ATG12 Conjugation to ATG3 Regulates
Mitochondrial Homeostasis and Cell Death
Lilliana Radoshevich,1,2Lyndsay Murrow,1,2Nan Chen,1Estefania Fernandez,1Srirupa Roy,1Christopher Fung,1
and Jayanta Debnath1,2,*
1Department of Pathology and Helen Diller Family Comprehensive Cancer Center
2Biomedical Sciences Graduate Program
University of California San Francisco, San Francisco, CA 94143, USA
ATG12, an ubiquitin-like modifier required for macro-
autophagy, has a single known conjugation target,
another autophagy regulator called ATG5. Here, we
identify ATG3 as a substrate for ATG12 conjugation.
ATG3 is the E2-like enzyme necessary for ATG8/LC3
lipidation during autophagy. ATG12-ATG3 complex
formation requires ATG7 as the E1 enzyme and
ATG3 autocatalytic activity as the E2, resulting in
the covalent linkage of ATG12 onto a single lysine
on ATG3. Surprisingly, disruptingATG12 conjugation
to ATG3 does not affect starvation-induced auto-
phagy. Rather, the lack of ATG12-ATG3 complex
formation produces an expansion in mitochondrial
mass and inhibits cell death mediated by mitochon-
drial pathways. Overall, these results unveil a role
for ATG12-ATG3 in mitochondrial homeostasis and
implicate the ATG12 conjugation system in cellular
functions distinct from the early steps of autophago-
Ubiquitin-like protein conjugations (UBLs), such as ubiquitina-
including protein targeting, organelle trafficking, cell division,
signal transduction, and transcription (Welchman et al., 2005).
These posttranslational modifications, which typically result in
the covalent attachment of the UBL tag to the 3-amine group
of lysine residues in target substrates, are highly dynamic,
reversible, and tightly regulated by well-established biochemical
cascades. First, the UBL is activated in an ATP-dependent
manner by an E1-activating enzyme in which the C-terminal
glycine residue of the UBL moiety forms a high-energy thioester
bond with a cysteine residue in the active site of the E1. Second,
a trans-esterification reaction. Finally, the UBL is transferred
onto a lysine of a target substrate; in many cases, this final trans-
fer requires an E3 ligase enzyme (Kerscher et al., 2006). To date,
two family members, ubiquitin and small ubiquitin-like modifier
(SUMO), have received the greatest attention; nonetheless,
other UBLs undoubtedly have undiscovered functions in biology
and disease (Welchman et al., 2005).
Two UBLs, ATG12 and ATG8 (ATG stands for autophagy
regulator), play critical roles in macroautophagy (hereafter called
autophagy), a tightly controlled lysosomal degradation process
in which a cell digests its own proteins and organelles during
starvation or stress (Levine and Kroemer, 2008; Ohsumi, 2001).
Although ATG12 and ATG8 possess little primary sequence
homology to ubiquitin, both contain an ‘‘ubiquitin superfold’’
and the C-terminal glycine required for isopeptide linkage
(Hanada and Ohsumi, 2005; Sugawara et al., 2004; Suzuki
et al., 2005). Importantly, the early steps of autophagosome
formation require these two ubiquitin-like conjugation pro-
cesses, which covalently attach ATG12 to the target protein
ATG5 (Mizushima et al., 1998a, 1998b) and ATG8 (for which
microtubule-associated protein light chain 3 [LC3] is a chief
mammalian ortholog) to the lipid phosphotidylethanolamine
(PE) (Ichimura et al., 2000; Kabeya et al., 2000). Both ATG8
and ATG12 are activated in an ATP-dependent process by an
ubiquitin-like E1-activating enzyme, called ATG7 (Tanida et al.,
1999). Subsequently, ATG12 is conjugated to ATG5 by ATG10,
an E2-like conjugating enzyme (Nemoto et al., 2003), whereas
ATG8/LC3 is conjugated to PE via another E2-like molecule,
ATG3 (Tanida et al., 2002b; Yamada et al., 2007). Furthermore,
recent biochemical evidence supports a model in which the
ATG12-ATG5 complex possesses an E3-like activity for efficient
PE lipidation of ATG8 (Fujita et al., 2008; Hanada et al., 2007).
Unlike other UBLs, ATG12 is proposed to modify a single
target, ATG5. However, it remains unclear why a complex
energy-consuming enzymatic cascade hasevolved to conjugate
a single substrate. Furthermore, certain stimuli, namely mito-
chondrial outer membrane permeabilization (MOMP) and endo-
plasmic reticulum (ER) stress, produce an increase in ATG12
transcript or protein levels but no concomitant rise in ATG5;
such discordant regulation is enigmatic given that ATG12 is
solely proposed to function as part of a 1:1 stoichiometric
complex with ATG5 (Colell et al., 2007; Kouroku et al., 2007).
One potential explanation for these previous findings is that,
similar to other UBLs, ATG12 modifies additional substrates
that regulate autophagy or other cellular functions.
Hence, we sought to test the hypothesis that ATG12 is not an
isolated UBL modification confined to a single target. Here, we
590 Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc.
identify ATG3, the E2 enzyme necessary for ATG8/LC3 lipidation
during autophagy, as a substrate for ATG12 conjugation.
ATG12-ATG3 complex formation requires ATG7 as the E1-acti-
vating enzyme and an autocatalytic function of ATG3 as the
E2, resulting in the covalent attachment of ATG12 onto a single
lysine on ATG3. Remarkably, disrupting ATG12-ATG3 complex
formation has no discernable effect on nonselective autophagy.
Rather, upon disrupting ATG12 conjugation to ATG3, cells
display increased mitochondrial mass and enhanced survival
in response to agents that activate mitochondrial cell death
pathways. Overall, these results demonstrate a previously
unrecognized role for the ATG12-ATG3 complex in mitochon-
drial homeostasis and cell death. We propose that the ATG12
conjugation system directs cellular functions distinct from the
early steps of autophagosome formation.
ATG12 Covalently Modifies Multiple Protein Targets
in Addition to ATG5 in Mammalian Cells
To test whether ATG12 covalently modifies protein targets in
addition to ATG5, we created a retroviral construct encoding
epitope-tagged mouse ATG12 (FHA-ATG12); as a negative
Multiple Protein Targets in Mammalian
(A) Schematic of ATG12 constructs: FHA-ATG12
is mouse ATG12 tandem tagged at the N terminus
with FLAG and HA epitopes. In FHA-Stop, the
C-terminal glycine in ATG12 required for conjuga-
tion is replaced with a Stop codon.
(B) MCF10A cells stably expressing empty vector
(MSCV), FHA-ATG12, and FHA-Stop were lysed
and immunoblotted with a-ATG12 and a-HA
antibodies. Asterisk (*) indicates nonspecific
band during a-HA immunoblotting.
(C) MCF10A cells stably expressing the indicated
constructs were lysed and immunoprecipitated
with a-FLAG; immune complexes were resolved
(D) HeLa cells stably expressing the indicated con-
structs were lysed and immunoblotted with a-HA.
(E) atg7+/+(WT) and atg7?/?mouse embryonic
fibroblasts (MEFs) stably expressing the indicated
constructs were lysed and immunoblotted with
a-HA. Asterisk (*) indicates nonspecific band.
(F) Left: atg5+/+(WT) and atg5?/?MEFs stably
expressing the indicated constructs were lysed
and immunoblotted with a-HA or a-ATG5. Right:
WT and atg5?/?MEFs expressing FHA-ATG12
were lysed and immunoprecipitated with a-FLAG;
immune complexes were resolved and immuno-
blotted with a-HA.
1. ATG12 CovalentlyModifies
control, we generated a conjugation-
C-terminal glycine in ATG12, required
for the isopeptide linkage to target lysine
residues,was replaced withastopcodon
(FHA-Stop) (Figure 1A) (Mizushima et al.,
1998b). Upon stable expression in MCF10A human mammary
epithelial cells, we observed a slower-migrating ATG12-ATG5
complex, consistent with epitope tag addition, as well as
additional higher molecular weight conjugates in FHA-ATG12-
expressing cells but not in FHA-Stop cells (Figure 1B). Impor-
tantly, these higher molecular weight proteins were observed
using denaturing and reducing conditions, suggesting that
ATG12 was covalently attached to multiple protein targets.
Furthermore, upon a-FLAG immunoprecipitation, we observed
numerous additional ATG12-X species that were unique to cells
expressing conjugatable ATG12 (Figure 1C). Similar results were
obtained upon stable FHA-ATG12 expression in HeLa cervical
carcinoma cells, indicating that the ATG12-X proteins are
present in multiple human cell types (Figure 1D).
We next asked whether the E1-like enzyme ATG7 is required
for the activation of ATG12 and its subsequent conjugation to
these targets (Tanida et al., 1999, 2001). We stably infected
ATG7-deficient fibroblasts with FHA-ATG12 or FHA-Stop and
found that all ATG12 conjugations were eliminated in the
absence of ATG7 (Figure 1E). Thus, ATG12 requires ATG7 as
an E1-activating enzyme to be conjugated to any of its multiple
protein targets. As ATG5 is the only known substrate of
ATG12, we tested if the additional higher molecular weight
Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc. 591
ATG12-X proteins were unique ATG12-modified proteins versus
multiple ATG12 conjugations onto the ATG12-ATG5 complex
(Mizushima et al., 1998a, 1998b). To distinguish among these
possibilities, we stably expressed FHA-ATG12 and FHA-Stop
in both wild-type (atg5+/+)and ATG5 null (atg5?/?) mouse embry-
onic fibroblasts (MEFs). Similar to our results in human cells, we
observed numerous ATG12 conjugates in both atg5+/+and?/?
mouse fibroblasts. In atg5?/?cells, only the band corresponding
ATG12-X proteins continued to be produced, supporting that
these species are unique conjugation targets that can be formed
in the absence of ATG5 (Figure 1F).
ATG12 and ATG3 Form a Covalent Complex
To identify putative ATG12-X proteins, lysates from atg5?/?
fibroblasts stably expressing either FHA-ATG12 or FHA-Stop
were subject to large-scale tandem affinity purification with
a-FLAG and a-HA antibodies. Upon resolving the eluted pro-
teins, each protein in the predominant doublet on a Coomassie
Figure 2. ATG12 and ATG3 Form a Covalent
(A) Lysates from atg5?/?MEFs stably expressing
FHA-ATG12 or FHA-Stop were subject to affinity
purification with a-FLAG followed by a-HA anti-
bodies. Eluted proteins were resolved using
SDS-PAGE and immunoblotted with a-HA (left)
or stained with Coomassie (middle). Each protein
in the indicated doublet (arrows) was individually
subject to MS/MS analysis and both were identi-
fied as ATG3. Right: Amino acid sequence of
mouse ATG3; underlined sequences correspond
to independent peptides identified by mass
(B) Lysates from MEFs expressing the indicated
constructs were immunoprecipitated with a-FLAG;
immune complexes (FLAG IP) were resolved using
SDS-PAGE and immunoblotted with a-ATG12 or
(C) MCF10A cells stably expressing FHA-ATG12
were infected with lentiviruses encoding nontar-
geting control shRNA or shRNA targeted to
ATG3 (shATG3). Lysates were immunoblotted as
(D) Wild-type and atg3?/?MEFs stably expressing
the indicated constructs were lysed and subject to
(E) Wild-type and atg3?/?MEFs stably expressing
FHA-ATG12 were lysed and subject to a-FLAG
immunoprecipitation; immune complexes were
resolved and immunoblotted with a-HA.
(F) Lysates from wild-type and atg3?/?MEFs were
immunoprecipated with a-ATG3; immune com-
plexes were resolved using SDS-PAGE and
subject to a-ATG3 or a-ATG12 immunoblotting.
Asterisk (*) indicates immunoglobulin heavy chain.
See also Figure S1.
brilliant blue stained gel (Figure 2A,
arrows) was trypsin-digested and sub-
ject totandem mass
(MS/MS). Both proteins in this doublet
were identified as ATG12 conjugated to ATG3 (Figure 2A).
ATG3 is the E2-like enzyme responsible for ATG8/LC3 lipidation
and has previously been demonstrated to interact with ATG12
(Tanida et al., 2002a, 2002b).
In a-FLAG immunoprecipitates prepared from MEFs express-
ing FHA-ATG12, we detected the 65 kDa doublet using anti-
bodies against either ATG12 or ATG3, confirming that this
protein corresponded to ectopic FHA-ATG12 conjugated to
endogenous ATG3 (Figure 2B). Remarkably, this doublet
collapsed to a single protein when treated with calf intestinal
phosphatase, suggesting that the ATG12-ATG3 complex
undergoes phosphorylation (Figure S1 available online). Further-
more, MCF10A cells stably expressing shRNA against ATG3
exhibited specific depletion of the ATG12-ATG3 complex, rela-
tive to a nontargeting shRNA control, whereas the ATG12-
ATG5 complex continued to be produced (Figure 2C). The
ATG12-ATG3 complex was not observed in atg3?/?MEFs
expressing FHA-ATG12, in contrast to wild-type controls (Fig-
ure 2D), whereas ATG12-ATG5 and other higher molecular
592 Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc.
weight species, corresponding to other ATG12-X conjugates,
persisted in atg3?/?cells (Figure 2E) (Sou et al., 2008). Finally,
to assess if the ATG12-ATG3 complex was present at endoge-
nous expression levels, protein extracts were immunoprecipi-
tated with a-ATG3 antibodies. We detected a 51 kDa protein
complex in wild-type fibroblasts that immunoblotted with both
a-ATG3 and a-ATG12 antibodies; this complex was not present
in protein lysates prepared from atg3?/?MEFs (Figure 2F).
Overall, these data support that ATG12 forms a covalent
(with ATG12-ATG5 being the first) of several potential ATG12
Autoconjugation of ATG12 onto a Single Lysine on ATG3
Similar to other UBLs, ATG12 is covalently attached to the
3-amine group of a lysine residue of its target (Kerscher et al.,
2006; Mizushima et al., 1998a). To elucidate potential functions
of the ATG12-ATG3 complex, we sought to identify the target
lysine(s) in ATG3 required for ATG12 conjugation. First, we
compared the amino acid sequence of ATG3 from multiple
species and found five lysines to be highly conserved (Fig-
ure S2A). Using site-directed mutagenesis, we systematically
eliminated each lysine, or groups of lysines, and transiently
coexpressed each mutant version of ATG3 along with ATG12
and ATG7 in HEK293T cells (Figure 3A and Figure S2B). Using
this strategy, we reconstituted ATG12-ATG3 complex formation
and identified lysine 243 (K243) as the primary lysine required
for ATG12 conjugation. Upon mutation of this single lysine to
arginine, complex formation was virtually eliminated. Remark-
ably, mutating an adjacent lysine at position 242 of ATG3 had
no effect on ATG12 conjugation. In contrast, mutating the
ATG3 catalytic cysteine to alanine (C264A) potently inhibited
formation of the ATG12-ATG3 complex (Figure 3A) (Tanida
et al., 2002b). To corroborate these results, we stably comple-
mented ATG3-deficient fibroblasts with epitope-tagged wild-
type (WT) ATG3, the nonconjugatable ATG3 mutant K243R,
and the catalytically inactive mutant C264A (Sou et al., 2008).
Unlike cells reconstituted with WTATG3, ATG12-ATG3 complex
formation was not observed in cells coexpressing FHA-ATG12
and either K243R or C264A (Figure 3B).
As ATG12-ATG3 formation required ATG3 catalytic activity,
we hypothesized that an autocatalytic activity of ATG3 served
as the E2 enzyme for this conjugation. Several E2 enzymes
undergo self-ubiquitination (Gwozd et al., 1995; Machida et al.,
2006; Walter et al., 2001). Typically, these are cis-acting intra-
molecular reactions, in which the target lysine of the E2 enzyme
lies in close proximity to the active site cysteine; upon inspection
of its tertiary structure, this also appeared to be the case for
ATG3 (Figure 3C) (Yamada et al., 2007). To test whether
ATG12 conjugation to ATG3 could occur in trans, we coex-
pressed two distinct tagged versions of ATG3, along with
ATG12 and ATG7, in 293T cells. The first was catalytically inac-
tive ATG3 (C264A) possessing the target lysine, whereas the
second was catalytically active ATG3 that lacked the target
lysine (K243R). In contrast to WTATG3, no ATG12-ATG3
complex was formed upon coexpressing these two mutant
forms of ATG3, supporting that ATG12 conjugation to ATG3
was a cis-acting reaction (Figure 3D). Finally, we assessed
if ATG10, the E2 enzyme responsible for ATG12 conjugation to
ATG5, contributed to the formation of ATG12-ATG3. However,
ATG10 was unable to mediate ATG12-ATG3 complex formation,
and the expression of catalytically inactive ATG10 (C165A) was
unable to dominantly inhibit the autoconjugation of ATG12 onto
ATG3 (Figure 3E and Figure S2C). Overall, these results indicate
Figure 3. Autoconjugation of ATG12 onto a
Single Lysine of ATG3
(A) HEK293T cells expressing YFP-ATG12, Myc-
tagged ATG7, and V5-tagged WTATG3 or the
indicated ATG3 mutants.
(B) atg3?/?MEFs stably reconstituted with empty
vector (BABE), wild-type ATG3, or the indicated
ATG3 mutants were then transduced with empty
vector (MSCV) or FHA-ATG12. Lysates were
a-HA immunoblotted todetect ATG12-conjugated
(C) Crystal structure of yeast ATG3 with labeled
a helices (blue) and b sheets (purple). Positions
of the conserved catalytic cysteine (C264 in
mouse ATG3, green) and the principal lysine
conjugated with ATG12 (K243, yellow) are shown.
Diagram prepared using PyMOL.
(D) 293T cells transfected with YFP-ATG12,
myc-tagged ATG7, and mutants of either HA- or
V5-tagged ATG3, as indicated.
(E) HEK293T cells transfected with Myc-tagged
ATG7, V5-tagged ATG10, YFP-ATG12, and either
HA-tagged ATG5 or ATG3 C264A; catalytically
inactive ATG3 (C264A) was used to distinguish
the E2 activity of ATG10 from ATG3 during
See also Figure S2.
Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc. 593
that ATG3 autocatalyzes the conjugation of ATG12 onto a single
conserved lysine residue (K243) of this E2-like enzyme required
Starvation-Induced Autophagy Remains Intact upon
Disrupting ATG12 Conjugation to ATG3
We next tested if ATG12-ATG3 modulates known ATG3 biolog-
ical activities, namely PElipidation ofATG8/LC3 andautophago-
experiments, we reconstituted atg3?/?fibroblasts with a control
empty vector (BABE), wild-type ATG3 (WTATG3), or the K243R
mutant (KR) that was incapable of ATG12-ATG3 complex forma-
using Hank’s buffered saline (HBSS) or rapamycin-mediated
mTORC1 inactivation, we found equivalent levels of LC3 lipida-
tion (LC3-II) between WTATG3 and KR cells and efficient
LC3-II turnover in the lysosome (Figures 4A and 4B). We also
observed no significant differences in the numberor morphology
of GFP-LC3 puncta, a widely utilized marker for autophagosome
formation, upon HBSS starvation, although we did note an
increase in punctate GFP-LC3 in KR cells cultured in nutrient-
rich conditions (Figures 4C and 4D and Figure S3B) (Kabeya
et al., 2000). Furthermore, WTATG3 and KR cells exhibited no
differences in the lipidation of other mammalian ATG8 orthologs,
including GABARAP and GATE-16 (Figure S3C).
To extend these results, we evaluated the accumulation of
p62SQSTM, an ubiquitin-binding scaffold protein selectively
degraded by autophagy, via immunofluorescence (Bjorkoy
et al., 2005). In both nutrient-rich and starvation conditions,
p62 cytoplasmic bodies amassed in autophagy-deficient cells
lacking ATG3; this accumulation was profoundly decreased in
cells rescued with either WTATG3 or the KR mutant (Figure 4E).
Finally, we assessed the processing of an ectopically expressed
autophagy cargo protein betaine homocysteine methyltransfer-
ase (BHMT) (Dennis and Mercer, 2009). Efficient proteolytic
cleavage of BHMT during HBSS starvation required both ATG3
and lysosomal function; this autophagy-dependent cleavage
was restored at equivalent levels upon rescue with either
WTATG3 or KR (Figure 4F). Overall, these data support
that disrupting ATG12 conjugation to ATG3 does not impair
ATG8/LC3 lipidation, autophagosome formation, or autophagic
proteolysis in response to nutrient starvation.
Remains Intact upon Disrupting ATG12
Conjugation to ATG3
Stable pools of atg3?/?fibroblasts expressing an
empty vector (BABE), wild-type mouse ATG3
(WTATG3), or mutant ATG3 unable to be conju-
gated by ATG12 (KR) were used for experiments
(A and B) Cells were grown in complete media,
starved in Hank’s buffered salt solution (HBSS)
for 4 hr, or treated with 10 nM rapamycin (B) for
6 hr. Cells were lysed and immunoblotted with
indicated antibodies. Phosphorylated ribosomal
S6 (P-S6) was used to verify rapamycin-mediated
mTORC1 inhibition. When indicated, bafilomycin
A (BafA, 10 nM) was added to cells 1 hr prior to
(C) Indicated cells types expressing GFP-LC3
were grown in complete media (control) or
HBSS-starved for 4 hr; boxed areas from center
panels are enlarged below. Bars, 25 mm.
(D) Quantification of punctate GFP-LC3 or GFP-
LC3DG (mean ± standard error of the mean
[SEM] puncta per cell).
(E) Indicated cell types grown in complete media
(control) or HBSS-starved for 4 hr and then
fixed and immunostained with a-p62 antibody.
Bar, 25 mm.
(F) Indicated cell types were transfected with
a GST-BHMT fusion construct, HBSS-starved for
6 hr, lysed, and immunoblotted with a-GST.
Asterisk (*) indicates full-length GST-BHMT and
arrow indicates cleaved BHMT produced in
autolysosomes. When indicated, bafilomycin A
(BafA, 10 nM) was used to inhibit lysosomal
function. a-Myc was used to detect GFP-myc
(expressed from an IRES sequence) to control
for transfection efficiency (Dennis and Mercer,
See also Figure S3.
4. Starvation-Induced Autophagy
594 Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc.
Cells Expressing Nonconjugatable ATG3 (KR) Exhibit
Increased Mitochondrial Mass and a Fragmented
Recent studies support that autophagy is required to maintain
mitochondrial homeostasis (Chen and Chan, 2009; Lemasters,
2005). Flow cytometric analysis for Mitotracker Green (MTG), a
mitochondria specific intravital dye, demonstrated that cells
reconstituted with WTATG3 exhibit an approximately 15% reduc-
tion in total mitochondrial mass compared to atg3?/?(BABE)
controls; similarly, atg3+/+cells possessed reduced MTG staining
compared to atg3?/?cells. In contrast, the mitochondrial mass
of cells reconstituted with KR was unchanged relative to atg3?/?
(BABE) cells (Figure 5A). Protein levels of the mitochondrial inner
membrane protein, cytochrome c oxidase subunit IV (COX IV),
corroborated these differences in MTG staining (Figure 5B).
Wenextevaluatedmitochondrial morphologyinthe various cell
types by immunostaining for TOM20, a mitochondrial import
receptor located in the outer membrane, and cytochrome c,
Conjugation to ATG3 on Mitochondrial
Mass and Morphology
(A) Left: Mitotracker Green (MTG) fluorescence
intensity (mean ± SEM from five experiments) for
the indicated cell types relative to atg3?/?cells
significance calculated using ANOVA, followed
by Tukey’s HSD test. Right: MTG fluorescence
intensity (mean ± SEM from three experiments)
for atg3+/+cells relative to atg3?/?cells.
(B) Lysates from indicated cell types were immu-
noblotted with a-COX IV, a-tubulin, and a-V5.
(C) Indicated cell types were immunostained with
TOM20 (top and middle) or cytochrome c (bottom)
(D) Percent of cells with purely fragmented/round
morphology or purely tubular morphology was
quantified from TOM20-immunostained images.
Results are the mean ± SEM from five experi-
ments, where at least 250 cells were scored per
condition for each individual experiment. Statis-
tical significance calculated using ANOVA, fol-
lowed by Tukey’s HSD test.
(E) Cells expressing either mitochondria-targeted
dsRed (mt-dsRed, red) or CFP (mt-CFP, green)
were hybridized using PEG to assess mitochon-
images of cell hybrids from each cell type are
shown; the colocalization of these signals (yellow)
within cell hybrids indicates mitochondrial fusion
(Chen et al., 2003). Each bottom panel is an
enlargement of the boxed inset in the correspond-
ing panel above. Bar, 25 mm.
See also Figure S4 and Figure S5.
5. Effects ofDisrupting ATG12
located in the inner membrane. In both
ATG3 null (BABE) and KR cultures,
numerous cells possessed fragmented
and round mitochondria, whereas in
Because the mitochondria in individual cells in these cultures
exhibited a range of morphologies (Figure S4A), we enumerated
cells from each condition that showed purely fragmented/round
versus purely tubular mitochondrial morphology. We confirmed
sponding decrease in cells with purely tubular mitochondria
(Figure 5D). In contrast, we observed no obvious morphological
peroxisomes, or Golgi apparatus (Figure S4B).
Fragmentation can arise as an early consequence of declining
mitochondrial function or cell viability (Youle and Karbowski,
2005). Hence, to determine whether the excess mitochondria
inKR cellsweredepolarized, wecostained cellswithMitotracker
Red (MTR), whose accumulation in mitochondria depends on
intact membrane potential, and MTG, which labels the lipid
membranes of all mitochondria (Tal et al., 2009). A small popula-
tion of ATG3 null cells had depolarized mitochondria (MTR
Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc. 595
negative, MTG positive), which was not significantly reduced
upon rescue with either WTATG3 or KR (Figure S4C). We also
performed growth curves for the three cell types and found no
overt differences in proliferation or viability (Figure S4D).
Alternatively, defects in mitochondrial fusion also promote
fragmentation (Chen and Chan, 2009). Accordingly, we evalu-
ated mitochondrial fusion activity more directly using a polyeth-
ylene glycol (PEG) fusion assay (Chen et al., 2003). For these
assays, either mitochondria-targeted red (mt-dsRed) or cyan
(mt-CFP) fluorescent proteins were ectopically expressed in
each cell type; subsequently, hybrids between the differentially
labeled populations were scored for mitochondrial fusion.
Although we observed robust mitochondrial fusion among
hybrids derived from WTATG3 cells, such events were reduced
in both ATG3 null and KR-expressing hybrids, indicating de-
creased fusion activity (Figure 5E and Figures S5A–S5B). Alto-
gether, these results support that disrupting ATG12 conjugation
to ATG3 produces significant increases in mitochondrial mass
and fragmentation, which correlates with a reduction in mito-
chondrial fusion activity. Most importantly, these phenotypes
arise in cells capable of robust autophagy, indicating that the
effects of the ATG12-ATG3 complex on mitochondria are
distinct and separable from the well-established functions of
ATG3 in ATG8/LC3 lipidation and autophagosome formation.
Effects of CCCP Uncoupling on Mitochondria in Cells
Expressing Nonconjugatable ATG3 (KR)
Next, by stressing cells with carbonyl cyanide m-chlorophenyl-
hydrazone (CCCP), a proton ionophore that uncouples mito-
chondria, we sought to clarify how the ATG12-ATG3 complex
regulates mitochondrial homeostasis. CCCP-induced depolar-
ization elicits a multifaceted cellular response that includes an
overall increase in mitochondrial biogenesis coupled to the
autophagic degradation of damaged mitochondria, termed
mitophagy. (Lemasters, 2005; Narendra et al., 2008; Rohas
et al., 2007). Indeed, CCCP treatment resulted in increased
MTG intensity compared to DMSO-treated controls in all cell
types, corroborating that overall mitochondrial mass increased
during chemical uncoupling (Rohas et al., 2007). Notably, this
CCCP-induced increase in MTG staining intensity was more
pronounced in both ATG3 null (BABE, 27% increase) and KR
cells (25% increase) in comparison to WTATG3 cells (14%
increase) (Figure S6A). When directly compared to atg3?/?
(BABE) cells, CCCP-treated WTATG3 cells displayed 20% lower
MTG staining intensity; similar differences were observed
between atg3+/+and atg3?/?cells. In contrast, KR cells pos-
sessed a 15% and 35% increase in mitochondria compared
to BABE and WTATG3 cells, respectively (Figure 6A). TOM20
immunostaining of CCCP-treated cultures also supported that
mitochondria were more abundant in ATG3 null and KR cells
compared to WTATG3 cultures (Figure S6B). These results indi-
cate that ATG12 conjugation to ATG3 restricts the expansion of
mitochondrial mass during CCCP-induced depolarization.
We next assessed the protein levels of two mitochondrial resi-
dent proteins, COX IV and TOM40, and found that, compared to
WTATG3,KRcellshadhigherlevelsofboth proteinsincontrol as
well as CCCP-treated cultures. Interestingly, in WTATG3 cells,
both proteins were slightly reduced during CCCP treatment,
whereas in KR cells, these levels remained unchanged, suggest-
ing that the rate of mitochondrial degradation may be reduced in
Figure 6. Effects of CCCP Treatment on
Mitochondria inCellsExpressing a Noncon-
jugatable ATG3 Mutant (KR)
(A) Left: Cells treated with 10 mM CCCP for 24 hr
(mean ± SEM from eight experiments) relative to
atg3?/?cells expressing empty vector (BABE) is
shown. Statistical significance was calculated
using ANOVA, followed by Tukey’s HSD test.
Right: MTG fluorescence intensity (mean ± SEM
from three experiments) for CCCP-treated atg3+/+
cells relative to atg3?/?cells.
(B) Cells were CCCP-treated as indicated, lysed,
and immunoblotted with antibodies against the
resident mitochondrial proteins TOM40 and COX
IV, V5, and tubulin (loading control).
(C) Indicated cell types expressing GFP-LC3 were
transfected with mito-dsRed and treated with
10 mM CCCP for 24 hr. White arrows indicate
colocalization of GFP-LC3 and mito-dsRed. Bar,
(D) Quantification of mito-dsRed and GFP-LC3
colocalizations per cell (mean ± SEM).
(mean ± SEM from five experiments) during
See also Figure S6.
596 Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc.
cells lacking the ATG12-ATG3 complex (Figure 6B). As a result,
we sought to more closely analyze if KR cells exhibited
decreased mitophagy. We first compared the rates of CCCP-
induced autophagosome induction in WTATG3 and KR cells
but found no significant differences in punctate GFP-LC3
between these cells following CCCP treatment (Figures S6C
and S6D). To measure mitochondrial targeting to autophago-
somes during CCCP treatment, we assessed the level of
colocalization between mito-dsRed and GFP-LC3 (Figure 6C).
Importantly, the number of colocalizations was significantly
higher in WTATG3 cells compared to KR cells (Figures 6C
and 6D). Hence, upon loss of ATG12 conjugation to ATG3,
mitochondrial targeting to autophagosomes is reduced.
To further assess if mitophagy was impaired in KR cells, we
tested if 3-methyladenine (3MA), a pharmacological autophagy
inhibitor, was able to augment mitochondrial mass during
CCCP treatment (Seglen and Gordon, 1982). In WTATG3 cells,
we observed a 27.5% increase in mitochondrial mass upon
treatment with CCCP + 3MA compared to CCCP alone, which
we attributed to 3MA-sensitive mitochondrial degradation. To
control for the nonspecific effects of 3MA, which can promote
significant protein degradation in an autophagy-independent
manner (Mizushima et al., 2001), we tested the effects of 3MA
on atg3?/?(BABE) cells; 3MA-sensitive mitochondrial degrada-
tion in these autophagy-incompetent cells was reduced to
20% (Figure 6E). Similar to atg3?/?(BABE) cells, 3MA-sensitive
mitochondrial degradation in KR cells was decreased to 22%.
Overall, these results support that ATG12 conjugation to ATG3
Exhibit Decreased Cell Death Mediated by
Cells were treated for 24 hr with the following
agents: (A) 100 mM CCCP; (B) 100 nM staurospor-
ine; and (C) 20 ng/ml TNF-a + 2.5 mg/ml cyclohex-
imide. Percent cell death (mean ± SEM) was
assayed by propidium iodide uptake using flow
(D) Lysates prepared from WTATG3 and KR cells
were immunoblotted for the indicated markers.
(E) Cells were treated for 24 hr with 500 nM
obatoclax. Cell death (mean ± SEM) was quanti-
fied using trypan blue exclusion. Statistical signif-
icance calculated using t test.
(F) Summary model of results.
7. Cells LackingATG12-ATG3
facilitates mitochondrial degradation in
the presence of CCCP. Importantly, this
separable from the ability of ATG3 to
induce autophagosome formation during
Cells Lacking ATG12-ATG3 Exhibit
Reduced Cell Death Mediated
by Mitochondrial Pathways
Mitochondria play critical roles in many
forms of cell death, including apoptosis
mediated by the intrinsic pathway (Bren-
ner and Mak, 2009). Accordingly, we determined how the
the effects of CCCP on cell death in WTATG3 and KR cells;
similar to other mitochondrial uncoupling agents, high doses
of CCCP induce death that can be inhibited by antiapoptotic
Bcl-2 family members (de Graaf et al., 2004). In response to
CCCP treatment, KR cells exhibited a 2-fold reduction in death
compared to WTATG3 controls (Figure 7A). Similarly, cell death
inhibitor that potently activates the intrinsic apoptosis pathway
(Figure 7B). We also observed reduced cleavage of the execu-
tioner caspase, caspase-3, in KR cells in response to both
agents, supporting that apoptosis was reduced compared to
WTATG3 (data not shown). In contrast, both cell types exhibited
robust and equivalent levels of cell death in response to tumor
necrosis factor-a (TNF-a). TNF-a induces apoptosis via the
extrinsic pathway, in which death receptors directly activate
executioner caspases independently of mitochondrial pathways
(Figure 7C) (Brenner and Mak, 2009). Thus, ATG12 conjugation
to ATG3 sensitizes cells to death downstream of mitochondrial
pathways but has no effect on death-receptor-mediated
Antiapoptotic members of the Bcl-2 family, such as Bcl-xL,
function as potent inhibitors of cell death; these proteins are
mainly located on mitochondria, which is vital for their prosur-
vival functions (Brenner and Mak, 2009). Increased Bcl-xLpro-
tein levels were present in KR cells compared to WTATG3,
whereas no differences in the protein levels of the proapoptotic
Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc. 597
Bcl-2 family members, Bax and Bak, were observed (Figure 7D).
Accordingly, we predicted that both WTATG3 and KR cells
would be sensitive to the potent chemical inhibition of antiapop-
totic Bcl-2 family members. To test this hypothesis, we utilized
obatoclax, a small molecule BH3 mimetic that antagonizes
multiple antiapoptotic Bcl-2 family proteins, including Bcl-xL,
Bcl-2, and Mcl-1 (Nguyen et al., 2007). As a single agent, obato-
clax was able to robustly induce cell death in both WTATG3
and KR cells; most importantly, we found no significant differ-
ences in obatoclax-mediated death between these two cell
types (Figure 7E). In conclusion, cells lacking ATG12-ATG3
exhibit reduced cell death mediated by mitochondrial pathways;
this protection correlates with an increase in antiapoptotic Bcl-2
proteins secondary to the lack of ATG12 conjugation to ATG3.
system, ATG5 remains as the only known target of ATG12
(Mizushima et al., 1998a, 1998b). Here, we provide evidence
that multiple ATG12 substrates exist and identify ATG3, the E2
enzyme that mediates ATG8/LC3 lipidation during autophagy,
this complex links two components of the autophagy conjuga-
tion machinery, we reasoned that ATG12 modification of ATG3
would primarily regulate the early steps of autophagosome
formation (Ohsumi, 2001). However, our results argue against
this hypothesis. Disrupting ATG12 conjugation to ATG3 has no
discernable effect on LC3/ATG8 lipidation or nonselective
autophagy in response to three different stresses—nutrient
starvation, rapamycin-mediated mTOR inhibition, and CCCP-
induced mitochondrial depolarization. Instead, the most obvious
consequences of disrupting the ATG12-ATG3 complex are in-
creased mitochondrial mass, fragmentation of the mitochondrial
network, and resistance to cell death mediated by mitochondrial
pathways (Figure 7F).
Mitochondrial homeostasis requires the careful balance and
integration of numerous processes, namely fission, fusion,
biogenesis, and degradation (Chen and Chan, 2009). Although
we observe decreased levels of mitophagy upon disrupting
ATG12-ATG3 complex formation, we do not believe our results
can be solely explained by the defective autophagy of damaged
mitochondria. First, the reduction in mitophagy in cells lacking
ATG12-ATG3 is modest. Second, the increase in mitochondrial
mass in KR cells is not accompanied by mitochondrial depolar-
ization, suggesting that the excess mitochondria are functional.
Third, we have uncovered that during CCCP-induced uncou-
pling, mitochondrial mass is actually higher in autophagy-
competent KR cells than in ATG3 null cells, which are autophagy
deficient. Based on these results, we hypothesize that the
ATG12-ATG3 complex may also restrict mitochondrial expan-
sion using mitophagy-independent mechanisms. Notably, other
proteins, like Parkin, have versatile effects on mitochondria. In
addition to promoting mitophagy, Parkin has been found to
control mitochondrial dynamics and to induce biogenesis; the
precise interrelationships between these diverse biological
outcomes remain unclear (Deng et al., 2008; Kuroda et al.,
2006; Lutz et al., 2009; Narendra et al., 2008). Notably, we
have uncovered increased basal levels of Bcl-XLin KR cells,
which may contribute to changes in both mitochondrial mass
and morphology. Recent work in neurons demonstrates that
Bcl-XLplays a vital role in mitochondrial homeostasis and is
able to increase mitochondrial fission, fusion, and biomass
(Berman et al., 2009). Future studies are required to more
precisely dissect how this complex between two ATGs uniquely
affects mitochondrial expansion and morphology, as well as to
identify potential interconnections with the mitochondrial fission
and fusion machinery.
Surprisingly, despite possessing fragmented mitochondrial
morphology, cells lacking ATG12-ATG3 exhibit resistance to
death mediated by mitochondrial pathways. Overall, the effects
of mitochondrial fission and fusion on cell death are variable and
context dependent (Suen et al., 2008). We hypothesize that the
protection found in KR cells may result, at least in part, from
increased basal levels of antiapoptotic Bcl-2 family proteins. In
support, specific and potent antagonism of antiapoptotic Bcl-2
proteins causes equivalent amounts of cell death in WTATG3
and KR cells. A future challenge will be to discern which of these
dramatic perturbations in mitochondrial
function in KR cells is causal, as mitochondrial homeostasis,
dynamics, and cell death are in delicate equilibrium (Figure 7F).
Importantly, our results indicate that the effects of the
ATG12-ATG3 complex on mitochondrial homeostasis and cell
death are unique functions that can be completely separated
from the established roles of either ATG in autophagosome
formation. Other studies demonstrate that pleiotropic roles for
ATGs beyond autophagy may exist; for example, recent work
delineates a macroautophagy-independent mechanism of LC3
recruitment to the mammalian phagosome, which is dependent
on Beclin-1 and ATG5 (Sanjuan et al., 2007). Moreover, our
studies support that ATG12, like ubiquitin and SUMO, may
serve as a broad-based ubiquitin-like protein conjugation with
far-reaching implications in biology and humandisease. Accord-
ingly, we are actively pursuing the identification and validation
of additional substrates subject to ‘‘12-ylation.’’
Dr. Noburu Mizushima (Tokyo Medical and Dental University) provided atg5+/+
and atg5?/?MEFs and Dr. Masaaki Komatsu (Tokyo Metropolitan Institute)
provided atg7+/+, atg7?/?, atg3+/+, and atg3?/?MEFs. Fibroblasts and HeLa
cells were cultured in DMEM (Invitrogen) supplemented with 10% FBS,
penicillin, and streptomycin. A peptide corresponding to the N terminus
common to human, mouse, and rat MAP1LC3 was used to create a-LC3
rabbit polyclonal antibody (Fung et al., 2008). See Extended Experimental
Procedures for commercial antibodies and chemicals.
Generation of Stable Pools
cDNAs in this study are described in the Extended Experimental Procedures.
For retroviral transduction, VSV-G-pseudotyped retroviruses were generated,
and cells were infected and selected as previously described (Debnath et al.,
2003). Following infection and drug selection, early passage stable pools
(maximum of 6–8 passages) were utilized for experiments to avoid clonal
selection or drift.
monoclonal a-HA conjugated to agarose and eluted with 33 FLAG peptide or
598 Cell 142, 590–600, August 20, 2010 ª2010 Elsevier Inc.
HA peptide, respectively (Sigma). The final eluate was separated by
SDS-PAGE and visualized with Novex Colloidal Blue Stain Kit (Invitrogen).
Bands of interest were in-gel tryptically digested following destain and subject
to tandem mass spectrometry (MS/MS) using electrosprayionization (ESI) and
aquadropole quadropole time-of-flight(QqTOF)massspectrometerlocatedin
the UCSF Mass Spectroscopy Core (QStarXL).
Immunofluorescent staining was carried out as previously described
(Debnath et al., 2003) with the following modifications during fixation and
permeabilization. Fibroblasts were fixed with 2% paraformaldehyde at 4?C
for 10 min, followed by cold methanol (?20?C) for 10 min, and then permeabi-
lized for 10 min at 20?C with 0.5% Triton X-100 in PBS.
Widefield immunofluorescence imagingwasperformedusingthe633(1.4 NA)
or 1003 (1.3 NA) objectives of a Zeiss Axiovert 200 microscope equipped with
a Spot RT camera (Diagnostics Instruments) and mercury lamp; images were
acquired using Metamorph (v6.0) software (Molecular Devices). Confocal
analysis was performed using the 603 (1.4 NA) objective of a Nikon C1si
Spectral Confocal System equipped with an argon laser (488 line) and two
solid-state diodes (405 and 546 lines). Images were color-combined in
Metamorph (v6.0) and arranged in Adobe Photoshop (v7.0).
Analysis of Punctate GFP-LC3
Fibroblasts expressing GFP-LC3 were grown overnight on fibronectin-coated
coverslips prior to HBSS starvation. Cells were fixed with 4% paraformalde-
hyde, washed with PBS, mounted using Immunomount (Thermo), and
analyzed by widefield immunofluorescent microscopy as described above;
images were acquired and punctate GFP-LC3 was quantified using Meta-
morph (v6.0) software. At least 250 cells from four independent experiments
expressing GFP-LC3 were transiently transfected with mito-dsRed, plated
onto fibronectin-coated coverslips, and treated with 10 mM CCCP for 24 hr.
Cells were fixed, washed, and imaged as above and colocalization, defined
as the complete overlap of a GFP-LC3 punctum with mito-dsRed, was
quantified per cell; at least 50 cells from three independent experiments
Flow Cytometry for Mitochondrial Mass
As indicated, cells were grown in full media or subject to the indicated
treatments for 24 hr. Cells were stained with 100 nM Mitotracker Green
(MTG) for 25 min at 37?C. Cells were trypsinized, collected by centrifugation,
washed twice in PBS, and analyzed using a FACSCalibur (BD) and CellQuest
Pro software. When indicated, cells were costained with 100 nM Mitotracker
Red CMXRos (MTR). For assays of 3MA-sensitive mitochondrial degradation
during CCCP treatment, cells were treated for 24 hr with 10 mM CCCP in the
presence or absence of 5 mM 3MA and then stained with MTG as described.
3MA-sensitive degradation was calculated as follows:
ðMFIðMean MTG fluorescence intensityÞfor CCCP+3MA treated cells-MFI for CCCP aloneÞ
ðMFI for CCCP aloneÞ
Cell Death Assays
Cells were treated with death agents for 24 hr. Cells were then trypsinized,
pooled with detached cells (floaters), pelleted by centrifugation, PBS washed,
and stained with propidium iodide (0.5 mg/ml) in PBS for 10 min at 20?C. Cells
were analyzed using a FACSCalibur (BD) and CellQuest Pro software. Obato-
clax-treated cells were analyzed using trypan blue exclusion because nonspe-
cific fluorescence associated with drug treatment technically interfered with PI
detection by flow cytometry.
Experimental groups were compared using t test for pairwise comparisons
or ANOVA (followed by Tukey’s HSD test). For all experiments, statistical
significance is indicated as follows: N.S., nonsignificant; *p < 0.05; **p < 0.01;
***p < 0.001.
Supplemental Information includes Extended Experimental Procedures and
six figures and can be found with this article online at doi:10.1016/j.cell.
We thank Drs. Noboru Mizushima, Masaaki Komatsu, and Patrick Dennis
for generously providing reagents and Drs. Gerard Evan, Don Ganem, Abul
Abbas, and Feroz Papa for critically reading the manuscript. Confocal micros-
copy was performed in the Biological Imaging Development Center at UCSF.
Grant support to J.D. includes the NIH (RO1CA126792, KO8CA098419),
a Culpeper Medical Scholar Award (Partnership For Cures), an AACR-Genen-
a Stewart Family Trust Award. L.R. was a Genentech/Sandler Graduate
Student Fellow, and E.F. was an HHMI Summer Undergraduate Research
Received: November 10, 2009
Revised: March 22, 2010
Accepted: June 10, 2010
Published: August 19, 2010
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