Báo cáo khoa học: Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3 docx

Atg8L/Apg8L is the fourth mammalian modifier of
mammalian Atg8 conjugation mediated by human Atg4B,
Atg7 and Atg3
Isei Tanida, Yu-shin Sou, Naoko Minematsu-Ikeguchi, Takashi Ueno and Eiki Kominami
Molecular Cell Biology, Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan
Ubiquitylation and ubiquitylation-like reactions are
post-translational modifications that play indispensable
roles in many cellular events. Atg8 ⁄ Apg8 ⁄ Aut7 is a
ubiquitin-like (Ubl) protein essential for autophagy in
the yeast Saccharomyces cerevisiae [1]. Genetic analyses
of yeast ATG gene mutants have suggested that the
C-terminus of Atg8 is cleaved by Atg4 or a protease
activated by Atg4 ⁄ Apg4 to expose the C-terminal Gly
of Atg8 [2–4]. Subsequently, Atg8 is activated by
Atg7 ⁄ Apg7 ⁄ Gsa7 ⁄ Cvt2, an E1-like enzyme [5–8],
transferred to Atg3 ⁄ Apg3 ⁄ Aut1, an E2-like enzyme [3],
and finally conjugated to phosphatidylethanolamine
[3]. This conjugation reaction is essential for auto-
phagy under conditions of starvation and in the cyto-
plasm for the vacuole-targeting (Cvt) pathway under
nutrient-rich conditions.
To date, three Atg8 homologs have been character-
ized in mammals: LC3 (microtubule-associated protein 1
light chain 3, MAP1-LC3) [9,10], 4-aminobutyrate
A
-
receptor associated protein (GABARAP) [11–13], and
Golgi-associated ATPase enhancer of 16 kDa (GATE-
16) [12–14]. Following cleavage by human Atg4B ⁄
autophagin 1, the C-terminal Gly of each of these Atg8
homologs is exposed, as is also observed for yeast Atg8

[13,15]. Human Atg4A ⁄ autophagin 2 also cleaves the
C-terminus of GATE-16 [16]. Following C-terminal
cleavage, each Atg8 homolog is activated by human
Atg7, an E1-like enzyme, to form a transient E1-sub-
strate intermediate [12,17,18]. Each Atg8 homolog is
subsequently transferred to human Atg3, an E2-like
enzyme, to form a transient E2-substrate intermediate,
and modified to the membrane-bound forms, LC3-II,
GABARAP-PL (GABARAP-II), and GATE-16-II
[12,19]. Recently, it has been shown that LC3-II and
GABARAP-PL are protein–phospholipid conjugates
[15], with phosphatidylethanolamine thought to be the
Keywords
autophagy; GABARAP; GATE-16; LC3;
ubiquitylation-like modification
Correspondence
E. Kominami, Department of Biochemistry,
Juntendo University School of Medicine,
2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421,
Japan
Fax: +81 3 5802 5889
Tel: +81 3 5802 1031
E-mail:
(Received 23 December 2005, revised
4 April 2006, accepted 5 April 2006)
doi:10.1111/j.1742-4658.2006.05260.x
Murine Atg8L ⁄ Apg8L has significant homology with the other known
mammalian Atg8 homologs, LC3, GABARAP and GATE-16. However, it
is unclear whether murine Atg8L modification is mediated by human
Atg4B, Atg7 and Atg3. Expression of Atg8L in HEK293 cells led to clea-

vage of its C-terminus. In vitro, the C-terminus of Atg8L was cleaved by
human Atg4B, but not human Atg4A or Atg4C. Atg8L-I formed an
E1-substrate intermediate with Atg7
C572S
, and an E2-substrate intermediate
with Atg3
C264S
. A modified form of Atg8L was detected in the pelletable
fraction in the presence of lysosomal protease inhibitors under nutrient-rich
conditions. Cyan fluorescent protein (CFP)–Atg8L colocalized with yellow
fluorescent protein (YFP)–LC3 in HeLa cells in the presence of the inhibi-
tors. However, little accumulation of the modified form of Atg8L was
observed under conditions of starvation. These results indicate that Atg8L
is the fourth modifier of mammalian Atg8 conjugation.
Abbreviations
CFP, cyan fluorescent protein; GABARAP, 4-aminobutyrate
A
receptor-associated protein; GABARAP-PL, GABARAP–phospholipid conjugate;
GFP, green fluorescent protein; LC3, human microtubule-associated protein 1 light chain 3; LC3-I, soluble unmodified form of LC3; LC3-II,
LC3–phospholipid conjugate; PL, phospholipid; PVDF, poly(vinylidene difluoride); TRX, thioredoxin; YFP, yellow fluorescent protein.
FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS 2553
phospholipid bound to LC3 [13,20]. LC3-II and
GABARAP-PL are both deconjugated by human
Atg4B [15]. It is unclear whether the target of GATE-
16 is a phospholipid.
Recently, a fourth mammalian Atg8 homolog has
been reported, mouse Atg8L ⁄ Apg8L [21]. The amino
acid sequence of mouse Atg8L ⁄ Apg8L shows 100%
and 54% identity to those of human Atg8L and yeast
Atg8, respectively, and 33%, 60% and 86% identity to

the amino acid sequences of LC3, GATE-16 and
GABARAP, respectively. In addition, Atg8L shares a
conserved Gly at its C-terminus. Following chemical
modification of Atg8L with a C-terminal vinyl sulfone,
mouse Atg8L–vinyl sulfone was shown to react with
Atg4B, suggesting that mouse Atg8L is a substrate of
Atg4B [21]. However, no direct evidence that mouse
Atg8L is cleaved by Atg4B has yet been reported. Fur-
thermore, both the homology between Atg8L and the
three other mammalian Atg8 homologs and the con-
served C-terminal Gly in all four proteins suggest that
Atg8L may also be a substrate of mammalian Atg7
and Atg3. Therefore, we examined whether Atg8L is a
substrate of these three enzymes involved in mamma-
lian Atg8 conjugation.
Results
The C-terminus of Atg8L is cleaved in HEK293
cells, and the Gly116 of Atg8L is essential for
cleavage
The C-termini of yeast Atg8, mammalian LC3, GABA-
RAP and GATE-16 are post-translationally cleaved to
expose a Gly residue, which is essential for ubiquitin-like
modification [3,10,12]. This consensus Gly116 is con-
served in Atg8 and its mammalian homologs [21], and is
present in Atg8L. To determine whether the C-terminus
of Atg8L is post-translationally cleaved, we constructed
an Atg8L expression vector tagged with a Myc epitope
at its N-terminus and a 3xFLAG epitope at its C-termi-
nus (Fig. 1A, Myc–Atg8Lwt)3xFLAG). Following
transfection of HEK293 cells with this construct, the

lysates were analyzed by SDS ⁄ PAGE. A protein of
18 kDa corresponding to Myc–Atg8L, a C-terminal
cleaved form of Myc–Atg8Lwt)3xFLAG, was recog-
nized by immunoblotting with anti-Myc, but not with
anti-FLAG (Fig. 1B).
We next investigated whether the Gly116 residue of
Atg8L is essential for cleavage of its C-terminus by
changing Gly116 of Myc–Atg8Lwt)3xFLAG to Ala
by site-directed mutagenesis (Fig. 1A, Myc–Atg8L-
GA)3xFLAG), and expressing the mutant protein in
HEK293 cells (Fig. 1B, GA). The mobility of mutant
Myc–Atg8LGA)3xFLAG on SDS ⁄ PAGE was slower
than that of wild-type Myc–Atg8Lwt)3xFLAG. More-
over, the mutant protein was recognized by immuno-
blotting with both anti-Myc and anti-FLAG (Fig. 1B).
Similar results were obtained when the Gly116 residue
was deleted by site-directed mutagenesis (Fig. 1A,
Myc–Atg8LDG)3xFLAG; Fig. 1B, DG). These results
suggest that the C-terminus of Atg8L is post-transla-
tionally cleaved in HEK293 cells, and that Gly116 of
Atg8L is essential for the cleavage of its C-terminus.
The cleaved form of Atg8L was designated Atg8L-I.
Atg4B cleaves the C-terminus of Atg8L in vitro
The three previously identified mammalian Atg8 homo-
logs, LC3, GABARAP, and GATE-16, were shown to
be cleaved by human Atg4B (hAtg4B) in vitro [13,15].
Although we showed that the C-terminus of Atg8L was
cleaved soon after its translation in HEK293 cells, the
enzyme responsible for this activity could not be identi-
fied. Therefore, we examined whether Atg4B has pro-

teolytic activity on the C-terminus of Atg8L. FLAG–
hAtg4B was expressed in HEK293 cells, the cells were
lysed, and the lysate was fractionated by ultracentrifuga-
tion, with the resulting supernatant used as the enzyme
mixture. FLAG–hAtg4B in the supernatant was recog-
nized by immunoblotting with anti-FLAG (Fig. 1C,
FLAG–hAtg4B). The substrate, wild-type thioredoxin
(TRX)–Atg8L)3xFLAG, consisting of Atg8L with
TRX at its N-terminus and the 3xFLAG epitope at its
C-terminus, was expressed in Escherichia coli, and the
supernatant of this cell lysate was used as the substrate
solution. When we incubated the two supernatants con-
taining FLAG–hAtg4B with TRX–Atg8L)3xFLAG,
we found that the C-terminus of Atg8L was cleaved, as
shown by immunoblotting with anti-FLAG (Fig. 1C).
Using anti-TRX, we confirmed that the N-terminal
TRX tag within each substrate remains unchanged,
indicating that the C-terminal FLAG tag was cleaved
by hAtg4B (Fig. 1C). When an active site mutant of
hAtg4B, hAtg4B
C74A
[15], was used instead of wild-type
hAtg4B, little cleavage occurred (Fig. 1D, lane 2 versus
lane 1). When a mutant in which the Gly116 residue of
Atg8L had been deleted, TRX–Atg8LDG)3xFLAG,
was employed instead of the wild type, little C-terminal
cleavage occurred (Fig. 1D, lane 5 versus lane 1).
In addition to hAtg4B, hAtg4A ⁄ hApg4A ⁄ autophag-
in-2, hAtg4C ⁄ hAutl1 ⁄ autophagin-3 and hAtg4D ⁄
autophagin-4 have also been reported [16,22]. Of these

three Atg4 homologs, hAtg4A has been shown to
cleave the C-terminus of GATE-16 [16], and autophag-
in-3 ⁄ hAtg4C ⁄ hAutl1, but not hAtg4D, has been shown
to exert N-ethylmaleimide-sensitive proteolytic activity
Ubiquitylation-like modification of murine Atg8L I. Tanida et al.
2554 FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS
on the synthetic substrate Mca-Thr-Phe-Gly-Met-Dpa-
NH
2
[22]. Therefore, hAtg4A and hAtg4C may also
cleave the C-terminus of Atg8L. When hAtg4A or
hAtg4C was used instead of hAtg4B for in vitro diges-
tion, little cleavage of the C-terminal 3xFLAG tag of
TRX–Atg8L)3xFLAG occurred (Fig. 1, lanes 3 and 4
versus lane 1). These results indicate that Atg4B
cleaves the C-terminus of Atg8L in vitro.
Atg8L-I forms an E1-substrate intermediate with
human Atg7
C572S
In the ubiquitlyation-like modification steps, an active
site Cys within an E1 enzyme temporally conjugates to
a substrate to form an E1-substrate intermediate via a
thiol–ester bond. Owing to the rapid turnover of an
E1 reaction, it is difficult to recognize such an interme-
diate in sufficient quantity. When an active site Cys
residue of human Atg7 is changed to Ser, a stable
O-ester bond instead of a thiol–ester bond will be
formed between the enzyme and substrate(s). Previ-
ously, we showed that the mammalian Atg8 homologs
LC3, GATE-16 and GABARAP form E1-substrate

intermediates with an active site mutant human
Atg7
C572S
, in which the active site Cys572 of human
Atg7 was changed to Ser12. If Atg8L-I is a substrate
of human Atg7, Atg8L-I will form an E1-substrate
intermediate with human Atg7
C572S
. Therefore, we
investigated whether Atg8L-I also forms an intermedi-
ate with human Atg7
C572S
. When Myc–Atg8Lwt)3x-
FLAG and Atg7
C572S
were expressed together, an
Atg7
C572S
–Myc–Atg8L (E1-substrate) intermediate was
observed (Fig. 2). This intermediate was also detected
by immunoblotting with anti-Myc, but not with anti-
Fig. 1. The C-terminus of Atg8L is cleaved in vivo and in vitro.(A)
Schematic representation of mutant proteins of Myc–Atg8L)3x-
FLAG. The arrowhead indicates a Gly residue predicted to be
essential for ubiquitylation-like reactions. Myc–Atg8Lwt)3xFLAG
represents wild-type Atg8L protein tagged with the Myc epitope at
its N-terminus and with the 3xFLAG epitope at the C-terminus.
Myc–Atg8LGA)3xFLAG represents a mutant protein in which the
Gly116 of Atg8L was changed to Ala, and Myc–Atg8LDG)3xFLAG
represents a mutant protein in which the Gly116 of Atg8L was

deleted. (B) Cleavage of the C-terminus of Atg8L. Wild-type or
mutant Myc–Atg8L)3xFLAG proteins were transiently expressed in
HEK293 cells. The cells were lysed, total proteins were separated
by SDS ⁄ PAGE, and wild-type and mutant Myc–Atg8L)3xFLAG
were recognized by immunoblotting with anti-Myc (a-Myc) and anti-
FLAG (a-FLAG). wt, GA, and DG were the same as in (A). (C) In vit-
ro assay for Atg4B cleavage of the C-terminus of Atg8L. Wild-type
TRX–Atg8L)3xFLAG was expressed in Escherichia coli; the cells
were lysed and centrifuged, and the supernatants were used as
the substrate. The supernatants of HEK293 cells transfected with
pTag2B–hATG4B (hAtg4B wild) were used as enzymes; the negat-
ive control consisted of the supernatants of HEK293 cells transfect-
ed with pCMV–Tag2B (control). Following incubation of 1 lgof
enzyme solution with 10 lg of substrate solution for the indicated
times (incubation time), the reactions were stopped, and the total
proteins in each mixture were separated by SDS ⁄ PAGE. TRX–
Atg8L)3xFLAG was recognized by immunoblotting with anti-thiore-
doxin (TRX), and FLAG–hAtg4B and the C-terminal 3-FLAG tag of
TRX–Atg8L)3xFLAG were recognized with anti-FLAG. TRX–
Atg8L)3xFLAG, uncleaved form of TRX–Atg8L)3xFLAG; TRX–
Atg8L-I, cleaved form of TRX–Atg8L)3xFLAG; FLAG–hAtg4B,
FLAG-tagged Atg4B cysteine protease. (D) In vitro assay for the
cleavage of the C-terminus of Atg8L by an active site mutant
hAtg4
C74A
or other Atg4 homologs. As an enzyme solution, supern-
atants of HEK293 cells expressing an active site mutant
hAtg4B
C74A
(hAtg4B C74A), wild-type hAtg4A (hAtg4A wild) or

wild-type hAtg4C (hAtg4C) were employed instead of wild-type
hAtg4B (hAtg4B wild). Supernatant containing mutant TRX–
Atg8LDG)3xFLAG (DG), in which the Gly116 of Atg8L was deleted,
was used as the substrate. The enzyme solution and the substrate
solution were mixed and incubated for 30 min.
A
B
C
D
I. Tanida et al. Ubiquitylation-like modification of murine Atg8L
FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS 2555
FLAG. These results indicate that, like the other Atg8
homologs, Atg8L-I forms an E1-substrate intermediate
with human Atg7
C572S
.
Atg8L-I forms an E2-substrate intermediate with
green fluorescent protein (GFP)–Atg3
C264S
; this is
dependent on Atg7
Owing to the rapid turnover of the E2 reaction, it is
difficult to recognize such an E2-substrate intermediate
with an E2-like enzyme. When the active site Cys resi-
due of the E2-like enzyme is replaced by Ser, a stable
O-ester bond instead of a thiol–ester bond will be
formed between the enzyme and substrate(s); this is
dependent on an E1-like enzyme. Previously, we
showed that LC3, GABARAP and GATE-16 form
E2-substrate intermediates with an active site mutant

human Atg3
C264S
and that this activity was dependent
on human Atg7 [19]. If Atg8-L is a substrate of Atg3,
Atg8L-I will form an E2-substrate intermediate with
human Atg3
C264S
that is dependent on Atg7. There-
fore, we investigated whether Atg8L-I also forms an
Atg7-dependent E2-substrate intermediate with human
Atg3 and whether formation of this intermediate
occurs via the Gly116 in Atg8L. When wild-type
Myc–Atg8Lwt)3xFLAG, GFP–Atg3
C264S
and wild-
type Atg7 were expressed in HEK293 cells, a GFP–
Atg3
C264S
–Myc–Atg8L (E2-substrate) intermediate
was formed (Fig. 3, lane 2). When mutant Myc–
Atg8LDG)3xFLAG was substituted for wild-type
Atg8L (Fig. 3, lane 3), no intermediate was observed,
as demonstrated by immunoblotting with anti-GFP.
When Atg7 was not overexpressed (endogenous Atg7
alone), no intermediate was observed, while endo-
genous Atg7 was detected by anti-Atg7 (Fig. 3, lane
Fig. 2. Atg8L-I forms an E1-substrate intermediate with human
Atg7
C572S
. Myc-tagged Atg8L)3xFLAG (Myc–Atg8L)3xFLAG) was

transiently expressed with wild-type (Atg7 wt) or mutant human
Atg7 (Atg7 C572S). The cells were lysed, the proteins were separ-
ated by SDS ⁄ PAGE, and human Atg7 was recognized by immuno-
blotting with anti-human Atg7 (WB:a-Atg7), while Myc–Atg8L-I was
recognized by immunoblotting with anti-Myc (WB:a-Myc). Atg7–
Atg8L-I intermediate, E1-substrate intermediate between human
Atg7
C572S
and Atg8L; Atg7, human Atg7; Myc–Atg8L-I, Myc-tagged
Atg8L-I.
Fig. 3. Atg8L-I forms an E2-substrate intermediate with human
Atg3
C264S
, which is dependent on human Atg7. Myc–Atg8Lwt)3x-
FLAG (wt) was transiently expressed together with green fluores-
cent protein (GFP)–Atg3
C264S
in the presence (Atg7+) or absence
(Atg7–) of human Atg7. DG represents mutant Myc–Atg8LDG)3x-
FLAG lacking the Gly116 of Atg8L (see Fig. 1A). After lysing the
cells, total proteins were separated by SDS ⁄ PAGE. Atg7 was
recognized by immunoblotting with anti-human Atg7 (WB:a-Atg7),
GFP–Atg3
C264S
and its E2-substrate intermediate were recognized
with anti-GFP (WB:a-GFP), and Myc-tagged Atg8L proteins were
recognized by immunoblotting with anti-Myc (WB:a-Myc). The faint
band in lane 3 (DG) of the middle panel (WB:a-GFP) is GFP–
Atg3
C264S

-endogenous Atg8 homolog intermediate(s).
Ubiquitylation-like modification of murine Atg8L I. Tanida et al.
2556 FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS
1). These results indicate that Atg8L-I forms an Atg7-
dependent E2-substrate intermediate with human Atg3
via its Gly116.
Atg8L-II is increased in the presence of E64d and
pepstatin A, inhibitors of lysosomal proteases,
in HeLa cells
LC3-I and GABARAP-I have been shown to be modi-
fied by Atg7 and Atg3 to form the respective protein–
phospholipid conjugates, LC3-II and GABARAP-PL
[15]. We have reported the accumulation of LC3-II
and GABARAP-PL in HeLa cells incubated with
E64d [23], a membrane-permeable inhibitor of cathep-
sins B, H, and L, and pepstatin A [24], an inhibitor of
cathepsins D and E, for 24 h under nutrient-rich con-
ditions [15,25]. These results suggest that a modified
form of Atg8L-I may accumulate under the same con-
ditions. When HeLa cells expressing Myc–Atg8L were
incubated with E64d and pepstatin A for 24 h under
nutrient-rich conditions, LC3-II and GABARAP-PL
accumulated (Fig. 4A, WB:a-LC3 and a-GABARAP).
Under these conditions, we observed two bands by
immunoblotting with anti-Myc (Fig. 4A, WB:a-Myc).
One band corresponded to Myc–Atg8L-I, while the
other showed faster mobility. This second band, repre-
senting a modified form of Atg8L, was designated
Atg8L-II.
We have also shown that the modified forms of

LC3 and GABARAP, LC3-II and GABARAP-PL,
are present in the pellet after subcellular fraction-
ation [15]. Therefore, we hypothesized that, if
Atg8L-II is a modified form and not a degradation
product of Atg8L-I, it would be present in the pellet
of inhibitor-treated HeLa cells. Therefore, we centri-
fuged cell lysates at 100 000 g for 1 h and examined
the subcellular localization of Myc–Atg8L-I and
Myc–Atg8L-II by immunoblotting with anti-Myc.
Our results indicated that Myc–Atg8L-II was present
in the pellet, whereas Myc–Atg8L-I was present in
the supernatant (Fig. 4B), suggesting that Myc–
Atg8L-II is a modified form of Myc–Atg8L-I, and
not a degradation product.
We next investigated the intracellular localization of
Atg8L-II under these conditions using cyan fluorescent
protein (CFP)–Atg8L. HeLa cells expressing both
CFP–Atg8L and yellow fluorescent protein (YFP)–
LC3 were cultured under nutrient-rich conditions in
the presence of E64d and pepstatin A for 24 h, and
punctate signals of both CFP–Atg8L (Fig. 4C,e,h,k)
and YFP–LC3 (Fig. 4C,d,g,j) were observed by fluor-
escent microscopy. Like Myc–Atg8L, CFP–Atg8L can
form E1-substrate and E2-substrate intermediates with
human Atg7
C572S
and human Atg3
C264S
, respectively
(Fig. 4D,E), and little degradation of CFP–Atg8L was

observed even in the presence of these inhibitors for
24 h under nutrient-rich conditions (Fig. 4F), indica-
ting that the fluorescence signal reflects intact tagged
protein. Merging of the images indicated that most of
the puncta of CFP–Atg8L were colocalized with those
of YFP–LC3 (Fig. 4,f,I,l). In the absence of inhibitors,
only a few puncta of either type were observed
(Fig. 4C,a–c).
Little Atg8L-II accumulates under conditions of
starvation even in the presence of E64d and
pepstatin A in HeLa cells
LC3-I is significantly lipidated to form LC3-II under
conditions of starvation [10], and LC3-II is degraded
in the lysosome [25]. Therefore, in the presence of
E64d and pepstatin A, LC3-II shows significant accu-
mulation under conditions of starvation [25]. It is poss-
ible that, like LC3, Atg8L is modified to Atg8L-II
under conditions of starvation. Therefore, we investi-
gated whether Atg8L-II accumulates in HeLa cells
expressing Myc–Atg8L)3xFLAG incubated in Krebs–
Ringer buffered medium for 4 h under conditions of
starvation in the presence of E64d and pepstatin A,
conditions under which LC3-II has been reported to
accumulate [25] (Fig. 5, LC3-II). However, little
Atg8L-II accumulation was observed (Fig. 5, lane 4
versus lane 3). Even when cells were incubated with
the inhibitors under nutrient-rich conditions for a
short time—4 h compared with 24 h (Fig. 4A)—little
accumulation of Atg8L-II occurred (Fig. 5, lane 2).
Discussion

Here, we have shown that Atg8L is a substrate of reac-
tions mediated by human Atg4B, Atg7, and Atg3. We
found that the C-terminus of Atg8L is post-transla-
tionally cleaved and that the Gly116 in Atg8L is essen-
tial for this reaction, which is mediated by human
Atg4B, a cysteine protease, in vitro. We also found
that Atg8L forms an E1-substrate intermediate with an
E1-like enzyme, the active site mutant Atg7
C572S
, and
an E2-substrate intermediate with an E2-like enzyme,
the active site mutant Atg3
C264S
, with the latter reac-
tion dependent on Atg7. All these reactions are similar
to a series of reactions of three other mammalian Atg8
homologs: LC3, GABARAP, and GATE-16. In addi-
tion, we showed that a modified form of Atg8L,
Atg8L-II, accumulates in HeLa cells in the presence of
lysosomal protease inhibitors under nutrient-rich con-
ditions, comparable to the accumulation of LC3-II
I. Tanida et al. Ubiquitylation-like modification of murine Atg8L
FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS 2557
A
B
C
abc
def
ghi
jkl

Fig. 4. Atg8L-II, a modified form of Atg8L, accumulates in HeLa cells in the presence of inhibitors of lysosomal proteases, E64d and
pepstatin A. (A) Accumulation of Atg8L-II in the presence of E64d and pepstatin A. Myc–Atg8L)3xFLAG was transiently expressed in
HeLa cells, and the transfectants were incubated for 24 h in the presence (+) or absence (–) of E64d and pepstatin A. The cells were
lysed, and total proteins were separated by SDS ⁄ PAGE. Endogenous LC3 and GABARAP were recognized by immunoblotting with anti-
LC3 and 4-aminobutyrate
A
-receptor associated protein (GABARAP), respectively (WB:a-LC3 and a-GABARAP). Myc–Atg8L was recognized
with anti-Myc (WB:a-Myc). (B) Subcellular localization of Myc–Atg8L-II. Cell lysates of inhibitor-treated HeLa cells (A, E64d and pepstatin
A+) were fractionated into pellet (Ppt) and soluble (Sup) fractions by ultracentrifugation at 100 000 g for 1 h [12,15]. LC3-I, soluble unlip-
idated form of LC3; LC3-II, lipidated membrane-bound form of LC3; GABARAP-I, unlipidated form of GABARAP; GABARAP-PL, lipidated
form of GABARAP; Myc–Atg8L-I, soluble form of Myc–Atg8L; Myc–Atg8L-II, membrane-bound form of Myc–Atg8L. (C) Intracellular local-
ization of cyan fluorescent protein (CFP)–Atg8L. HeLa cells expressing both CFP–Atg8L and yellow fluorescent protein (YFP)–LC3 were
cultured in the absence (dimethylsulfoxide (DMSO)) or presence of E64d and pepstatin A (E64d and pepstatin A) for 24 h. After fixing,
cyan and yellow fluorescence in HeLa cells were observed with a Zeiss Axioplan2 fluorescence microscope with filters XF114-2 and
XF104-2. The deconvoluted images are shown in (C). In a, d, g, and j, yellow fluorescent images correspond to YFP–LC3. In b, e, h, and
k, cyan fluorescent images correspond to CFP–Atg8L. Merged images (Merge) are shown in e, f, i, and l. Arrowheads indicate colocaliz-
ation of YFP–LC3 and CFP–Atg8L. (D) Formation of an E1-substrate intermediate with Atg7
C572S
and CFP–Atg8L. CFP–Atg8L was trans-
iently expressed with wild-type (Atg7 wt) or mutant human Atg7 (Atg7 C572S). The cells were lysed, the proteins were separated by
SDS ⁄ PAGE, and human Atg7 was recognized by immunoblotting with anti-human Atg7 (WB:a-Atg7), and CFP–Atg8L was recognized by
immunoblotting with anti-GFP (WB:a-GFP). Atg7–Atg8L intermediate, E1-substrate intermediate between human Atg7
C572S
and Atg8L;
Atg7, human Atg7; CFP–Atg8L, CFP-tagged Atg8L. (E) Formation of an E2-substrate intermediate with Atg3
C264S S
and CFP–Atg8L. CFP–
Atg8L was transiently expressed together with GFP–Atg3
C264S
in the presence of human Atg7 (Atg7+). After lysing the cells, total pro-

teins were separated by SDS ⁄ PAGE. Atg7 was recognized by immunoblotting with anti-human Atg7 (WB:a-Atg7), GFP–Atg3
C264S
and its
E2-substrate intermediate were recognized with anti-Atg3 (WB:a-Atg3), and CFP–Atg8L was recognized by immunoblotting with anti-Atg8L
(WB:a-Atg8L). (F) There was little degradation of CFP–Atg8L in the presence of E64d and pepstatin A for 24 h under nutrient-rich condit-
ions. CFP–Atg8L and YFP–LC3 were transiently expressed in HeLa cells, and the transfectant was incubated for 24 h in the presence (+)
or absence (–) of E64d and pepstatin A. The cells were lysed, and total proteins were separated by SDS ⁄ PAGE. YFP–LC3 and CFP–Atg8L
were recognized by immunoblotting with anti-GFP (WB:a-GFP). CFP–Atg8L was recognized with anti-Atg8L (WB:a-Atg8L). Positions of
molecular weight markers for SDS ⁄ PAGE are indicated on the right of the panel.
Ubiquitylation-like modification of murine Atg8L I. Tanida et al.
2558 FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS
and GABARAP-PL under identical conditions. More-
over, we found that Atg8L-II was fractionated in the
pellet. These results indicate that Atg8L is a substrate
of Atg4B, Atg7, and Atg3, and that Atg8L is the
authentic fourth modifier in the mammalian Atg8 con-
jugation system. Previously, we showed that LC3-II
and GABARAP-PL are protein–phospholipid conju-
gates in vivo [15], and that these two conjugates, as
well as GATE-16-II, are localized to a membrane
compartment [10,12]. Like LC3-II and GABARAP-
PL, Atg8L-II was fractionated in the pelletable
fraction, suggesting that Atg8L-II may be a
protein–phospholipid conjugate that is localized to a
membrane compartment.
The modified form, Myc–Atg8L-II, was observed
only in the presence of E64d and pepstatin A for 24 h
under nutrient-rich conditions (Fig. 4A), whereas little
Myc–Atg8L was observed for a shorter period (4 h)
even in the presence of these inhibitors. In contrast,

LC3-II accumulates in the presence of E64 and pepstatin
A within only 4 h under both nutrient-rich and starva-
tion conditions. There are two possible reasons for the
low level of Myc–Atg8L-II accumulation for 4 h in the
presence of these inhibitors. The first is that modifica-
DE
F
Fig. 4. (Continued).
I. Tanida et al. Ubiquitylation-like modification of murine Atg8L
FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS 2559
tion of Atg8L-I to Atg8L-II may occur much more
slowly than that of LC3-I to LC3-II. Therefore, Myc–
Atg8L-II was observed only after a longer incubation
time (24 h) in the presence of E64d and pepstatin A.
The other possibility is that Myc–Atg8L-II may be very
unstable compared with LC3-II. We have reported that
Atg4B is a delipidating enzyme for LC3-II and GABA-
RAP-PL in addition to being a cysteine protease that
cleaves the C-termini of mammalian Atg8 homologs.
Therefore, if Atg4B delipidates Myc–Atg8L-II more
effectively than LC3-II, there will be less accumulation
of Myc–Atg8L-II compared with LC3-II. These points
will be clarified in future studies by in vitro assays of
lipidation and delipidation of Atg8L.
Experimental procedures
Materials, and biochemical and molecular
biological techniques
Molecular biological and biochemical techniques were per-
formed as described [25]. To clone mouse Atg8L cDNA by
PCR, we used high-fidelity KOD plus DNA polymerase

(Toyobo, Osaka, Japan). A plasmid containing mouse
Atg8L cDNA was transfected into HEK293 cells with
Lipofectamine 2000 reagent according to the manufac-
turer’s protocol (Invitrogen, Carlsbad, CA). E. coli strain
DH5a cells, the hosts for plasmids and protein expression,
were grown in Luria broth medium in the presence of anti-
biotics as required. Restriction enzymes were purchased
from Toyobo and New England Biolabs (Beverly, MA).
Oligonucleotides were synthesized by Invitrogen. pGEM-T
was purchased from Promega (Madison, WI), and
p3xFLAG–CMV14 was obtained from Sigma-Aldrich (St
Louis, MO).
Cloning of mouse Atg8L cDNA and site-directed
mutagenesis
A DNA fragment encoding the entire open reading frame
of ATG8L, according to a cDNA sequence in GenBank
(accession number: BG244294) [21], was amplified by high-
fidelity PCR from a Marathon ready mouse brain cDNA
library (BD Biosciences Clontech, Palo Alto, CA) and
inserted into pGEM-T. The resulting plasmid was designa-
ted pGEM–mATG8L.
Using the Gene-Editor in vitro site-directed mutagenesis
system (Promega) and the plasmid pGEM–mATG8L,
Gly116 in mouse Atg8L was replaced by Ala in accordance
with the manufacturer’s protocol, and the resulting plasmid
was designated pGEM–mATG8L
G116A
. Gly116 in mouse
Atg8L of pGEM–mATG8L was deleted by site-directed
mutagenesis, and the resulting plasmid was designated

pGEM–mATG8LDG. Mammalian expression vectors for
N-terminal Myc-tagged and C-terminal 3xFLAG-tagged
versions of Atg8L, Myc–Atg8Lwt)3xFLAG, Myc–Atg8L-
GA)3xFLAG and Myc–Atg8LDG)3xFLAG were con-
structed from pGEM–mATG8L and p3xFLAG–CMV14 by
high-fidelity PCR, and designated pMyc–mATG8L)
3xFLAG, pMyc–mATG8LGA)3xFLAG, and pMyc–
mATG8LDG)3xFLAG, respectively. An expression vector
for N-terminal TRX-tagged and C-terminal 3xFLAG-
tagged Atg8L in E. coli, TRX–Atg8L)3xFLAG, was con-
structed based on pThioHisA (Invitrogen) and designated
pTRX–ATG8L)3xFLAG. A mammalian expression vector
for N-terminal CFP-tagged Atg8L designated pCFP–
ATG8L was generated by inserting the open reading frame
of mAtg8L excised from pGEM–mATG8L into pCFP-C1
(BD Biosciences Clontech). The DNA sequences of new
constructs were confirmed using a ABI PRISM
TM
310 Gen-
etic Analyzer (Applied Biosystems, Foster City, CA). The
expression vectors pCMV–hAPG7, pCMV–hAPG7CS,
pGFP–hAPG3, pGFP–hAPG3CS, pTag2B–HsATG4B,
pTag2B–HsATG4B
C74A
, pTag2B–HsATG4A and pTag2B–
HsAUTL1 for human Atg7, mutant Atg7
C572S
, GFP–Atg3,
mutant GFP–Atg3
C264S

, wild-type FLAG–hAtg4B, mutant
FLAG–hAtg4B
C74A
, wild-type FLAG–hAtg4A and wild-
Fig. 5. There was little modification of Atg8L under conditions of
starvation, even in the presence of inhibitors of lysosomal proteases.
Myc–Atg8L)3xFLAG was transiently expressed in HeLa cells. The
transfectant was transferred to Krebs–Ringer buffered medium
(KRB, 4 h, starvation) or Dulbecco’s modified Eagle’s medium
(DMEM) containing 10% fetal bovine serum (DMEM, 10% fetal
bovine serum, nutrient-rich), and incubated for 4 h in the presence
(+) or absence (–) of inhibitors. Little Myc–Atg8L-II was detected by
immunoblotting with anti-Myc under either condition. Endogenous
LC3 was recognized by immunoblotting with antibodies to LC3. As a
control, the membrane was stained with Coomassie Brilliant Blue.
LC3-II, LC3–phospholipid conjugate.
Ubiquitylation-like modification of murine Atg8L I. Tanida et al.
2560 FEBS Journal 273 (2006) 2553–2562 ª 2006 The Authors Journal compilation ª 2006 FEBS
type FLAG–hAtg4C ⁄ AutL1, respectively, were as described
[15,17,19].
Cell culture
The HEK293 and HeLa cell lines were purchased from the
ATCC (Manassas, VA) and cultured in 60-mm dishes in
Dulbeccos’s modified Eagles’s medium (DMEM, Invitro-
gen) containing 10% fetal bovine serum (Invitrogen) in a
humidified 5% CO
2
atmosphere at 37 °C.
Antibodies
For preparation of antiserum against mouse Atg8L, rabbits

were immunized with a TRX–Atg8L fusion protein. Anti-
human GABARAP, LC3, Atg7 and Atg3 were prepared
and purified as described [12,17,19,26]. Anti-Myc was pur-
chased from Cell Signaling (Beverly, MA), anti-FLAG
M2 was purchased from Sigma-Aldrich, anti-TRX was pur-
chased from Santa Cruz Biotechnology (Santa Cruz, CA),
and anti-GFP was purchased from BD Biosciences Clontech.
Immunoblotting analyses
Protein concentrations were determined using the bicincho-
ninic acid protein assay reagent (Pierce, Rockford, IL).
Immunoblotting analyses were carried out according to
standard protocols using a chemiluminescent method with
SuperSignal West Dura Extended Duration Substrate or
SuperSignal West Pico Chemiluminescent Substrate (Pierce).
In vitro assay for cleavage of the C-terminus of
TRX–Atg8L)3xFLAG by Atg4B
The in vitro assay for cleavage of the C-terminus of mouse
Atg8L was performed as previously described [15].
Fluorescence microscopy
HeLa cells expressing YFP–hLC3 and CFP–mAtg8L were
fixed according to the manufacturer’s protocol (BD Bio-
sciences Clontech). Briefly, after transfection of pYFP–LC3
and pCFP–ATG8L into HeLa cells using Lipofecta-
mine2000 (Invitrogen), cells were incubated for 24 h under
nutrient-rich conditions in the presence or absence of E64d
and pepstatin A. Thereafter, cells were washed twice with
NaCl ⁄ P
i
, and fixed in NaCl ⁄ P
i

containing 4% paraformal-
dehyde for 10 min at room temperature. Fixed cells were
washed three times with NaCl ⁄ P
i
, and then mounted on
slide-glasses with SlowFade Light antifade reagent (50%
glycerol solution) (Invitrogen). Fluorescence of YFP and
CFP in the cells was monitored with a Zeiss Axioplan2
fluorescence microscope (Carl Zeiss, Thornwood, NY)
equipped with an ORCA-ER CCD camera (Hamamatsu
Photonics, Tokyo, Japan). For deconvolution of the ima-
ges, a Zeiss Axioplan2 fluorescence microscope (Carl Zeiss)
and an Aqua C-imaging system (Hamamatsu Photonics)
were employed.
Acknowledgements
This work was supported in part by grants-in-aid
15590254 (to IT), 09680629 (to TU) and 12470040 (to
EK) for Scientific Research, and grant-in-aid 12146205
(to EK) for Scientific Research on Priority Areas from
the Ministry of Education, Science, Sports, and Cul-
ture of Japan, and The Science Research Promotion
Fund from the Japan Private School Promotion Foun-
dation (to EK).
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