Molecular Biology of the Cell
Vol. 19, 4651–4659, November 2008
An Atg4B Mutant Hampers the Lipidation of LC3
Paralogues and Causes Defects in Autophagosome Closure
Naonobu Fujita,*†Mitsuko Hayashi-Nishino,‡§Hiromi Fukumoto,*
Hiroko Omori,* Akitsugu Yamamoto,‡Takeshi Noda,* and Tamotsu Yoshimori*?
*Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka
565-0871, Japan;†Department of Genetics, The Graduate University for Advanced Studies, Mishima 455-8540,
Japan;‡Department of Cell Biology, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga
526-0829, Japan; and?CREST, Japan Science and Technology Agency, Kawaguchi-Saitama 332-0012, Japan
Submitted March 25, 2008; Revised July 9, 2008; Accepted August 26, 2008
Monitoring Editor: Suresh Subramani
In the process of autophagy, a ubiquitin-like molecule, LC3/Atg8, is conjugated to phosphatidylethanolamine (PE) and
associates with forming autophagosomes. In mammalian cells, the existence of multiple Atg8 homologues (referred to as
LC3 paralogues) has hampered genetic analysis of the lipidation of LC3 paralogues. Here, we show that overexpression
of an inactive mutant of Atg4B, a protease that processes pro-LC3 paralogues, inhibits autophagic degradation and
lipidation of LC3 paralogues. Inhibition was caused by sequestration of free LC3 paralogues in stable complexes with the
Atg4B mutant. In mutant overexpressing cells, Atg5- and ULK1-positive intermediate autophagic structures accumulated.
The length of these membrane structures was comparable to that in control cells; however, a significant number were not
closed. These results show that the lipidation of LC3 paralogues is involved in the completion of autophagosome
formation in mammalian cells. This study also provides a powerful tool for a wide variety of studies of autophagy in the
Macroautophagy, referred to here as autophagy, is an intra-
cellular process in which cytosol and organelles are seques-
tered within double-membrane–bound structures, called
autophagosomes, which deliver their contents to the lyso-
some/vacuole for degradation. In addition to its well un-
derstood physiological role in recycling intracellular mate-
rials as a starvation response, there is growing evidence for
the participation of autophagy in other cellular processes
including cellular differentiation, tissue remodeling, growth
control, adaptation to adverse environments, and cellular
immunity (Cuervo, 2004; Levine and Klionsky, 2004; Mi-
zushima, 2007; Yoshimori, 2004). The mode of autophago-
some formation stands apart from vesicle formation in other
membrane trafficking processes, such as endocytosis and the
secretory pathway (Noda et al., 2002). In autophagy, a flat-
tened membrane sac, the so-called isolation membrane in
mammals, is generated de novo, elongates, and encloses a
cargo to form the autophagosome.
The LC3 (mammalian Atg8 homologue) protein is a ubiq-
uitin-like molecule involved in autophagy. After synthesis,
the C-terminal 22 residues of precursor LC3 are immediately
removed by a protease, Atg4, to produce the LC3-I form.
The C-terminal carboxyl base of LC3-I/Atg8 is conjugated to
the head group amine of phosphatidylethanolamine (PE)
through an amide bond by a sequence of ubiquitination-like
reactions that involves an E1 (Atg7), an E2 (Atg3), and an E3
(protein complex including Atg5, Atg12, and Atg16L;
Ichimura et al., 2000; Hanada et al., 2007; Fujita et al., 2008).
The lipidated form of LC3 (LC3-II) and Atg8-PE are associ-
ated with the autophagosomal membrane (Kabeya et al.,
2000; Kirisako et al., 2000). The LC3 lipidation process is
reversible, because the Atg4 proteases can also catalyze the
reverse modification reaction, termed delipidation, of LC3/
Atg8 (Kabeya et al., 2004; Kirisako et al., 2000). In fact, most
of the LC3/Atg8 is liberated from the membrane at, or
before, the final stage of autophagy: fusion between auto-
phagosomes and lysosomes (Kimura et al., 2007; Kirisako
et al., 1999).
In yeast, Atg8 is proposed to function in expansion of the
autophagosomal membrane (Nakatogawa et al., 2007; Xie
et al., 2008). Atg8-PE causes the hemifusion of vesicles in
vitro, and this property may be related to the membrane
expansion step of autophagosome formation (Nakatogawa
et al., 2007). In mammals, the existence of multiple Atg8
homologues (referred to as LC3 paralogues), including LC3,
LC3A, LC3B, GABARAP, GATE16, and Atg8L, has been an
impediment to genetic analysis of the lipidation of LC3
paralogues (Tanida et al., 2006; Wu et al., 2006).
In mammalian cells, four Atg4 homologues have been re-
ported: Atg4A/autophagin-2, Atg4B/autophagin-1, Atg4C/
autophagin-3, and autophagin-4 (Marino et al., 2003).
Among these, Atg4B has a broad specificity for LC3 paral-
ogues (Hemelaar et al., 2003; Kabeya et al., 2004; Tanida et al.,
2004). Human Atg4B is a cysteine protease whose active
This article was published online ahead of print in MBC in Press
on September 3, 2008.
§Present address: Institute of Scientific and Industrial Research,
Osaka, University, Ibraki, Osaka 567-0047, Japan.
Address correspondence to: Tamotsu Yoshimori (tamyoshi@
Abbreviations used: HBSS, Hanks’ balanced salt solution; MEF,
mouse embryonic fibroblast; PE, phosphatidylethanolamine; TCA,
trichloroacetic acid; ULK, uncoordinated 51-like kinase.
© 2008 by The American Society for Cell Biology4651
catalytic triad consists of Cys74, His280, and Asp278 (Sug-
awara et al., 2005; Kumanomidou et al., 2006).
Here, we found that overexpression of a protease activity-
deficient mutant of Atg4B strongly inhibits autophagosome
formation. Through a mechanistic analysis, we show that
excess inactive Atg4B blocks lipidation of LC3 paralogues,
resulting in inhibition of autophagy. We believe this study
not only demonstrates the role of the LC3 paralogues in
autophagy, but also provides a powerful tool for inhibiting
autophagy than will be useful in a wide variety of future
MATERIALS AND METHODS
Reagents and Antibodies
Cell culture reagents were purchased from Invitrogen (Carlsbad, CA). The
following antibodies were used: rabbit polyclonal anti-rat LC3 (Kabeya et al.,
2000); anti-human Atg5 (Mizushima et al., 2001); anti-mouse Atg16L (Mi-
zushima et al., 2003); anti-p62 (BIOMOL Research Laboratories, Plymouth
Meeting, PA); anti-GABARAP (MBL, Nagoya, Japan); anti-GATE16 (MBL);
anti-monomeric red fluorescent protein, which reacts with mStrawberry
(MBL); mouse monoclonal anti-GFP (clone 7.1 and 13.1; Roche, Indianapolis,
IN); anti-c-myc (clone 9E10; Gentaur Molecular Products, Kobe, Japan); anti-
?-tubulin (clone B5-1-2; Sigma, St. Louis, MO). Wortmannin (Calbiochem,
La Jolla, CA) was prepared as a 100 ?M stock in Me2SO. All other reagents
were purchased from Sigma-Aldrich.
DNA Engineering, Recombinant Adenoviruses, and
The plasmid encoding monomeric red fluorescent protein (mStrawberry)
was a generous gift from Dr. Roger Y. Tsien (University of California, San
Diego, CA; Shaner et al., 2004). Expression vectors for green fluorescent
protein (GFP)-LC3, Myc-LC3-HA (hemagglutinin), Myc-LC3G120A-HA, and
mStrawberry have previously been described (Kabeya et al., 2000; Mizushima
et al., 2001; Kimura et al., 2007). To construct the mStrawberry-Atg4BC74A
plasmid, the Atg4B cDNA was cloned from genomic DNA isolated from
mouse embryonic fibroblast (MEF) cells and was inserted into pmStraw-
berry-C1 using engineered BamHI and KpnI sites; the point mutation (C74A
or C74S) was introduced using the QuikChange Site-Directed mutagenesis
system (Stratagene, La Jolla, CA). To construct the anti-human LC3 shRNA-
plasmid, two oligonucleotides, 5?-GATCCGCTGAGATCGATCAGTTCATT-
TCAAGAGAATGAACTGATCGATCTCAGTTTTTTGGAAA-3? and 5?-AGCTT
ATCTCAGCG-3? were synthesized and annealed, and the double-stranded
fragment was subcloned into the pRNA-H1/neo vector (GenScript, Piscat-
away, NJ) at the BamHI/HindIII sites. To produce recombinant adenoviruses,
the cDNAs corresponding to mStrawberry, mStrawberry-tagged-Atg4BWT,
-Atg4BC74A, or -Atg4BC74Swere subcloned into the pENTR 1A plasmid (In-
vitrogen). The cDNA inserts in pENTR-1A were transferred to the pAd/
CMV/V5-DEST vector (Invitrogen) by means of the Gateway system using
LR clonase (Invitrogen). Recombinant adenoviruses were prepared with the
ViraPower Adenovirus Expression System (Invitrogen) according to the man-
ufacturer’s instructions. pMRX-IRES-puro and pMRX-IRES-bsr were donated
by Dr. S. Yamaoka (Tokyo Medical and Dental University, Japan; Saitoh et al.,
2003). For production of recombinant retroviruses, the cDNAs corresponding
to enhanced green fluorescent protein (EGFP)-LC3, EGFP-Atg5, or mStraw-
berry-Atg4BC74Awere transferred to pMRX-IRES-puro or pMRX-IRES-bsr.
Recombinant retroviruses were prepared as described previously (Saitoh et
Cell Culture, Plasmid Transfections, and Adenovirus
Plat-E cells were generously provided by Dr. T. Kitamura (The University of
Tokyo; Morita et al., 2000). MCF7, 293A, NIH3T3, and Plat-E cells were grown
in DMEM supplemented with 10% fetal bovine serum, 2 mM l-glutamine,
and appropriate antibiotics in a 5% CO2incubator at 37°C. For nutrient-
starvation, cells were cultured in Hanks’ balanced salt solution (HBSS; In-
vitrogen) for 1 or 2 h. Transient transfections were carried out using Lipo-
fectAMINE 2000 reagent (Invitrogen) according to the manufacturer’s
protocol. Stable transformants were selected in growth medium with 500
?g/ml G418, 1 ?g/ml puromycin, or 10 ?g/ml blastcidin. Adenovirus infec-
tions were carried out as follows: on the day before infection, ?2 ? 105cells
were plated into six-well plates and incubated at 37°C overnight in a CO2
incubator. The medium was replaced with 1.5 ml of culture medium that
contained recombinant adenoviruses. After 16-h of incubation, the medium
containing adenoviruses was replaced with 1.5 ml culture medium. After an
additional 24-h incubation, the cells were used for experiments.
Cells were rinsed with ice-cold PBS, scraped, and collected by centrifugation
at 4°C. Cells were lysed in PBS containing 2% Triton X-100, 1 mM phenyl-
methylsulfonyl fluoride, and Protease inhibitor cocktail (Roche; Sou et al.,
2006). Cell lysates were centrifuged at 15,000 ? g for 15 min at 4°C, and
supernatants were collected. Samples were separated by SDS-PAGE and
transferred to polyvinylidene difluoride membrane. The membranes were
blocked with 1% skim milk in 0.1% Tween 20/TBS and incubated with
primary antibodies. Immunoreactive bands were detected using horseradish
peroxidase–conjugated secondary antibodies (The Jackson Laboratory, Bar
Harbor, ME) and luminol solution (1.25 mM luminol, 65 mM Tris-HCl, pH
8.0, 0.2 mM coumaric acid, and 0.01% H2O2).
Cells cultured on coverslips were fixed with 4% paraformaldehyde in PBS.
Samples were examined under a fluorescence laser scanning confocal micro-
scope, FV1000 (Olympus, Tokyo, Japan) or Olympus IX81 microscope
equipped with a mercury lamp and cooled charge-coupled device camera
(Cool Snap HQ; Roper Scientific, Tucson, AZ), under control of SlideBook
software (Intelligent Imaging Innovations, Denver, CO).
Gel filtration analysis was performed as previously described (Mizushima et al.,
2003). Briefly, 293A cells were homogenized in homogenization buffer (50 mM
Tris-HCl, pH 7.5, 150 mM NaCl, and Protease inhibitor cocktail; Roche) by
homogenate was centrifuged at 10,000 ? g for 10 min, and the supernatant was
further centrifuged at 100,000 ? g for 60 min. The resulting supernatants (cytosol
fraction) were separated by size exclusion chromatography on a Superose 6
column (GE Healthcare, Waukesha, WI).
Bulk Protein Degradation Assay
Cells were seeded in 24-well dishes and incubated overnight. On the follow-
ing day, the cells were exchanged into labeling medium containing14C-valine
(1.5 ?Ci/ml) and incubated overnight. Cells were exchanged into chase
medium (DMEM supplemented with 10% FBS and 10 mM unlabeled valine)
and further incubated for 4 h to remove the contribution of short-lived
proteins. After the chase period, cells were exchanged into growth medium
containing 10 mM valine or HBSS containing 10 mM valine to induce auto-
phagy. After a 2-h incubation, the media were collected and the trichloroacetic
acid (TCA)-soluble fraction was analyzed by scintillation counting. The cells
were lysed in ice-cold RIPA buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA, and Protease
inhibitor cocktail; (Roche) and the TCA-insoluble fraction was isolated and
analyzed by scintillation counting. To determine the rate of long-lived protein
degradation, the count in the TCA-soluble fraction in the medium was di-
vided by the equivalent TCA-insoluble count in the cell.
Conventional electron microscopy was performed as previously described
(Yoshimori et al., 2000) except that NIH 3T3 cells were postfixed with 1% OsO4
in 1% K4Fe(CN)6and 0.1 M phosphate buffer, pH 7.4, for 1 h. Immunoelectron
microscopy using the gold enhancement method was also performed, as
described previously (Luo et al., 2006).
All values shown in figures are represented with SD. Statistical signifi-
cance (p value) is determined by Student’s t test.
Atg4B Overexpression Inhibits Autophagic Flux
Independent of Its Catalytic Activity
It has been reported that overexpression of Atg4B, a protease
that processes LC3 paralogues, negatively affects the mem-
brane localization and PE conjugation of LC3 (Tanida et al.,
2004). In our experimental system, GFP-LC3 puncta were
not observed after overexpression of Atg4B (Figure 1A). One
possible mechanism underlying this negative effect is that
excess Atg4B efficiently deconjugates the LC3 from PE and
decreases membrane localized LC3. To test this hypothesis,
inactive Atg4B mutants (Atg4BC74Aor Atg4BC74S) that lack
protease activity due to mutation of the catalytic cysteine
residue (Cys74) were overexpressed. Unexpectedly, both
mutants showed similar effects as wild-type Atg4B on mem-
brane targeting and PE conjugation of LC3 (Figure 1, A and
N. Fujita et al.
Molecular Biology of the Cell4652
B), indicating that the negative effect is independent of the
Next, we examined the effect of Atg4BC74Aoverexpression
on autophagic flux in 293A cells. As it has been reported that
p62/SQSTM1 is a selective substrate of autophagy (Bjorkoy
et al., 2005; Mizushima and Yoshimori, 2007), we investi-
gated autophagic degradation by monitoring endogenous
p62 protein levels. At steady state, the p62 level in
Atg4BC74Aoverexpressing cells was much higher than in
mock cells, suggesting a defect in constitutive autophagic
clearance (Figure 1C). In mock cells, nutrient starvation,
which induces autophagy, resulted in a slight decrease of
p62 protein level; this was blocked by 100 nM wortmannin,
an inhibitor of phosphatidylinositol 3-kinase that blocks au-
tophagic activity (Blommaart et al., 1997). A similar reduc-
tion was not observed in Atg4BC74A-overexpressing cells
(Figure 1C). We then monitored long-lived protein degrada-
tion upon nutrient starvation in mock cells, cells stably
expressing mStrawberry-Atg4BC74A, and cells stably ex-
pressing shRNA against LC3 (Figure 1D). In mock and LC3-
knockdown cells, long-lived protein degradation was ele-
vated by nutrient starvation, and the elevation was blocked
by wortmannin (Figure 1E). This suggests that LC3 defi-
ciency has little effect on autophagy flux. It is possible that
this is due to the presence of LC3 paralogues, such as
GABARAP, that may have redundant function. On the other
hand, in cells stably expressing mStrawberry-Atg4BC74A, the
degradation of long-lived proteins was significantly inhib-
ited (Figure 1E), indicating a defect in autophagy.
Overexpression of Atg4BC74AInhibits PE Conjugation by
We next assessed which step in the PE conjugation pathway
was affected by the Atg4BC74Amutant. In the LC3/Atg8
conjugation reaction, the Atg12-Atg5 conjugate and Atg16L
form an 800-kDa super complex (referred as to the Atg16L
complex; Mizushima et al., 2003) that plays an E3-like role by
recruiting an E2 (Atg3)-LC3 intermediate to the site of con-
jugation (Hanada et al., 2007; Fujita et al., 2008). In 293A cells
stably expressing mStrawberry-Atg4BC74A, the formation of
the Atg12-Atg5 conjugate was not affected (Figure 2A). To
examine the size of the Atg16L complex, cytosolic fractions
of mock 293A or 293A cells stably expressing mStrawberry-
Atg4BC74Awere separated by size exclusion chromatogra-
phy and immunoblotted with anti-Atg5 or anti-Atg16L an-
tibody. As shown in Figure 2B, the molecular mass of the
Atg16L complex was not shifted by mStrawberry-Atg4BC74A
overexpression. From these results, we conclude that the
Atg16L complex formation is not affected by overexpression
LC3 paralogues are synthesized as a proform that must be
cleaved by Atg4 to expose a glycine residue that is engaged
in conjugation. It is possible that this initial processing step
is competitively inhibited by excess inactive Atg4B, resulting
in failure to expose the glycine residue. To determine
whether cleavage was inhibited, we used a Myc-LC3-HA
construct, in which the HA tag is fused at the C-terminus of
LC3. This construct enables us to easily distinguish between
pro-LC3 and LC3-I by monitoring size (Kabeya et al., 2000).
As shown in Figure 3A, the negative control Myc-
LC3G120A-HA protein was not processed. However, Myc-
LC3-I was detected both in 293A cells stably expressing
mStrawberry-Atg4BC74Aand in mock cells. This result
shows that pro-LC3 processing is not affected by overexpres-
sion of Atg4BC74A.
GFP-LC3 is usually detected throughout the cytoplasm
and within the nucleus when it does not form puncta. Nu-
clear localization of GFP-LC3 likely depends on the nature
of GFP, because indirect immunofluorescence analysis of
endogenous LC3 using an anti-LC3 antibody does not show
a nuclear localization pattern (Komatsu et al., 2005). Inter-
estingly, in Atg4B-overexpressing cells, GFP-LC3 dot forma-
tion was suppressed and GFP-LC3 signal was detected only
in the cytoplasm (Figure 1A). The absence of nuclear GFP-
LC3 suggests that it is trapped by excess Atg4B that localizes
only in the cytoplasm. In support of this hypothesis, we
ascertained that Atg4B and its inactive mutants form stable
degradation. (A) MCF7 cells stably expressing GFP-LC3 were in-
fected with adenovirus bearing mStrawberry (Mock), mStrawberry-
Atg4BWT(WT), or mStrawberry-Atg4BC74A(C74A) and incubated
for 40 h. The cells were then cultured in HBSS for 2 h, fixed, and
observed using fluorescence microscopy. Bar, 10 ?m. (B) PC12 cells
were infected with adenovirus bearing GFP (?), 3xFlag-tagged
wild-type Atg4B (WT), Atg4BC74A(CA), or Atg4BC74S(CS). Cells
were cultured either in growth or starvation medium, and lysates
were examined by Western blotting using each antibody. Top panel,
anti-Flag; middle panel, anti-LC3; bottom panel, anti-?-tubulin. (C)
293A cells stably expressing empty vector (Mock) or mStrawberry-
Atg4BC74A(Atg4BC74A) were grown in growth medium (F), HBSS (S),
or HBSS with 100 nM wortmannin (W) for 2 h. Cell lysates were
examined by Western blotting using each antibody. From top panel,
anti-RFP, anti-p62, anti-LC3, and anti-?-tubulin. (D) 293A cells sta-
bly expressing empty vector (Mock) or shRNA against LC3 (LC3
KD) were cultured in HBSS for 2 h and collected. Cell lysates were
examined by Western blotting using each antibody. From top panel,
anti-LC3, anti-GABARAP, and anti-?-tubulin. (E) Mock, mStraw-
berry-Atg4BC74A-expressing (Atg4BC74A), or LC3-knockdown (LC3-
KD) 293A cells were grown in growth medium (F), HBSS (S), or
HBSS with 100 nM wortmannin (W) for 2 h. Long-lived protein
degradation was scored as described in Materials and Methods.
Cells expressing Atg4BC74Aexhibit a block in autophagic
Role of the LC3 Paralogues in Autophagy
Vol. 19, November 20084653
complexes with LC3 paralogues. 3xFlag-Atg4B mutants ef-
ficiently pulled down LC3-I and GATE16-I (Figure 3B). Ac-
cordingly, the formation of the LC3-Atg7 intermediate
should be inhibited by overexpression of Atg4BC74A. To
confirm this, we used a mutant E1 (Atg7C567S) enzyme
whose active-site cysteine residue is replaced by serine. This
mutant stabilizes the high-molecular-mass LC3-Atg7C567S
intermediate, because a stable ester bond is formed between
the enzyme and substrate instead of a labile thioester bond
(Tanida et al., 2001). Although the LC3-Atg7 intermediate
was detected in mock 293A cells, it was not detected in 293A
cells stably expressing mStrawberry-Atg4BC74A(Figure 3C).
We also observed that the LC3-Atg7 intermediate was not
detected in wild-type Atg4B, as well as Atg4BC74Amutant,
overexpressing cells (Supplemental Figure S1). These results
indicate that the formation of Atg7-LC3 intermediate is the
step that is inhibited by overexpression of Atg4B.
Atg4BC74Amutant inhibits LC3 lipidation in a dose-de-
pendent manner (Figure 4A). Accumulating data set suggest
that the cause of the inhibitory effect is sequestration of LC3
paralogues by excess Atg4B mutant. If so, the inhibitory
effect should be dependent on the molecular ratio of Atg4B
to LC3 paralogues. To test this model, we expressed GFP-
tagged LC3 paralogues in NIH3T3 cells stably expressing
mStrawberry-Atg4BC74A. As expected, the inhibitory effect
of Atg4B mutant on LC3 lipidation was suppressed by ex-
ogenous LC3 or other LC3 paralogues in a dose-dependent
manner (Figure 4B and Supplemental Figure S2). Collec-
tively, we conclude that sequestration of LC3 paralogues by
excess Atg4BC74Aprevents access of LC3 paralogues to Atg7
and leads to a defect in autophagic degradation.
The LC3 Paralogues Are Involved in Closing the Isolation
As the Atg4B mutant sequesters LC3 paralogues, cells over-
expressing this protein provide a useful system for analysis
of the role of the LC3 paralogues in autophagosome forma-
tion. To this end, we utilized NIH3T3 cells, which are suited
to morphological analysis of the autophagic membrane, be-
cause of well-spread cytoplasm. The inhibitory effect of
Atg4B mutant overexpression on PE conjugation of LC3
paralogues and membrane targeting of LC3 were also ob-
served in this cell line (Figure 4, Supplemental Figure S3 and
S4A). Moreover, the number of p62 bodies was significantly
increased in mStrawberry-Atg4BC74A–expressing cells (Sup-
plemental Figure S4C). In wild-type cells, isolation mem-
branes were detected as punctate GFP-Atg5 signals. When
the isolation membrane elongates and fuses to form the
autophagosome, Atg5 detaches from the membrane. There-
fore, GFP-Atg5 can be detected only on nascent autophago-
somes, but not on completely formed ones (Mizushima et al.,
2001). In a cell line in which GFP-Atg5 and mStrawberry-
Atg4BC74Awere stably coexpressed, the number of punctate
GFP-Atg5 signals was increased in both nutrient-rich and
starvation conditions (Figure 5, A and B). We obtained sim-
ilar results by immunostaining with anti-Atg16L antibody
(Supplemental Figure S4B). The membrane localization of
the Atg16L complex is dependent on the phosphatidylino-
sitol 3-kinase (Fujita et al., 2008; Mizushima et al., 2001). The
punctate GFP-Atg5 signals in mStrawberry-Atg4BC74A–ex-
pressing cells were also dispersed by wortmannin treatment,
as in control cells (Figure 5, A and B). The average lifetime of
the GFP-Atg5 structure, as measured by time-lapse video-
microscopy, was ?5 min in control cells, but was prolonged
to ?20 min in Atg4BC74A-overexpressing cells (Supplemen-
tal Movies S1 and S2, and Figure 5C).
To further characterize the Atg5-positive membrane struc-
ture, we examined the localization of Atg9L1, which is a
mammalian homolog of yeast Atg9, a membrane protein
engaged in autophagosome formation (Yamada et al., 2005).
In mammalian cells, Atg9L is reported to cycle between the
trans-Golgi network and the Golgi (Young et al., 2006). As
shown in Supplemental Figure S5A, Atg9L1 did not show
high colocalization with GFP-Atg5 in the presence or ab-
Atg16L complex. (A) 293A cells stably expressing empty
vector (Mock) or mStrawberry-Atg4BC74A(Atg4BC74A)
were cultured in growth medium or HBSS for 2 h, and
Western blotting was performed using each antibody.
From top panel, anti-RFP, anti-Atg5, anti-LC3, anti-?-
tubulin. Anti-RFP antibody reacts with mStrawberry.
(B) Cytosolic fractions of 293A cells stably expressing
empty vector or mStrawberry-Atg4BC74Awere sepa-
rated by size exclusion chromatography. Fractions were
subjected to Western blotting using the indicated anti-
bodies. The positions of the molecular-mass standards
are shown. Vo, void fraction.
Effect of Atg4BC74Aoverexpression on the
N. Fujita et al.
Molecular Biology of the Cell4654
sence of Atg4BC74A, suggesting that the defect seen in
Atg4BC74A-overexpressing cells is not related to Atg9L lo-
calization. We then analyzed the localization of the uncoor-
dinated 51-like kinase1 (ULK1), the putative mammalian
orthologue of yeast Atg1, a protein kinase also engaged in
autophagosome formation (Chan et al., 2007; Hara et al.,
2008). In control cells, myc-TEV-Flag–tagged ULK1 colocal-
ized with GFP-Atg5 (Supplemental Figure S5B). In Atg5
knockout cells expressing the conjugation-deficient mutant
of Atg5K130R, small crescent-like Atg5-positive membrane
compartments accumulated (Mizushima et al., 2001). In Atg5
knockout MEF cells expressing GFP-Atg5K130R, ULK1 is also
recruited to the structure (Supplemental Figure S6). It was
reported that ULK1 and GABARAP, an LC3 paralogue,
physically interact (Okazaki et al., 2000); therefore, it is pos-
sible that Atg4BC74Aoverexpression leads to a defect in
ULK1 localization. However, colocalization of ULK1 and
Atg5 was observed in Atg4BC74A-overexpressing cells (Sup-
plemental Figure S5B), showing that the GFP-Atg5–positive
structures that lack LC3-PE contain ULK1.
LC3 prevents access to Atg7. (A) 293A cells stably expressing empty
vector (Mock) or mStrawberry-Atg4BC74A(Atg4BC74A) were trans-
fected with Myc-LC3-HA (WT) or Myc-LC3G120A-HA (GA). Thirty-
six hours after transfection, cells were cultured in growth medium
or HBSS for 2 h, and Western blotting were performed. From top
panel, anti-RFP, anti-Myc, and anti-?-tubulin. (B) PC12 cells were
infected with adenovirus bearing 3xFlag-tagged wild-type Atg4B
(WT), Atg4BC74A(CA), or Atg4BC74S(CS). After 40-h incubation, cell
lysates were subjected to immunoprecipitation with anti-Flag M2-
conjugated agarose beads. Coimmunoprecipitated molecules were
examined by Western blotting using each antibody. From top panel,
anti-Flag, anti-LC3, and anti-GATE16. Total cell lysate (Input) and
immunoprecipitated proteins (IP) are shown. (C) 293A cells sta-
bly expressing empty vector (Mock) or mStrawberry-Atg4BC74A
(Atg4BC74A) were transfected with GFP-LC3 and Myc-Atg7 as
indicated. Thirty-six hours after transfection, cell lysates were
examined by Western blotting using each antibody. From top
panel, anti-RFP, anti-myc, anti-GFP, and anti-?-tubulin.
Stable complex formation between excess Atg4BC74Aand
is suppressed by exogenous LC3 in a dose-dependent manner. (A)
NIH3T3 cells were infected with different amounts of retroviruses
bearing mStrawberry-Atg4BC74A, and stable transformants were se-
lected. The stable cells were cultured in HBSS (Starved) for 1 h, and
cell lysates were examined by Western blotting using each antibody.
From top panel, anti-Atg4B, anti-RFP, anti-LC3, and anti-?-tubulin.
(B) NIH3T3 cells stably expressing mStrawberry-Atg4BC74Awere
infected with different amounts of retroviruses bearing GFP-LC3
and then double stable transformants were selected. Parent NIH3T3
cells and the stable transformants were cultured in HBSS (Starved)
for 1 h, and cell lysates were examined by Western blotting using
each antibody. From top panel, anti-RFP, anti-GFP, anti-LC3, and
The inhibitory effect of Atg4B mutant on LC3 lipidation
Role of the LC3 Paralogues in Autophagy
Vol. 19, November 20084655
Next, we examined the Atg5-positive structures at the
ultrastructural level. In mock cells, isolation membranes
(Figure 6B), autophagosomes (Figure 6, C–E), and many
autolysosome-like structures, which are characterized by
highly electron-dense signals, were observed by electron
microscopy (Figure 6A). In addition to autolysosomes, there
are other electron-dense structures within cells, and there-
fore certain structures, such as amphisomes or autolyso-
somes, cannot be absolutely distinguished without specific
markers (Eskelinen, 2008). Because membrane localization
of LC3, the sole specific marker for autophagosomes and
autolysosomes, was severely suppressed by Atg4BC74A
overexpression (Supplemental Figure S4A), we cannot inter-
pret the electron-dense structures further. Therefore, we
counted the isolation membranes and double-membraned
autophagic membranes. In contrast to mock cells, many
isolation membranes (Figure 6, G and H) and autophago-
some-like structures (Figure 6, I and J) were observed in cells
stably overexpressing Atg4BC74A(Figure 6, F and K). Be-
cause we observed cross-sections of the cells, it was some-
times difficult to determine whether the autophagosome-like
structures were really closed. However, the ratio of com-
pletely open structures to total autophagic structures was
significantly higher in Atg4BC74A-expressing cells than in
mock cells (Figure 6L). We also observed that the elevation
in the number of autophagic structures and the ratio of open
structures to total autophagic structures by excess Atg4B
was diminished by overexpression of GFP-LC3 (Supplemen-
tal Figure S7). Although there was no significant difference
in the length of open-autophagic membranes between
mock and mStrawberry-Atg4BC74A–overexpressing cell,
the length of the closed-autophagic membranes in Atg4BC74A-
overexpressing cells was slightly shorter than the length in
autophagic membrane in Atg4BC74A-overexpressing cells was
significantly higher than in mock cells (Figure 6N), suggest-
ing that the defect exists at a late stage in the autophagosome
formation. Finally, to correlate these structures with fluores-
cence microscopy, we performed immunoelectron micros-
copy. NIH3T3 cells expressing GFP-Atg5 and mStrawberry-
Atg4BC74Awere grown in HBSS for 1 h, and the localization
of GFP-Atg5 was examined by gold-enhanced immunogold
electron microscopy using an anti-GFP antibody. As shown
in Figure 6O, the isolation membranes, which elongate rel-
atively well, were positive for GFP-Atg5 in Atg4BC74A-over-
expressing cells. These lines of evidence indicate that the
LC3 paralogues are involved in the completion of autopha-
gosome formation in mammalian cells.
We found that overexpression of Atg4B has an inhibitory
effect on autophagy independent of its delipidation activity
in mammalian cells. The inactive Atg4BC74Amutant specif-
ically blocks the lipidation of LC3 paralogues by sequester-
ing LC3 paralogues in stable complexes, resulting in block-
ade of the Atg7-LC3 reaction. It is interesting that the Atg4B
enzyme has high affinity for its product, processed LC3
paralogues. This is in sharp contrast to overexpression of
inactive ubiquitin-deconjugating enzymes, which act in a
dominant-negative manner, elevating levels of their sub-
strate, ubiquitin-conjugated proteins (Hang and Dasso, 2002;
Li et al., 2002). In the course of the enzymatic reaction, Atg4
and LC3 paralogues should associate, but once cleavage has
occurred, the proteins should dissociate for the next step, the
Atg7-LC3 reaction. It is possible that an unknown factor is
Atg5–positive membrane structures. (A and B) NIH3T3
cells stably expressing GFP-Atg5 or both GFP-Atg5 and
mStrawberry-Atg4BC74Awere grown in growth me-
dium (F), HBSS (S), or HBSS with 100 nM wortmannin
(W) for 1 h and then fixed. Three-dimensional image
stacks were obtained from sequential optical sections
acquired 0.3 ?m apart by confocal laser scanning mi-
croscopy (FV1000, Olympus) (A). Bar, 10 ?m. The num-
ber of GFP-Atg5 puncta was counted in more than 100
cells. The value indicated is the mean ? SD (B). (C)
NIH3T3 cells stably expressing GFP-Atg5 or both GFP-
Atg5 and mStrawberry-Atg4BC74Awere grown in HBSS
for 1 h and directly observed by time-lapse video mi-
croscopy. The duration of each GFP-Atg5 puncta was
measured for more than 50 cases. The value indicated is
the mean ? SD.
Effect of Atg4BC74Aoverexpression on GFP-
N. Fujita et al.
Molecular Biology of the Cell4656
necessary for dissociating LC3 from Atg4, and Atg4 overex-
pression might result in limitation of this factor.
In cells overexpressing Atg4BC74A, a large number of the
autophagic structures were not closed, although the length
of these membranes was comparable to the length of auto-
phagosomal membranes in control cells (Figure 6M). This
observation fits with the results that Atg5-positive mem-
brane structures accumulated and had prolonged lifetimes
(Figure 5), based on the previous report that the Atg16L
complex detach once the autophagosome formation is com-
branes in Atg4BC74A-overexpressing cells. NIH3T3
cells stably expressing empty vector (Mock) (A–E) or
mStrawberry-Atg4BC74A(F–J) were cultured in HBSS
for 1 h, fixed, and subjected to conventional electron
microscopic analysis. (B–E) Typical autophagic struc-
tures in mock cells; isolation membrane (B), autopha-
gosomes (C–E). (G–J) Typical autophagic structures
in mStrawberry-Atg4BC74A-expressing cells; isolation
membranes (G and H), closed double-membrane struc-
tures (I and J). Examples of electron-dense structures
(black arrows), closed autophagic membranes (white
arrows), and open autophagic membranes (white ar-
rowheads) are indicated. Bar, 500 nm. (K) The number
of autophagic structures in mock and mStrawberry-
Atg4BC74A-expressing cells. u, open autophagic struc-
tures; f, closed autophagic structures. Data are the
means ? SD of triplicates from representative experi-
ments. (L) The ratio of open structures to total autoph-
agic structures. Data are the means ? SD of triplicates
from representative experiments. *p ? 0.05. (M) The
length of autophagic membranes in mock and mStraw-
berry-Atg4BC74A-expressing cells. u, open autophagic
structures; f, closed autophagic structures. For the
length of autophagic membranes determination, ImageJ
version 1.40 was used (http://rsb.info.nih.gov/ij/). The
value indicated is the mean ? SD. At least 20 samples
were examined for each structure. *p ? 0.05; NS, not
significant. (N) The ratio of the length of open to closed
autophagic structures in mock and mStrawberry-
Atg4BC74A-expressing cells. *p ? 0.05. (O) NIH3T3 cells
expressing both GFP-Atg5 and mStrawberry-Atg4BC74A
were grown in HBSS for 1 h and fixed. The localization
of GFP-Atg5 was examined by gold-enhanced immu-
nogold electron microscopy using an anti-GFP antibody.
Bar, 500 nm.
Ultrastructual analysis of autophagic mem-
Role of the LC3 Paralogues in Autophagy
Vol. 19, November 20084657
pleted (Mizushima et al., 2001). In Atg5 knockout cells ex-
pressing the GFP-Atg5K130Rmutant, in which Atg12-Atg5
conjugation does not occur, GFP-Atg5K130Rsignals also re-
main longer in membranous structures. These cells have
incomplete Atg16L complexes, which lack Atg12, whereas in
Atg4BC74Aoverexpressing cells, the Atg16L complex is in-
tact. Therefore, it seems that the trigger that liberates the
Atg16L complex from the membrane upon completion of
autophagosome formation is not within the Atg16L complex.
It is interesting that autophagosome formation proceeded
to a relatively late stage in Atg4BC74A-overexpressing cells;
formation of autophagosome-like structures takes place. In
addition, ULK1 is recruited to the structure in a manner
similar to that observed in the isolation membranes of con-
trol cells (Supplemental Figure S5). The apparent defect in
autophagosome completion is closure of the end of each
elongating membrane. This does not necessarily exclude the
proposal that Atg8 functions in expansion of autophagoso-
mal membranes in yeast (Xie et al., 2008), because we ob-
serve only terminal phenotype and cannot exclude the pos-
sibility that elongation speed is slower. Atg8-PE can cause
hemifusion of vesicles in vitro (Nakatogawa et al., 2007). One
possibility is that LC3 paralogues function to complete au-
tophagosome formation by fusing membranes in mamma-
lian cells. Interestingly, in atg8? yeast, autophagosome-like
structures were also detected by electron microscopy, how-
ever, at low frequency (Kirisako et al., 1999). There remains
a possibility that expansion of autophagosomal membrane is
not completely hampered by the deletion of Atg8.
We do not believe the phenotypes we observed are due to
incomplete inhibition of LC3 paralogues function, because
autophagosome-like structures were also observed in Atg3
(a specific E2 enzyme for Atg8 homologues) knockout MEF
cells, where PE conjugation of LC3 paralogues is defective
(Dr. Keiji Tanaka and Dr. Masaaki Komatsu, personal com-
munication). By utilizing an inactive mutant of Atg4B, we
could exclude secondary effects that might be brought about
by overexpression of wild-type Atg4B, such as hyperdelipi-
dation. Therefore, the phenotypes we observed likely re-
flect the physical sequestration and deficiency of the LC3
One strategy that may provide important information is
artificial inhibition of autophagy. Indeed, treatment with
drugs such as wortmannin or 3-methyladenine is widely
used in studies of autophagy, but these drugs have side
effects. RNA interference–mediated gene knockdown is a
potential approach; however, nearly complete suppression
of the ATG genes is needed to fully inhibit autophagy, and
this is often difficult to achieve (Hosokawa et al., 2006;
Yoshimura et al., 2006). The use of genetic knockouts of ATG
genes is an alternative option for complete inhibition, but
available cell types are restricted. In the case of Atg4BC74A
overexpression, it seems possible to fully inhibit autophagy
in any type of cell. Several studies have suggested that the
Atg12-Atg5 conjugate has other roles in addition to autoph-
agy (Pyo et al., 2005; Yousefi et al., 2006; Takeshita et al.,
2007). However, currently reported Atg5- or Atg7-deficient
cells do not distinguish between autophagic and nonauto-
phagic function of the Atg12-Atg5 conjugate, as it is lacking
in both cell types. As overexpression of the Atg4B mutant
inhibits formation of autophagosomes, but not generation
of the Atg12-Atg5 conjugate, such problems can be
avoided. We believe that the inactive Atg4B mutant will
provide a useful tool for a broad range of studies analyz-
The authors thank Dr. Kouichi Matsunaga (Yoshimori lab) for the gift of
MCF7 cells stably expressing GFP-LC3; Dr. Roger Y. Tsien (University of
California, San Diego, CA) for the gift of mStrawberry cDNA; Dr. Shoji
Yamaoka for the gifts of pMRX-IRES-puro and pMRX-IRES-bsr; Dr. Toshio
Kitamura (The University of Tokyo, Japan) for the gift of Plat-E cells; Dr.
Noboru Mizushima (Tokyo Medical and Dental University, Japan) for the gift
of anti-Atg16L antibody; Mr. Takuya Hayashi (Yoshimori lab) for the gift of
myc-TEV-Flag-tagged Atg9L1 construct; and Dr. Kyouhei Umebayashi (Yo-
shimori lab) for helpful discussion. The work described in this report was
supported in part by Special Coordination Funds for Promoting Science and
Technology of the Ministry of Education, Culture, Sports, Science and Tech-
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Role of the LC3 Paralogues in Autophagy
Vol. 19, November 20084659