REPORT
28503–28507, August 17, 2012
The Autophagy-related Protein
Kinase Atg1 Interacts with the
Ubiquitin-like Protein Atg8 via
the Atg8 Family Interacting
Motif to Facilitate
Autophagosome Formation*□
Received for publication, June 1, 2012, and in revised form, July 3, 2012
Published, JBC Papers in Press, July 9, 2012, DOI 10.1074/jbc.C112.387514
Hitoshi Nakatogawa‡1, Shiran Ohbayashi‡,
Machiko Sakoh-Nakatogawa‡, Soichiro Kakuta‡, Sho W. Suzuki‡,
Hiromi Kirisako‡, Chika Kondo-Kakuta‡, Nobuo N. Noda§,
Hayashi Yamamoto‡, and Yoshinori Ohsumi‡
From the ‡Frontier Research Center, Tokyo Institute of Technology,
Yokohama 226-8503 and the §Institute of Microbial Chemistry,
Tokyo 141-0021, Japan
Background: Atg1 is a protein kinase essential for the
initiation of autophagosome formation.
Results: Atg1 interacts with Atg8 to associate with form-
ing autophagosomal membranes; specific disruption of
this interaction causes a significant defect in autophagy.
Conclusion: Atg1 on forming autophagosomal mem-
branes promotes autophagosome formation, distinct
from its role for triggering the process.
Significance: This study provides novel insights into the
regulatory mechanisms of autophagy.
In autophagy, a cup-shaped membrane called the isolation
membrane is formed, expanded, and sealed to complete a dou-
ble membrane-bound vesicle called the autophagosome that
encapsulates cellular constituents to be transported to and
degraded in the lysosome/vacuole. The formation of the
autophagosome requires autophagy-related (Atg) proteins.
Atg8 is a ubiquitin-like protein that localizes to the isolation
membrane; a subpopulation of this protein remains inside the
autophagosome and is transported to the lysosome/vacuole. In
the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/
threonine kinase that functions in the initial step of autophago-
some formation and is also efficiently transported to the vacuole
via autophagy. Here, we explore the mechanism and significance
of this autophagic transport of Atg1. In selective types of
autophagy, receptor proteins recognize degradation targets and
also interact with Atg8, via the Atg8 family interacting motif
(AIM), to link the targets to the isolation membrane. We find
that Atg1 contains an AIM and directly interacts with Atg8.
Mutations in the AIM disrupt this interaction and abolish vac-
* This work was supported in part by the Funding Program for Next Genera-
tion World-Leading Researchers (to H. N.) and grants-in-aid for scientific
research (to Y. O.) from the Ministry of Education, Culture, Sports, Science
and Technology, Japan.
□S
This article contains supplemental Methods, Tables S1 and S2, Figs. S1–S7,
and Movie S1.
1
To whom correspondence should be addressed. Tel.: 81-45-924-5879; Fax:
81-45-924-5121; E-mail: hnakatogawa@iri.titech.ac.jp.
AUGUST 17, 2012 • VOLUME 287 • NUMBER 34
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 34, pp.
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
uolar transport of Atg1. These results suggest that Atg1 associ-
ates with the isolation membrane by binding to Atg8, resulting
in its incorporation into the autophagosome. We also show that
mutations in the Atg1 AIM cause a significant defect in
autophagy, without affecting the functions of Atg1 implicated in
triggering autophagosome formation. We propose that in addi-
tion to its essential function in the initial stage, Atg1 also asso-
ciates with the isolation membrane to promote its maturation
S
into the autophagosome.
Autophagy (macroautophagy) is a pathway for membrane
transport to the lysosome/vacuole (1). During this process, a
cup-shaped membrane called the isolation membrane is
formed and expanded into a double membrane-bound vesicle
called the autophagosome. The autophagosome eventually
fuses with those lytic organelles, allowing degradation of its
contents. Under starvation conditions, autophagosomes ran-
domly sequester portions of the cytoplasm. In contrast,
autophagosomes also selectively engulf degradation targets,
such as cytotoxic protein aggregates, and damaged or surplus
organelles (2, 3). These processes require autophagy-related
(Atg)2 proteins. The core Atgs mediate the formation of the
autophagosomal membrane, whereas receptor and adaptor
Atgs recognize degradation targets and recruit the core Atgs to
allow membrane formation along the surfaces of targets. The
ubiquitin-like protein Atg8 (microtubule-associated protein 1
light chain 3 (LC3) or gamma-aminobutyric acid A receptor-
associated protein (GABARAP) in mammals) is conjugated to
phosphatidylethanolamine (PE) and thereby anchored to the
isolation membrane, where it is involved in the expansion of
the membrane (4 –7). A proportion of Atg8 is retained inside
the complete autophagosomes and is transported to the lyso-
some/vacuole. In addition, Atg8 also plays an important role in
the incorporation of degradation targets into autophagosomes;
it interacts with receptor proteins that contain the Atg8 family
interacting motif (AIM) (or the LC3 interacting region), which
binds to highly conserved, hydrophobic pockets in Atg8 (2, 3, 8,
9). Atg8-PE conjugates on the isolation membrane may link the
target-receptor complex to the membrane to facilitate its
engulfment.
Atg1 (unc-51-like kinase 1 (ULK1) in mammals) is a serine/
threonine kinase essential for autophagy. In yeast, Atg1 forms a
complex with the regulatory proteins Atg13, Atg17, Atg29, and
Atg31 in response to autophagy-inducing signals (10, 11). This
complex serves as a scaffold to organize the pre-autophago-
somal structure (PAS), a dynamic assembly of Atg proteins,
during the initiation of autophagosome formation (1, 11–13).
The formation of the PAS-scaffolding complex also enhances
the kinase activity of Atg1 (10), which regulates the PAS local-
2
The abbreviations used are: Atg, autophagy-related; AIM, Atg8 family inter-
acting motif; PE, phosphatidylethanolamine; PAS, pre-autophagosomal
structure; ALP, alkaline phosphatase; OD, optical density; mut, mutant; SC,
synthetic complete.
JOURNAL OF BIOLOGICAL CHEMISTRY 28503
REPORT: Atg1 Interaction with Atg8 via the AIM
ization of downstream Atgs, probably via phosphorylation of
some of those proteins.
In a previous study, we found an intriguing behavior of Atg1
during autophagy (12). Although Atg1 has been implicated in
the early stage of autophagosome formation, a fraction of this
kinase is incorporated into the autophagosome and transported
to the vacuole. In addition, although the PAS localization of
Atg1 requires complex formation with the regulatory proteins,
only Atg1 among these proteins is significantly transported to
the vacuole.
In this study, we investigated the mechanism of the incorpo-
ration of Atg1 into the autophagosome. We found that Atg1
contains an AIM, with which it directly binds to Atg8. Muta-
tions that affected this interaction accordingly reduced the vac-
uolar transport of Atg1. Thus, interaction with Atg8 allows
Atg1 to associate with the isolation membrane, resulting in
Atg1 incorporation into the autophagosome. We also found
that mutations in the AIM of Atg1 attenuated autophagic activ-
ity, although they did not affect Atg1 functions involved in the
initiation of autophagosome formation. These results suggest
that Atg1 on the isolation membrane plays an important role in
the promotion of autophagosome formation, distinct from its
role in the initial stage.
EXPERIMENTAL PROCEDURES
Yeast Strains, Plasmids, and Media—The yeast strains and
oligonucleotides used in this study are listed in supplemental
Tables S1 and S2, respectively. Construction of these strains
and plasmids and the compositions of culture media are
described in supplemental Methods.
Fluorescence Microscopy—Fluorescence microscopy was
performed as described previously (14).
Immunoblotting—Immunoblotting analysis was performed
as described previously (14). Monoclonal antibodies against
GFP (Roche Applied Science) was used for detection of GFP-
fused proteins. Antibodies against Atg8 (anti-Atg8-2), Atg1
(anti-Atg1), Atg13 (anti-Atg13), and Atg17 (anti-GST-Atg17-
1) were described previously (10, 11, 14).
Yeast Two-hybrid Assay—The yeast two-hybrid assay was
performed using Matchmaker Gal4 Two-hybrid System 3
(Clontech). The AH109 strain was transformed with different
combinations of pGADT7-ATG1 and pGBD-C-ATG8 plas-
mids and their vectors, and the overnight cultures were spotted
onto SC-LT (control), SC-LTH (⫺His), and SC-LTA (⫺Ade)
agar plates followed by incubation at 30 °C for 1, 2, and 3 days,
respectively.
Immunoprecipitation—Yeast cells expressing Atg1-GFP and
3⫻FLAG-Atg8 were grown to mid-log phase, treated with
rapamycin for 2 h, and disrupted in HSE buffer (25 mM HEPES-
KOH (pH 7.2), 750 mM sorbitol, 5 mM EDTA) containing 0.5⫻
Complete protease inhibitor mixture (Roche Applied Science)
using Multi-beads shocker and 0.5-mm YZB zirconia beads
(Yasui Kikai). Sodium chloride and Triton X-100 were added to
the lysates to concentrations of 50 mM and 0.5%, respectively. A
monoclonal anti-FLAG M2 antibody (Sigma) and Dynabeads
protein G (Invitrogen) were incubated with the lysates at 4 °C
for 3 h. After washing the beads, the bound proteins were eluted
by incubating the beads in SDS sample buffer at 65 °C for 5 min.
28504 JOURNAL OF BIOLOGICAL CHEMISTRY
To examine the formation of the PAS-scaffolding complex, the
cell lysates were prepared from rapamycin-treated yeast cells
expressing Atg1-GFP by essentially the same method as
described above, except that TSG buffer (50 mM Tris-HCl (pH
8.0), 150 mM NaCl, 10% glycerol) was used instead of HSE
buffer. The lysates were treated with 0.2% n-dodecyl-␤-D-
maltoside at 4 °C for 30 min and incubated with GFP-Trap_M
beads (ChromoTek) at 4 °C for 2 h. After washing the beads, the
bound proteins were eluted as described above.
ALP Assay—To quantify autophagic activity of yeast cells, an
alkaline phosphatase (ALP) assay was performed as described
previously (15).
RESULTS
Atg1 Is Transported to and Degraded in the Vacuole via
Autophagy—In systematic analysis of the PAS localization of
Atg proteins (12), Atg1 was found to be transported to the vac-
uole via autophagy. When yeast cells expressing Atg1 tagged
with GFP were treated with the autophagy inducer rapamycin,
GFP fluorescence in the vacuole increased in a wild-type strain,
but not in atg mutants such as atg14⌬ (Fig. 1A). Immunoblot-
ting analysis using antibodies against GFP showed that wild-
type cells produce GFP-containing fragments under autophagy-
inducing conditions, but atg14⌬ cells do not (Fig. 1B). These
fragments were likely to have been produced by proteolysis of
Atg1-GFP in the vacuole because their accumulation was
blocked by the addition of phenylmethylsulfonyl fluoride
(PMSF), which inactivates the vacuolar protease Prb1 (supple-
mental Fig. S1A). In contrast, specific fragments were not
detected by immunoblotting using antibodies against Atg1
(supplemental Fig. S1B), suggesting that the Atg1 portion of the
fusion protein is degraded in the vacuole, whereas the GFP por-
tion is resistant to vacuolar proteases.
Atg1 Directly Binds to Atg8 via the AIM—We next addressed
the mechanism by which Atg1 is efficiently incorporated into
the autophagosome. First, we examined the involvement of fac-
tors known to function in selective types of autophagy in yeast
(16 –18). Deletion of genes encoding adaptor (Atg11) and
receptor proteins (Atg19, Atg34, and Atg32) did not signifi-
cantly affect the vacuolar transport of Atg1 (supplemental Fig.
S2). However, a double mutation in Atg8 (P52A/R67A), which
decreases its binding to the AIM but not its function in
autophagosome formation (8, 19), retarded Atg1 transport to
the vacuole (Fig. 1C). Although an as yet unidentified receptor
containing the AIM might mediate Atg1 incorporation into the
autophagosome, previous studies showed that not only recep-
tors but also some core Atgs use the AIM to interact with Atg8
(20). Therefore, we examined the possibility that Atg1 directly
interacts with Atg8 via the AIM. Atg1 consists of the N-termi-
nal kinase domain, the C-terminal region responsible for bind-
ing to Atg13, and the intervening middle region (Fig. 1D). The
yeast two-hybrid assay clearly showed that Atg1 binds to Atg8;
an indicator strain coexpressing Atg1 fused to the Gal4 tran-
scription activation domain and Atg8 fused to the Gal4 DNA-
binding domain could grow on agar medium lacking histidine
(Fig. 1E and supplemental Fig. S3). Furthermore, the Atg1 mid-
dle region alone interacted with Atg8, whereas Atg1 lacking this
region did not, suggesting that Atg1 interacts with Atg8 via its
VOLUME 287 • NUMBER 34 • AUGUST 17, 2012

FIGURE 1. Atg1 interacts with Atg8 via the AIM. A, wild-type (YNH512) and
atg14⌬ (YNH731) cells expressing Atg1-GFP were grown to mid-log phase,
treated with rapamycin for 6 h, and observed under a fluorescence micro-
scope. Arrowheads indicate vacuoles. Images obtained using differential
interference contrast (DIC) optics are also shown, with scale bars representing
5 ␮m. B, the same strains as in A were treated with rapamycin for the indicated
time periods and subjected to immunoblotting analysis using monoclonal
antibodies against GFP. GFP⬘ represents GFP-containing fragments gener-
ated by proteolysis of Atg1-GFP in the vacuole. C, ATG8WT (YNH695) and
ATG8P52A/R67A (YNH696) cells expressing Atg1-GFP were analyzed as
described in B. D, schematic diagram of Atg1. Atg1 consists of three regions:
the N-terminal kinase domain (N), the middle region (M), and the C-terminal
Atg13-binding region (C). The AIM mutant (AIM mut) of Atg1 contains two
alanine substitutions at Tyr429 and Val432. E, the indicator strain AH109 was
transformed with plasmids expressing a transcription activation domain (AD)
fused with full-length Atg1 (F) or the middle region of Atg1 (M), with or with-
out mutations at the AIM (AIM mut), in combination with plasmids expressing
a DNA-binding domain (BD) fused with either Atg8WT or the Atg8P52A/R67A
mutant. These strains were grown on SC-LTH (⫺His), SC-LTA (⫺Ade), and
SC-LT (control) agar plates. F, ATG8 (YNH740) and 3⫻FLAG-ATG8 (YNH741)
cells expressing Atg1WT-GFP or Atg1AIM mut-GFP from single-copy plasmids
were treated with rapamycin for 2 h and disrupted to prepare cell lysates
(input) for immunoprecipitation (IP) using a monoclonal antibody against the
FLAG sequence. The immunoprecipitates were analyzed by immunoblotting
with antibodies against GFP and Atg8.
AUGUST 17, 2012 • VOLUME 287 • NUMBER 34
REPORT: Atg1 Interaction with Atg8 via the AIM
middle region (Fig. 1E and supplemental Fig. S3). When com-
pared with the full-length protein, the Atg1 middle region
exhibited a higher affinity to Atg8; yeast strains coexpressing
this Atg1 variant could also grow in the absence of adenine (Fig.
1E). We found that the P52A/R67A double mutation in Atg8
negatively affected the interaction of Atg8 with the Atg1 middle
region (Fig. 1E), suggesting that the AIM binding ability of Atg8
is important for the interaction with Atg1.
Based on common features of known AIMs (20), we identi-
fied a putative AIM with the amino acid sequence Tyr429-
Val430-Val431-Val432 in the middle region of Atg1 (Fig. 1D).
When Tyr429 and Val432 were simultaneously replaced with ala-
nine residues, the interaction between Atg1 and Atg8 was
severely impaired (Fig. 1E). This was also confirmed by coim-
munoprecipitation experiments (Fig. 1F). Although immuno-
precipitation of Atg8 tagged with the 3⫻FLAG sequence copre-
cipitated wild-type Atg1-GFP, the efficiency was significantly
reduced by the Y429A/V432A double mutation. Thus, we con-
clude that this sequence in Atg1 functions as the AIM.
Association of Atg1 with the Isolation Membrane Facilitates
Autophagosome Formation—We showed that the mutations in
the AIM abolished the vacuolar transport of Atg1 (Fig. 2, A and
B). Given that Atg8 localizes to the isolation membrane and is
retained on the complete autophagosomal membrane, these
results suggest that Atg1 interacts with Atg8 using the AIM,
thereby associates with the isolation membrane, and is eventu-
ally encapsulated into the autophagosome.
To examine the autophagic activity of cells expressing the
AIM mutant of Atg1, we performed an ALP assay (Fig. 2C and
supplemental Fig. S4A) (15). The results revealed that the Atg1
AIM mutant cells exhibited a partial but significant defect in
autophagy. In accordance with this, the vacuolar transport of
GFP-Atg8 also decreased in the mutant cells (supplemental Fig.
S4, B and C). We also examined accumulation of autophagic
bodies, inner membrane vesicles released into the vacuolar
lumen upon fusion between autophagosomal outer membranes
and the vacuolar membrane, using vacuolar protease-deficient
strains (supplemental Movie S1). The Atg1 AIM mutant cells
accumulated autophagic bodies less than wild-type cells. These
results suggest that the mutations in the AIM of Atg1 decrease
the efficiency of autophagosome formation. Coimmunopre-
cipitation analysis showed that the Atg1 AIM mutant inter-
acted normally with Atg13 and Atg17, suggesting that the
mutations in the AIM did not affect the formation of the PAS-
scaffolding complex (Fig. 2D). In wild-type cells, Atg1 kinase
activity is up-regulated under autophagy-inducing conditions
(10), which can be assessed by the appearance of a slower
migrating band of autophosphorylated Atg1 in immunoblot-
ting analysis (21). That band was detected in the AIM mutant at
a level similar to wild-type Atg1 and disappeared upon deletion
of ATG13, which is essential for activation of Atg1 (Fig. 2E).
Thus, the stimulation of Atg1 kinase activity occurred normally
in the AIM mutant. Furthermore, the Atg1 AIM mutant local-
ized to the PAS as efficiently as the wild-type protein (Fig. 2F
and supplemental Fig. S5). It was also shown that the mutations
in the AIM of Atg1 did not reduce the PAS localization of Atg8;
it rather accumulated at the PAS, probably due to delayed
autophagosome formation (Fig. 2G and supplemental Fig. S5).
JOURNAL OF BIOLOGICAL CHEMISTRY 28505
REPORT: Atg1 Interaction with Atg8 via the AIM
FIGURE 2. Characterization of an AIM mutant of Atg1. A and B, atg1⌬ cells
(YNH204) expressing Atg1WT-GFP or Atg1AIM mut-GFP from single-copy plas-
mids were treated with rapamycin and analyzed by fluorescence microscopy
(A) or immunoblotting with anti-GFP antibodies (B). Scale bars represent 5
␮m. GFP⬘ represents GFP-containing fragments generated by proteolysis
of Atg1-GFP in the vacuole. AIM mut, AIM mutant. C, ATG1WT (YNH738),
ATG1AIM mut (YNH739), and atg1⌬ (YNH621) cells expressing a mutant form of
ALP were incubated in nitrogen-deprived medium and subjected to the ALP
assay. The same experiments were repeated three times; data are repre-
sented as means with standard deviations. AU, arbitrary units. D, YNH740 cells
that carried either plasmids expressing Atg1WT-GFP (W) or Atg1AIM mut-GFP
(A) or empty vector (⫺) were treated with rapamycin for 1 h and subjected to
coimmunoprecipitation (IP) analysis using anti-GFP antibodies. The immuno-
precipitates were analyzed by immunoblotting using antibodies against
Atg1, Atg13, and Atg17. E, ATG1WT (YNH736), ATG1AIM mut (YNH737), ATG1WT
atg13⌬ (YNH746), and ATG1AIM mut atg13⌬ (YNH747) cells were treated with
rapamycin for 1 h and examined by immunoblotting using anti-Atg1 anti-
bodies. F, ATG1WT-GFP (YNH742) and ATG1AIM mut-GFP (YNH743) cells treated
with rapamycin for 1 h were observed under a fluorescence microscope (the
numbers of cells examined were 272 and 221, respectively); the percentages
of cells with GFP dots are shown. G, dot formation of GFP-Atg8 in ATG1WT
(YNH744) and ATG1AIM mut (YNH745) cells was examined as described in F (the
numbers of cells examined were 307 and 246, respectively). H, the vacuolar
transport of Atg1WT-GFP and Atg1Y878A/R885A-GFP expressed from single-
copy plasmids in wild-type cells (BY4741) was examined by immunoblotting
analysis as described in legend for Fig. 1. I, the same experiments as in H were
repeated three times, and the intensities of the bands of Atg1-GFP and GFP⬘
at the time point of 6 h were measured to estimate the efficiency of the
production of processed GFP fragments (the intensities of GFP⬘ were divided
by the sum of those of Atg1-GFP and GFP⬘); results are represented as means
with standard deviations.
28506 JOURNAL OF BIOLOGICAL CHEMISTRY
Taken together, these results suggest that mutations in the AIM
of Atg1 cause a defect in autophagy by abolishing Atg1 associ-
ation with the isolation membrane rather than by affecting the
functions of Atg1 involved in the initiation of autophagosome
formation.
A double mutation in the C-terminal region of Atg1, Y878A/
R885A, impairs the interaction with Atg13, resulting in reduced
PAS localization of Atg1 (13). This Atg1 mutant also exhibited
a defect in PAS localization when it was coexpressed with wild-
type Atg1 (supplemental Fig. S6). We showed that this mutant
was less efficiently transported to the vacuole than wild-type
Atg1 (Fig. 2, H and I), although autophagy occurred normally
in these cells. These results suggest that PAS localization is a
prerequisite for Atg1 to associate with autophagosomal
membranes.
DISCUSSION
Previous studies have established the role of Atg1 kinase and
its regulatory proteins in the initiation of autophagosome for-
mation; in response to autophagy-inducing signals, Atg1 forms
a large complex with Atg13 and the Atg17-Atg29-Atg31 ter-
nary complex. This complex further forms a higher-order
assembly that serves as a scaffold, which organizes the PAS and
induces autophagosome formation. However, the autophagic
transport of Atg1 to the vacuole suggested that the kinase
remains associated with the autophagosomal membrane until a
late stage of its formation. In contrast, few of the regulatory
proteins are transported to the vacuole. These data indicated
that Atg1 plays an additional role, distinct from its function in
triggering autophagosome formation in association with the
regulators. In agreement with these observations, it has recently
been shown that Atg1, but not its regulators, localizes to the
isolation membrane.3 In the present study, our results suggest
that the association of Atg1 with the isolation membrane is
mediated by direct interaction with Atg8. Mutations that dis-
rupt this interaction, and thereby prevent Atg1 association with
the membrane, caused a significant defect in autophagy,
although they did not impair Atg1 functions involved in the
initiation of autophagosome formation. Thus, in accordance
with the above notion, our results suggest that Atg1 plays an
additional role in promoting autophagosome formation on the
isolation membrane.
The interaction between Atg1 and Atg8 involves an AIM
located in the middle region of Atg1. The AIM was first identi-
fied as a sequence commonly seen in receptors for selective
autophagy and subsequently found in core Atg proteins such as
yeast Atg3 and mammalian Atg4 (20). Although the AIM in
Atg3 is specifically required for the cytoplasm-to-vacuole tar-
geting pathway, which can be regarded as a type of selective
autophagy, this study identified for the first time the AIM
involved in starvation-induced, nonselective autophagy.
Most core Atg proteins localize to the PAS in a hierarchical
manner; the PAS-scaffolding complex assembles first, whereas
Atg8 is one of the most downstream factors (11–13). We
showed that AIM mutations do not significantly affect the PAS
localization of either Atg1 or Atg8. Therefore, it seems that the
3
K. Suzuki, M. Akioka, C. Kondo, and Y. Ohsumi, unpublished results.
VOLUME 287 • NUMBER 34 • AUGUST 17, 2012

AIM-mediated interaction between Atg1 and Atg8 is not
involved in PAS organization based on the hierarchical model.
We also showed that an Atg1 mutant defective in interaction
with Atg13, and thus in PAS localization, is not efficiently trans-
ported to the vacuole. These results suggest that Atg1 in com-
plex with the regulators first assembles to organize the PAS,
induces the formation of the isolation membrane by recruiting
downstream factors including Atg8, and is subsequently disso-
ciated from the complex and transferred to Atg8-PE on the
membrane. We propose that the role of Atg1 on the isolation
membrane is to facilitate membrane expansion by phosphory-
lating some protein(s) on the membrane. These target(s) of
Atg1 kinase may not be identical to those phosphorylated by
Atg1 in the early stage of autophagosome formation; identifica-
tion of these targets will require further study.
Interactions between Atg1 and Atg8 homologs have also
been reported in mammals and plants (22, 23). Although the
details of these interactions remain to be elucidated, the AIM in
Atg1 homologs may mediate their interactions with Atg8
homologs in higher eukaryotes as well. Indeed, amino acid
sequences predicted to function as an AIM are found in Arabi-
dopsis Atg1 homologs (supplemental Fig. S7). However, the sig-
nificance of the AIM-mediated interaction between Atg1 and
Atg8 may differ among organisms. In plant cells, unlike yeast,
Atg13 is also transported to the vacuole via autophagy (23). In
contrast, the autophagic transport of ULK1 to the lysosome has
not been observed in mammalian cells. These differences may
be relevant to the different organization of the Atg1 complex
and its behavior in response to autophagy-inducing signals in
these organisms (24). Therefore, it will be important to eluci-
date how the interaction of Atg1 with Atg8 contributes to
autophagy in mammals and plants. Our study provides a frame-
work to address these issues.
Acknowledgments—We thank the members of our laboratories for
materials, helpful discussions, and technical support, and Yoko Hara
for her secretarial support.
REFERENCES
1. Nakatogawa, H., Suzuki, K., Kamada, Y., and Ohsumi, Y. (2009) Dynamics
and diversity in autophagy mechanisms: lessons from yeast. Nat. Rev. Mol.
Cell Biol. 10, 458 – 467
2. Weidberg, H., Shvets, E., and Elazar, Z. (2011) Biogenesis and cargo selec-
tivity of autophagosomes. Annu. Rev. Biochem. 80, 125–156
3. Johansen, T., and Lamark, T. (2011) Selective autophagy mediated by
autophagic adapter proteins. Autophagy 7, 279 –296
4. Ichimura, Y., Kirisako, T., Takao, T., Satomi, Y., Shimonishi, Y., Ishihara,
N., Mizushima, N., Tanida, I., Kominami, E., Ohsumi, M., Noda, T., and
Ohsumi, Y. (2000) A ubiquitin-like system mediates protein lipidation.
Nature 408, 488 – 492
5. Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori,
T., Noda, T., and Ohsumi, Y. (1999) Formation process of autophagosome
is traced with Apg8/Aut7p in yeast. J. Cell Biol. 147, 435– 446
AUGUST 17, 2012 • VOLUME 287 • NUMBER 34
REPORT: Atg1 Interaction with Atg8 via the AIM
6. Nakatogawa, H., Ichimura, Y., and Ohsumi, Y. (2007) Atg8, a ubiquitin-
like protein required for autophagosome formation, mediates membrane
tethering and hemifusion. Cell 130, 165–178
7. Xie, Z., Nair, U., and Klionsky, D. J. (2008) Atg8 controls phagophore
expansion during autophagosome formation. Mol. Biol. Cell 19,
3290 –3298
8. Noda, N. N., Kumeta, H., Nakatogawa, H., Satoo, K., Adachi, W., Ishii, J.,
Fujioka, Y., Ohsumi, Y., and Inagaki, F. (2008) Structural basis of target
recognition by Atg8/LC3 during selective autophagy. Genes Cells 13,
1211–1218
9. Ichimura, Y., Kumanomidou, T., Sou, Y. S., Mizushima, T., Ezaki, J., Ueno,
T., Kominami, E., Yamane, T., Tanaka, K., and Komatsu, M. (2008) Struc-
tural basis for sorting mechanism of p62 in selective autophagy. J. Biol.
Chem. 283, 22847–22857
10. Kamada, Y., Funakoshi, T., Shintani, T., Nagano, K., Ohsumi, M., and
Ohsumi, Y. (2000) Tor-mediated induction of autophagy via an Apg1
protein kinase complex. J. Cell Biol. 150, 1507–1513
11. Kawamata, T., Kamada, Y., Kabeya, Y., Sekito, T., and Ohsumi, Y. (2008)
Organization of the pre-autophagosomal structure responsible for au-
tophagosome formation. Mol. Biol. Cell 19, 2039 –2050
12. Suzuki, K., Kubota, Y., Sekito, T., and Ohsumi, Y. (2007) Hierarchy of Atg
proteins in pre-autophagosomal structure organization. Genes Cells 12,
209 –218
13. Cheong, H., Nair, U., Geng, J., and Klionsky, D. J. (2008) The Atg1 kinase
complex is involved in the regulation of protein recruitment to initiate
sequestering vesicle formation for nonspecific autophagy in Saccharomy-
ces cerevisiae. Mol. Biol. Cell 19, 668 – 681
14. Nakatogawa, H., Ishii, J., Asai, E., and Ohsumi, Y. (2012) Atg4 recycles
inappropriately lipidated Atg8 to promote autophagosome biogenesis.
Autophagy 8, 177–186
15. Noda, T., Matsuura, A., Wada, Y., and Ohsumi, Y. (1995) Novel system for
monitoring autophagy in the yeast Saccharomyces cerevisiae. Biochem.
Biophys. Res. Commun. 210, 126 –132
16. Lynch-Day, M. A., and Klionsky, D. J. (2010) The Cvt pathway as a model
for selective autophagy. FEBS Lett. 584, 1359 –1366
17. Kanki, T., Klionsky, D. J., and Okamoto, K. (2011) Mitochondria au-
tophagy in yeast. Antioxid. Redox Signal. 14, 1989 –2001
18. Suzuki, K., Kondo, C., Morimoto, M., and Ohsumi, Y. (2010) Selective
transport of alpha-mannosidase by autophagic pathways. J. Biol. Chem.
285, 30019 –30025
19. Kondo-Okamoto, N., Noda, N. N., Suzuki, S. W., Nakatogawa, H., Taka-
hashi, I., Matsunami, M., Hashimoto, A., Inagaki, F., Ohsumi, Y., and
Okamoto, K. (2012) Autophagy-related protein 32 acts as autophagic de-
gron and directly initiates mitophagy. J. Biol. Chem. 287, 10631–10638
20. Noda, N. N., Ohsumi, Y., and Inagaki, F. (2010) Atg8 family interacting
motif crucial for selective autophagy. FEBS Lett. 584, 1379 –1385
21. Yeh, Y. Y., Wrasman, K., and Herman, P. K. (2010) Autophosphorylation
within the Atg1 activation loop is required for both kinase activity and the
induction of autophagy in Saccharomyces cerevisiae. Genetics 185,
871– 882
22. Okazaki, N., Yan, J., Yuasa, S., Ueno, T., Kominami, E., Masuho, Y., Koga,
H., and Muramatsu, M. (2000) Interaction of the Unc-51-like kinase and
microtubule-associated protein light chain 3-related proteins in the brain:
possible role of vesicular transport in axonal elongation. Brain Res. Mol.
Brain Res. 85, 1–12
23. Suttangkakul, A., Li, F., Chung, T., and Vierstra, R. D. (2011) The ATG1/
ATG13 protein kinase complex is both a regulator and a target of au-
tophagic recycling in Arabidopsis. Plant Cell 23, 3761–3779
24. Mizushima, N. (2010) The role of the Atg1/ULK1 complex in autophagy
regulation. Curr. Opin. Cell Biol. 22, 132–139
JOURNAL OF BIOLOGICAL CHEMISTRY 28507