the second study, Zhang
et al. altered mitochondria by expressing a temperature-
sensitive, mutant mitochondrial DNA (mtDNA) polymerase
(mip1ts). By culturing this mutant strain at the nonpermissive
temperature, they blocked mtDNA replication and observed
rapid degradation of mtDNA via autophagy. As mtDNA en-
codes some of the electron transport chain components, de-
pletion of mtDNA affects the mitochondrial membrane
strain is likely due to a deficiency in the electron transport
chain (103). Additional studies suggest that mitophagy is
regulated independently of nonselective macroautophagy.
Kanki and Klionsky found that mitophagy is blocked under
strong macroautophagy-inducing nitrogen starvation con-
ditions, if the carbon source makes mitochondria essential
for metabolism (26). The Camougrand group found that N-
acetylcysteine (NAC), a compound that increases the cellular
reduced glutathione (GSH) pool, prevents mitophagy in-
duced by nitrogen starvation or rapamycin, presumably
because the cellular redox imbalance affects mitophagy in-
duction but has no effect on nonspecific macroautophagy (9,
selectively degraded by autophagy in yeast. During the same
period, accumulating evidence suggests selective mitochon-
dria autophagy in mammalian cells (10, 73, 90). More recently,
two groups simultaneously performed screens for mitophagy-
deficient mutants, and both groups identified the same gene,
ATG32, as encoding a mitophagy-specific factor (29, 68). This
function of Atg32 is discussed below in detail.
Mitophagy Requires Most of the Atg Proteins
Although the morphology of autophagy was first studied
in mammalian cells, most of the molecular components were
initially identified in yeast (37). Studies in Saccharomyces cer-
evisiae and other fungi have allowed the isolation of 34 ATG
genes. At least 15 of these genes are essential for both mac-
roautophagy and selective autophagy, and are categorized as
part of the core autophagic machinery (53). Other genes have
roles in certain types of autophagy. For example, Atg19, a
composed of precursor Ape1 (prApe1) and Ams1 to form the
Cvt complex. Atg11, an adaptor protein for selective auto-
phagy, interacts with Atg19 and recruits the Cvt complex to
the phagophore assembly site (PAS), where the phagophores,
the initial sequestering membrane structure, are generated
(80). Similarly, during pexophagy in Pichia pastoris, Atg30
localizes to peroxisomes, where it subsequently binds Atg11,
allowing recruitment of peroxisomes to the PAS (13).
In 2004, Kissova et al. reported that ATG5 is required for
ATG genes for mitophagy has been reported by various
1990KANKI ET AL.
groups (26, 35, 85, 103), and recently, all of ATG genes have
been surveyed for mitophagy (28,68).Table 1 summarizesthe
requirement of ATG genes for nonselective autophagy (mac-
roautophagy), the Cvt pathway, pexophagy, and mitophagy
in S. cerevisiae. ATG genes encoding the core machinery for
autophagic membrane formation, such as the Atg1 protein
kinase, the phosphatidylinositol 3-kinase complex I that is
required for vesicle nucleation (including ATG6 and ATG14),
the ubiquitin-like Atg8–phosphatidylethanolamine (PE) con-
jugation machinery (Atg3, Atg8, Atg4, and Atg7), the ubi-
quitin-like Atg12–Atg5 conjugation machinery (Atg5, Atg7,
Atg10, and Atg16), and components that are involved in
supplying lipids to the phagophore (Atg2, Atg9, and Atg18),
are essential for all types of autophagy, suggesting that both
nonselective and selective autophagy processes fundamen-
tally rely on the same membrane formation machineries. In-
terestingly, ATG17, ATG29, and ATG31, which encode
components of the Atg1 kinase complex and regulate Atg1
activity (53), are completely or partially required for pex-
ophagy, mitophagy, and macroautophagy, but not the Cvt
pathway. These findings led us to speculate that the Atg1
require a generally large autophagosome to enwrap the cargo
(e.g., 500–1000nm for mitophagy), but are not required for
small protein complexes (e.g., a 150-nm vesicle is used for the
for both the Cvt pathway and pexophagy but not for macro-
autophagy (60), are also required for mitophagy, further
suggesting that mitophagy is a type of selective autophagy. In
particular, because Atg11 is an adaptor protein that recog-
nizes and interacts with cargo-specific receptor proteins for
the Cvt pathway and pexophagy, the requirement of Atg11
for mitophagy strongly suggests that mitochondria are de-
graded by an autophagic process that involves a receptor–
Genomic Screen for Yeast Mutants Defective
To identify molecules acting in mitophagy, two groups
used nonessential gene deletion strains and performed a
genome-wide screen for mutants that were defective in
selective mitochondria degradation (28, 68). Okamoto et al.
visualized mitochondria by expressing green fluorescent
protein (GFP) with a mitochondrial targeting signal fused to
Table 1. Requirement of Autophagy-Related Genes for Macroautophagy, the Cytoplasm
to Vacuole Targeting Pathway, Pexophagy, and Mitophagy
ATG genesMacroautophagyCvt pathway PexophagyMitophagya
(18, 78, 85, 87, 103)
(68, 78, 87, 93)
(16, 68, 78, 87)
(16, 68, 78, 87)
(18, 34, 78, 87)
(78, 87, 103)
(17, 18, 32, 68, 87)
(17, 18, 87, 103)
(17, 26, 35, 87, 88)
(18, 68, 78, 87)
(16, 26, 33)
(78, 87, 103)
(28, 68, 78, 87)
(16, 18, 68, 78, 87)
(11, 12, 68, 86)
(16, 68, 78, 87)
(7, 22, 25, 26, 28)
(2, 15, 68)
(26, 60, 68)
(3, 28, 68)
(26, 28, 60, 68)
(68, 96, 99)
(26, 30, 68)
(23, 26, 68)
Phenotypes of the indicated gene knockout strain: + +, no defect; +, partial defect; -, severe defect.
aPhenotypes are shown based on Refs. (68) and (28).
bATG15 encodes a putative lipase required for intravacuolar lysis of autophagic and Cvt bodies.
cATG22 encodes a vacuolar membrane protein required for efflux of amino acids during autophagic body breakdown in the vacuole.
ATG, autophagy-related; Cvt, cytoplasm to vacuole targeting; ND, not determined.
MITOPHAGY IN YEAST1991
its N terminus (mito-GFP); mitochondrial transport into the
vacuole occurs when mitophagy is induced by culturing cells
to the postlog phase in a medium containing a nonfermentable
carbon source. They examined 5150 knockout strains and
found 36 mutants that impaired mitophagy,excluding existing
ATG gene null strains (68). Kanki et al. generated a different
chimera by tagging the C terminus of the mitochondrial outer-
membrane protein Om45 with GFP (Om45-GFP); GFP accu-
free GFP is generated by vacuolar processing of Om45-GFP as
observed by western blotting, when mitophagy is induced by
culturing cells to the postlog phase in a medium containing a
nonfermentable carbon source or under conditions of nitrogen
starvation. In this case they monitored 4667 knockout strains
and found 32 mutants with impaired mitophagy, again ex-
cluding the existing ATG gene null mutants (28). Genes iden-
tified from both screens are summarized in Table 2, which has
been updated based on recently obtained results (28, 68; un-
published data). Although both screens were based in part on
the observed accumulation of a mitochondrially targeted GFP
the two screens (16 among the 45 total genes). One reason for
this relative lack of overlap may be due to the fact that Kanki
et al. did not screen strains that cannot grow well in non-
fermentable medium. Another possibility may simply reflect
in characterizing mitophagy-positive and -negative strains.
Notably, >40% of the genes identified from both screens are
membrane trafficking-related genes (20 among the 45 total
genes). The requirement of most of these genes for macro-
autophagy and/or the Cvt pathway has been previously re-
ported, and it is widely thought that defects in membrane
trafficking pathways affect the lipid supply that is needed for
extension of the phagophore, the initial sequestering com-
partment that generates the autophagosome (21, 31, 49, 50, 57,
partly a verification of the success of the screens—that both
groups identified a certain number of membrane trafficking-
mitochondrial dynamin-related GTPase required for mito-
chondrial fission (28). This finding is in agreement with pre-
vious reports that the fragmentation of mitochondria is a
prerequisite for mitophagy in mammalian cells (90), and the
dnm1D strain inhibits the mitophagy induced by mdm38
conditional knockout in yeast (65). Although it remains un-
clear whether mitochondrial fusion and fission affect mito-
phagy, because of the limitation of the size of the
autophagosome it may be essential to split mitochondria by
fission to generate a sequestering vesicle of the size appro-
priate for mitophagy.
Characterization of the Mitochondrial Receptor Atg32
Among >30 genes identified from the above screen, both
YIL146C as a mitophagy-specific gene and designated it as
ATG32 (29, 68). The deletion of ATG32 does not affect nonse-
lective autophagy, the Cvt pathway, and pexophagy, but
completely inhibits mitophagy. Atg32, a protein composed of
529 amino acids, is predicted to have a single transmembrane
domain. A proteinase sensitivity assay conducted on crude
mitochondrial fractions suggested that Atg32 is located in the
mitochondrial outer membrane with its N- and C-terminal
domains oriented toward the cytosol and the intermembrane
space, respectively (68). Yeast two-hybrid and immunopre-
cipitationexperiments revealed that Atg32 can bindAtg11 and
Atg8. Because Atg11 is an adaptor protein that interacts with
cargo-specific receptor proteins for selective autophagy, Atg32
is thought to be a mitochondrial receptor for mitophagy. No-
tably, the Atg11–Atg32 interaction dramatically increases
under conditions of nitrogen starvation that can induce
mitophagy. Thus, it is thought that the Atg11–Atg32 interac-
tion is the first physical step for mitochondria degradation via
autophagy. Atg8 is a component of the autophagosome, and
the PE-conjugated form of Atg8 (Atg8–PE) is involved in
phagophore expansion (19, 52). Atg8 and LC3, a mammalian
homolog of Atg8, bind Atg19 and p62, respectively. Interest-
ingly, the cytosolic domain of Atg32 has a WXXI/L/V se-
quence, which is identified as an Atg8 binding motif present in
Atg19 and p62 (20, 62, 70). In fact, the WXXI/L/V motif of
contributes to mitophagy (67, 68). The reason for this partial
contribution might be that Atg32 also interacts with Atg11, a
primary factor for linking the autophagy machinery with mi-
tochondria at the early stage of mitophagy, whereas Atg32
interacts with Atg8 for assisting efficient formation of phago-
phoressurrounding mitochondria at a latestage ofmitophagy.
Alternatively, there might be other binding sites for Atg8–
Atg32 interaction in vivo, so that disruption of the WXXI/L/V
motif only results in partial defects in mitophagy.
The Cvt Pathway, Pexophagy, and Mitophagy Depend on
a Similar Molecular Process
The identification and characterization of Atg32 provided
some insight into the process of selecting and delivering mi-
tochondria to the vacuole in yeast. As shown in Figure 1, this
process resembles those of the Cvt pathway and pexophagy.
Different from other types of autophagy, the Cvt pathway
constitutively (i.e., regardless of nutrient conditions or other
Ape1 (prApe1) and Ams1 to the vacuole. PrApe1 is synthe-
sized in the cytosol, forms dodecamers, and is further as-
sembled into an Ape1 complex composed of multiple
dodecamers. The Ape1 complex is recognized and bound by
the receptor protein Atg19, which independently recruits
Ams1 and forms the prApe1–Atg19–Ams1 complex (the Cvt
complex) (77). The adaptor protein Atg11 recognizes and
binds Atg19 in the context of the Cvt complex and transports
the complex to the PAS, where most of the Atg proteins ac-
cumulate and where the initial sequestering membrane
structure (i.e.,the phagophore) is generated (80, 83). When the
Cvt complex reaches the PAS, the Cvt complex binds the
phagophore membrane through an interaction between
Atg19 and the lipid-conjugated Atg8–PE. The phagophore
membrane expands around the Cvt complex, excluding bulk
cytoplasm and forming the Cvt vesicle. Subsequently, the Cvt
outer membrane fuses with the vacuole, releasing the inner
vesicle, which is now termed a Cvt body, into the vacuole
of prApe1 is proteolytically removed and the Ape1 complex
dissociates into dodecamers (46, 101) (Fig. 1).
1992 KANKI ET AL.
Contrary to macroautophagy and the Cvt pathway,
which are well characterized in S. cerevisiae, pexophagy is
extensively studied in methylotropic yeast, such as P. pas-
toris and Hansenula polymorpha. When no longer required,
autophagic- or microautophagic-like process, called macro-
pexophagy and micropexophagy, respectively. In P. pastoris,
adaptation from methanol to glucose medium induces mi-
cropexophagy, whereas a switch from methanol to ethanol
medium induces macropexophagy (56, 58, 89). During
peroxisome proliferation in P. pastoris, PpAtg30 is induced
and binds the peroxisomal proteins PpPex3 and PpPex14.
Table 2. Genes Identified from Yeast Genome-Wide Screen for Mitophagy-Defective Mutants
Okamoto et al.
Kanki et al.
(initial screen) Function
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
Vacuolar protein sorting
Endoplasmic reticulum-to-Golgi transport
Endocytosis, vacuole fusion
Vacuolar protein sorting
Vacuolar protein sorting
Vacuolar protein sorting
Vacuolar protein sorting
Vacuolar protein sorting
Vacuolar protein sorting
Vacuolar protein sorting
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
Formation of mitochondrial cristae junction
Assembly of mitochondrial F1FoATP synthase
Mitochondrial magnesium transporter
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
N-terminal protein acetylation
Inositol polyphosphate multikinase
Chorismate synthase/flavin reductase
MAP kinase kinase kinase
Noncatalytic subunit of N-terminal
acetyltransferase of the NatC type
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
ND2 (+ +)
aPMR1 and HUR1 partially overlap.
MVB, multivesicular body; ND1, not determined; ND2, not determined because the initial screen was negative; ND3, not determined
because of slow growth of the knockout strain in lactate medium; + +, mitophagy is normal in the knockout strain; +, mitophagy is partially
defective in the knockout strain; -, mitophagy is completely defective in the knockout strain.
MITOPHAGY IN YEAST1993
Following the induction of pexophagy, PpAtg30 is phos-
phorylated and interacts with PpAtg11 and PpAtg17,
allowing recruitment of the peroxisome to the PAS for both
macro- and micropexophagy (13). At the PAS, peroxisomes
are sequestered by the phagophore membrane during
macropexophagy or are sequestered by the vacuolar se-
questering membrane and cup-shaped micropexophagy
apparatus during micropexophagy (13, 47) (Fig. 1).
Similar to the Cvt pathway and pexophagy, and as dis-
When mitophagy is induced, Atg11 binds the mitochondrial
resident protein Atg32. The Atg11–Atg32 interaction is needed
to recruit mitochondria to the PAS. When mitochondria reach
the PAS, the phagophore membrane begins to sequester the
organelle; this depends on the interaction between Atg32 and
Atg8–PE (67, 68). The last step of the mitophagy pathway is
controversial. Based on electron microsocopy, some research-
ers found that mitochondria are sequestered through a micro-
autophagy-like process at the vacuole limiting membrane (9,
35, 65), whereas others observed macroautophagy-like mito-
vary depending on the mitophagy triggers, similar to the sit-
uation with pexophagy.
In yeast, the PAS is usually formed next to the vacuole
surface and most of the Atg proteins accumulate at the PAS
during selective and nonselective autophagy (83). In the Cvt
the PAS via their interaction with Atg11 before they are se-
questered within vesicles. In an atg1D strain, cargoes accu-
mulate at the PAS; when different cargoes are labeled with
fluorescent proteins, and cargo localization at the PAS is ob-
served in an atg1D strain, CFP-Ape1 (Cvt pathway marker)
and GFP-Atg32 (mitophagy marker) accumulate at different
sites near the vacuole surface (29). This finding suggests that
ing (Cvt) pathway: Precursor
aminopeptidase I (prApe1) is
synthesized in the cytosol,
forms dodecamers, and is
further assembled into an
Ape1 complex composed of
prApe1 complex is recog-
nized and bound by the re-
related 19 (Atg19), which
complex). The adaptor pro-
tein Atg11 recognizes and
binds Atg19 in the Cvt com-
complex to the phagophore
assembly site (PAS). When
the Cvt complex reaches the
PAS, the complex binds the
through an interaction be-
tween Atg19 and the lipid-
dylethanolamine (PE). The
phagophore membrane ex-
pands around the Cvt com-
cytoplasm and forming the
Cvt vesicle. Pexophagy in
Pichia pastoris: During perox-
isome proliferation in P. pas-
toris, PpAtg30 is induced and
binds the peroxisomal proteins PpPex3 and PpPex14. Following the induction of pexophagy, PpAtg30 is phosphorylated and
interacts with PpAtg11 and PpAtg17, allowing recruitment of the peroxisome to the PAS for both macropexophagy and
micropexophagy. At the PAS, peroxisomes are sequestered by the phagophore membrane during macropexophagy or by the
vacuolar sequestering membrane (VSM) and cup-shaped micropexophagy apparatus (MIPA) during micropexophagy. Mi-
tophagy: When mitophagy is induced, Atg11 binds the mitochondrial resident protein Atg32. The Atg11-Atg32 complex
recruits mitochondria to the PAS. When mitochondria reach the PAS, mitochondria are surrounded by the phagophore
membranethrough aninteraction betweenAtg32 and Atg8–PE.The last stepofthemitophagy pathway is controversial.In this
figure, we show a macroautophagy-like process; however, some researchers observed a microautophagy-like process.
1994 KANKI ET AL.
the Cvt pathway and mitophagy use a different site for bio-
genesis of the PAS, although it is thought that macro-
autophagy and the Cvt pathway use the same site (27, 29).
Similarly, pexophagy utilizes a pexophagy-specific PAS (47).
These findings fit with the concept that cargoes are selectively
sequestered during specific types of autophagy, and different
cargo molecules or bulk cytoplasmic components are ex-
cluded from the resulting vesicles.
Mitophagy Induction and Regulation
mitochondria are eliminated by mitophagy in yeast. For ex-
ample, as discussed above, interference with FoF1-ATPase
biogenesis in a temperature-sensitive fmc1 deletion mutant
(71), or osmotic swelling of mitochondria caused by depletion
of the mitochondrial K+/H+exchanger Mdm38 (65), induces
mitophagy. On the other hand, mitochondrial depolariza-
tion caused by an uncoupler such as carbonyl cyanide m-
chlorophenylhydrazone (CCCP) does not induce mitophagy
in yeast (29, 34). At present, we know of very few conditions
can be induced in wild-type yeast cells by nitrogen starvation
or treating with the target of rapamycin (TOR) kinase inhibi-
tor rapamycin after preculturing yeast in a nonfermentable
medium that facilitates the proliferation of mitochondria (e.g.,
where lactate or glycerol is the sole carbon source), or it can
be induced at stationary phase when yeast cells are cultured
in a nonfermentable medium (26, 29, 34, 68, 85). Although
macroautophagy is also activated under these mitophagy-
inducing conditions, mitochondria are specifically selected
and degraded by mitophagy via the Atg11–Atg32 interaction
(29, 68). Because a certain amount of mitochondria,butnot the
majority, are degraded by mitophagy, there is presumably
some mechanism that can distinguish mitochondria that need
to be degraded from those that are functioning normally. Two
lines of evidence support this idea. Deffieu et al. and Okamoto
et al. reported that NAC, a compound that increases the cel-
lular GSH pool, prevents mitophagy induction (9, 68). This
finding suggests that the mitochondrial redox status or ROS
production level is one of the factors used to distinguish
between healthy and unhealthy mitochondria. Atg33 is a
mitochondrial outer-membrane protein identified as a mito-
phagy-related protein. Kanki et al. found that the deletion of
ATG33 blocks mitophagy to half the level of the wild type
when induced by starvation, but it blocks mitophagy almost
completely when mitophagy is induced at stationary phase
detect or presentaged mitochondria for mitophagy when cells
have reached the stationary phase. Taken together, Atg33 and
another unknown factor(s) may serve to detect damaged,
aged, or redox status-compromised mitochondria and pro-
mote their degradation by mitophagy in yeast.
Physiological Role of Mitophagy
In mammalian cells, two important roles of mitophagy
have been reported. One is mitochondrial quality control and
the other is mitochondria elimination during development as
occurs during erythropoiesis. Studies on Parkinson disease
reveal PINK1-Parkin–mediated degradation of depolarized
mitochondria by autophagy (14, 48, 54, 55, 91). Studies on
erythrocyte maturation demonstrate that the elimination of
mitochondria from reticulocytes is the result of Nix-mediated
mitophagy (74, 76).
In a similar way, mitophagy in yeast is thought to have a
mitochondria. As described in the foregoing section, there are
several lines of evidence supporting the idea that impaired
mitochondria are selected and degraded by mitophagy.
However, mitophagy-deficient yeast, such as the atg32D
strain, do not show any phenotype resulting from the accu-
mulation of impaired mitochondria. Cell growth on a non-
fermentable carbon source, the production of ROS, mtDNA
copy number, and the amount of electron transport chain
complex proteins are indistinguishable between wild-type
and the atg32D strains (29, 68). These findings are not easily
reconciled with the idea that mitophagy plays a critical role in
mitochondrial quality control in yeast, and further studies are
required to clarify this point. Nonetheless, it is clear that mi-
tophagy in yeast has a role in the elimination of excess mito-
chondria. Mitophagy is induced at the stationary phase in
media containing a nonfermentable carbon source. At sta-
accordingly, the requirement for mitochondria is decreased. It
is energetically costly to maintain mitochondria that are not
needed, and there is the potential for ROS production if these
organelles are not maintained. As a result, mitophagy is in-
is induced when cells are cultured in medium containing
lactate as the sole carbon source and then shifted to nitrogen
starvation medium supplemented with glucose; however,
mitophagy is blocked when cells are shifted to nitrogen star-
vation medium supplemented with lactate (26). In this case,
essential for energy production, and mitophagy is not in-
duced. In contrast, if mitochondria are present in excess in
glucose medium, mitochondria degradation is activated.
Mitophagy in Mammals and Yeast
Figure 2 summarizes current models of mitophagy in
mammals and yeast. The mitochondrial kinase PINK1 has a
mitochondrial targeting signal and is constitutively delivered
to the mitochondrial outer membrane. PINK1 on the surface
of mitochondria is, however, quickly cleaved by an unknown
mechanism and is degraded by the proteasome (55). Only
when mitochondria are depolarized, does PINK1 remain
stably localized on the mitochondrial outer membrane.
PINK1 then recruits Parkin, an E3 ubiquitin ligase, to the
mitochondrial surface. Subsequently, Parkin ubiquitinates
mitochondrial proteins (14, 48, 104). Although several sub-
strates of Parkin, such as voltage-dependent anion channel or
mitofusin, have been reported (14, 104), the specific substrate
required for mitophagy remains unclear. Finally, the multi-
signaling adaptor protein p62/SQSTM1 and/or the micro-
tubule-associated histone deacetylase 6 (HDAC6) interact
with the ubiquitinated mitochondrial proteins, and then au-
tophagosomes enwrap the mitochondria via the p62/
HDAC6–LC3 interaction (14, 44) (Fig. 2, upper panel). This
step remains controversial, as it has been reported that p62
knockout mouse embryonic fibroblasts show wild-type levels
of mitophagy (69).
Nix, a BH3-only member of the Bcl-2 family, is a mito-
chondrial outer-membrane protein and has a WXXL
MITOPHAGY IN YEAST1995
sequence that is identified as a yeast Atg8 and mammalian
LC3 binding motif (62, 63). During terminal erythroid
differentiation, an unknown stimulus triggers mitophagy.
The next step remains controversial; mitochondrial depo-
larization may occur in a Nix-dependent manner (74), or
mitochondrial depolarization may not occur at this time
(102). Nix then interacts with LC3, and this interaction
contributes to the formation of selective autophagosomes
(mitophagosomes) that surround mitochondria (63). Be-
cause Nix functions as a mitochondria tag, this BH3-only
protein is considered to be a functional counterpart of
Atg32. However, it is not known whether Nix requires an
1996 KANKI ET AL.
adaptor protein corresponding to Atg11 in yeast (Fig. 2,
middle panel, beige box).
Mitochondrial damage caused by mutations of fmc1,
mdm38,ormip1induces mitophagy inyeast,which is detected
and signaled to the mitophagy machinery through, at least in
part, Atg33. Mitophagy is also induced at stationary phase
when cells are grown in a nonfermentable medium. In the
latter case, some, if not all, mitochondria are aged and dam-
aged during the growing phase. These aged and damaged
mitochondria are alsodetected andsignaled tothemitophagy
machinery through Atg33. The expression level of Atg32 is
dramatically increased in nonfermentable medium, positively
affecting the induction of mitophagy. NAC, a scavenger of
free radicals, can suppress the expression of Atg32 and mi-
tophagy. Thus, oxidative stress positively affects mitophagy
induction (Fig. 2, lower panel). Nitrogen starvation or the
TOR kinase inhibitor rapamycin also induces mitophagy in
yeast pregrown on a nonfermentable medium. The cellular
pool of GSH, which affects the mitochondrial redox status,
negatively regulates mitophagy induced by nitrogen starva-
Atg32 interacts with Atg11, followed by recruitment of the
mitochondria to the PAS and mitophagy-specific uptake.
During the last few years, there has been significant prog-
ress in studies on mitophagy in yeast. In particular, the
identification of Atg32 has provided substantial insight into
the molecular aspects of mitophagy. There are, however,
many questions still to be addressed. (i) It is clear that the
specific interaction between Atg32 and Atg11 is an initial se-
lection step of mitochondria as a cargo. However, how these
proteins interact and what factors regulate this interaction are
not known. In particular, whether the mitophagy induction
signals are derived from mitochondria or the cytosol is an
important issue. In other words, the question is whether mi-
tochondria can dictate their own self-degradation by detect-
ing internal damage or, instead, whether some cytosolic
factors control mitochondria degradation by monitoring the
for example). (ii) The physiological role of mitophagy in yeast
remains unclear. It is apparent that mitophagy in mammalian
cells contributes to maintaining the quality of mitochondria.
Thus, we think that there should be a similar role of mito-
phagy in yeast. However, deletion of the mitophagy-specific
gene ATG32 does not affect cell growth on nonfermentable
medium or increase cellular ROS production (29, 68), which
leaves open the question of how yeast mitophagy contributes
to the quality control of mitochondria. Further studies fo-
cusing on a potential link between mitophagy and mito-
Unfortunately, mammalian homologs of the Atg proteins re-
quired for mitophagy (especially Atg11, 32, and 33) have not
yet been identified. Considering the complexity of the mam-
malian macroautophagy processes including conventional
Atg5- and Atg7-dependent macroautophagy and Atg5- and
Atg7-independent macroautophagy (61), mitophagy might
not be a simple process. If damaged mitochondria can be
specifically degraded by mitophagy, it is reasonable to think
that a yeast mitophagy-like receptor–adaptor interaction is
present in mammalian cells, and Nix is a good candidate for
the counterpart of Atg32.
In mammals, mitophagy governs the elimination of dam-
aged mitochondria. At present, except for Parkinson disease,
with mitochondrial diseases due to the dysfunction of the
respiratory chain. Mitochondrial disease is a collective des-
ignation including some types of neurodegenerative disease
and diabetes mellitus and is caused by many etiologies, such
as accumulation of mitochondria DNA mutations, nuclear
DNA mutations encoding mitochondrial proteins, nucleotide
pool imbalance, and so on. Notably, mitochondria in most
mitochondrial disease cells are heteroplasmic (e.g., functional
and impaired mitochondria are present heterogeneously in
the same cell). These types of mitochondrial diseases are po-
tential targets for therapeutic treatment using mitophagy,
which couldselectively eliminate compromisedmitochondria
from a heterogeneous population (27). Recently, it has been
reported that overexpression of Parkin can eliminate mito-
chondria with deleterious mtDNA mutations, but not those
with wild-type mtDNA, in heteroplasmic cybrid cells (82).
cess—When mitochondria are depolarized (e.g., by carbonyl cyanide m-chlorophenylhydrazone [CCCP] or paraquat treat-
ment), PINK1 can stably localize on the mitochondrial outer membrane. PINK1 recruits Parkin to the surface of mitochondria.
Subsequently, Parkin ubiquitinates mitochondrial proteins, although the specific substrate required for mitophagy remains
unclear. Finally, p62 and/or histone deacetylase 6 (HDAC6) interact with ubiquitinated mitochondrial proteins, and then
autophagosomes enwrap the mitochondria by p62/HADC6–LC3 interaction. Nix-related process—During terminal ery-
throid differentiation, an unknown stimulus triggers mitophagy. Nix interacts with LC3; this interaction contributes to the
formation of autophagosomes surrounding mitochondria. It is not known whether Nix requires an adaptor protein corre-
sponding to Atg11 in yeast (beige box). Mitophagy in yeast: Mitochondrial damage caused by mutations of fmc1, mdm38, or
mip1 induces mitophagy. Mitophagy is also induced at stationary phase when cells are grown in nonfermentable medium.
Appropriate signals are delivered to the mitophagic machinery through, in part, Atg33. At stationary phase, the expression
level of Atg32 is dramatically increased, positively affecting the induction of mitophagy. N-acetylcysteine (NAC), a scavenger
of free radicals, can suppress the expression of Atg32. Nitrogen starvation or the target of rapamycin kinase inhibitor
rapamycin also induces mitophagy. The cellular pool of reduced glutathione (GSH) negatively regulates mitophagy induced
by nitrogen starvation or rapamycin. Once mitophagy is induced, mitochondrial Atg32 interacts with Atg11. Atg11 recruits
mitochondria to the PAS, where mitophagy-specific uptake occurs. At the PAS, Atg32 interacts with Atg8 on the phagophore
membrane and promotes formation of the autophagosome surrounding mitochondria (the mitophagosome). Atg20, Atg24,
and most of the core autophagy machinery components form a complex at the PAS and cooperate to complete mitopha-
gosome formation. (To see this illustration in color the reader is referred to the web version of this article at www
Schematic models of mitophagy in mammals and in yeast. Mitophagy in mammalian cells: Parkin-related pro-
MITOPHAGY IN YEAST1997
This finding strongly supports the idea that mitophagy is a
valid therapeutic target for mitochondrial diseases. However,
further investigations are required prior to actual clinical
This work was supported in part by Grant-in-Aids for
22020028 to T.K. and K.O.) and Scientific Research (C)
(20570144 to K.O.) from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (MEXT), The Ue-
(T.K.), Osaka University Life Science Young Independent
Researcher Support Program through the Special Coordina-
tion Funds for Promoting Science and Technology from the
MEXT (to K.O.), and National Institutes of Health (Grant
GM53396 to D.J.K).
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Address correspondence to:
Asst. Prof. Tomotake Kanki
Department of Clinical Chemistry and Laboratory Medicine
Kyushu University Graduate School of Medical Sciences
Assoc. Prof. Koji Okamoto
Laboratory of Mitochondrial Dynamics
Graduate School of Frontier Biosciences
Date of first submission to ARS Central, November 5, 2010;
date of final revised submission, December 14, 2010; date of
acceptance, January 1, 2011
CCCP¼carbonyl cyanide m-chlorophenylhydrazone
Cvt¼cytoplasm to vacuole targeting
GFP¼green fluorescent protein
HDAC6¼histone deacetylase 6
PAS¼phagophore assembly site
PINK1¼PTEN-induced putative kinase 1
ROS¼reactive oxygen species
TOR¼target of rapamycin
VSM¼vacuolar sequestering membrane
MITOPHAGY IN YEAST 2001