DIVISION OF MOLECULAR CELL BIOLOGY †Professor

OHSUMI, Yoshinori

Assistant Professors:
Technical Staff:
Postdoctoral Fellows:

Graduate Students:
Technical Assistants:

Secretary:

KAMADA, Yoshiaki
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
KABEYA, Yukiko
HANADA, Takao
FUJIKI, Yuki
HARASHIMA, Toshiaki
YAMAMOTO, Hayashi
OKAMOTO, Koji
OKAMOTO, Noriko
KAKUTA, Soichiro
KOBAYASHI, Takafumi
KAGEYAMA, Takuya
OH-OKA, Kyoko
KONDO, Chika
NIIMI, KaoriISHII, Junko
ICHIKAWA, Rie
SUZUKI, Yuko

Most cellular activity is maintained by the balance between
the synthesis and degradation of related proteins.
Degradation processes, therefore, play important roles inmany physiological aspects as well as in the regulation ofgene expression. Autophagy is a bulk degradation system forcytosolic proteins and organelles in lysosomes/vacuoles thatis highly conserved in eukaryotic cells. Our division aims tounderstand the physiological roles and molecularmechanisms of autophagy in yeast and higher eukaryotes.

fraction of mitochondria are exclusively sequestered andtransported to the vacuole, a lytic compartment, in post-log
phase cells under respiratory conditions (Figure 1). Using agenome-wide visual screen, we identified Atg32, a protein
essential for mitophagy in budding yeast. During respiratory
growth, Atg32 is highly expressed, likely in response tooxidative stress, and anchored on the surface ofmitochondria. We also demonstrate that Atg32 interacts withAtg8 and Atg11, proteins critical for recognition of cargo
receptors. Notably, Atg32 contains WXXI/L/V, a conserved
motif that serves as a binding site for the Atg8 family
members. Atg32 exposes its N- and C-terminal domains tothe cytosol and mitochondrial intermembrane space,respectively. We propose that Atg32 is a transmembrane
receptor that directs autophagosome formation tomitochondria.

Figure 1. Electron microscopy reveals electron-dense bodies containing
mitochondria accumulated in vacuoles under respiratory conditions.
Vacuolar protease-deficient cells were grown in non-fermentable mediumfor 5 days. Notably, mitochondria are selectively incorporated into thevacuolar lumen in an Atg32-dependent manner.

I. BackgroundIn response to nutrient starvation, the autophagic process
starts as a building up of membrane structures called
autophagosomes in the cytoplasm. The autophagosome
sequesters a part of the cytosol and organelles nonselectively. It is then delivered to the vacuole/lysosome, andthe cytoplasmic materials inside are degraded by vacuolar/lysosomal hydrolases. We discovered autophagy in a simple
model organism, the budding yeast Saccharomyces
cerevisiae, and have morphologically and genetically defined
the whole process.

II. Mitochondria-specific autophagy requires
the transmembrane receptor Atg32

Autophagy-dependent degradation of mitochondria is afundamental process conserved from yeast to humans. In
contrast to starvation-induced, nonselective autophagy
responsible for nutrient recycling, selective autophagy, which
involves particular cues and receptors required for induction
and cargo recognition, respectively, mediates mitochondriaspecific breakdown. Although numerous studies highlightthat mitochondria-specific autophagy (mitophagy)
contributes to mitochondrial homeostasis, the molecularmechanisms underlying this selective clearance process arevery poorly understood. We establish that a substantialIII. A novel physiological role of autophagy inplants: plant autophagy puts the brakes oncell death by controlling salicylic acidsignaling

It has long been thought that autophagy in plants isimportant for nutrient recycling and plays a critical role in
the ability of plants to adapt to environmental variation suchas nutrient deprivation. Recent reverse genetic studies,
however, hint at other roles for autophagy, showing that
autophagy defects in higher plants result in early senescenceand excessive immunity-related programmed cell death(PCD), irrespective of nutrient conditions. Until now themechanisms by which cells die in the absence of autophagy
were unclear. In collaboration with RIKEN Plant Science
Center research groups, using biochemical, pharmacologicaland genetic approaches, we revealed a conserved
requirement for salicylic acid (SA) signaling for these
phenomena in autophagy-defective mutants (atg mutants).
The atg mutant phenotypes of accelerated PCD in senescenceand immunity were suppressed by inactivation of SA
signaling but not by inactivation of jasmonic acid (JA) orethylene (ET) signalings (Figure 2). Application of an SA
agonist restored the senescence/cell death phenotype in†: This laboratory was closed on 31 March, 2009.

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National Institute for Basic Biology

Cell Biology

SA-deficient atg mutants but not in atg npr1 plants,suggesting that the cell death phenotypes in the atg mutants
are dependent on the SA signal transducer NPR1. We also
showed that the SA signal can induce autophagy in plants.
These findings demonstrate a novel physiological function
for plant autophagy that operates a negative feedback loop tomodulate SA signaling.

reactions catalyzed by Atg7 and Atg3, E1 and E2 enzymes,respectively. Atg4 also serves as a deconjugation enzyme forAtg8; it cleaves the Atg8-PE conjugate and releases Atg8
from membranes. The crystal structure of LC3 (a mammalianAtg8 homolog) complexed with Atg4B (a mammalian Atg4homolog) has been determined. This provided insights intoFigure 2. Accelerated PCD phenotypes in autophagy-defective plants are
SA signaling dependent but do not require intact JA and ET signaling
pathways. The early senescence phenotype of atg5 mutant wassuppressed by reducing SA biosynthesis in sid2 mutant background, or byblocking SA signaling in npr1 mutant background. Shown arephotographs of five-week-old plants grown on rockwool supplied with arich nutrient solution under long-day conditions.

IV. Characterization of the Atg17-Atg29-Atg31
c o m p l e x s p e c i f i c a l l y re q u i re d f o r
starvation-induced autophagy in
Saccharomyces cerevisiae
In the yeast, Saccharomyces cerevisiae, Atg17, Atg29, andAtg31/Cis1 are specifically required for autophagosome
formation by acting as a scaffold complex essential for preautophagosomal structure (PAS) organization. We show thatthese proteins constitutively form an Atg17-Atg29-Atg31
ternary complex, in which phosphorylated Atg31 is included(Figure 3). Reconstitution analysis of the ternary complex inE. coli indicates that the three proteins are included inequimolar amounts in the complex. The molecular mass of amonomeric Atg17-Atg29-Atg31 complex is calculated at 97kDa; however, analytical ultracentrifugation shows that themolecular mass of the ternary complex is 198 kDa,
suggesting a dimeric complex. We propose that this ternarycomplex acts as a functional unit for autophagosomeformation.

Figure 3. Atg17, Atg29, and Atg31 form a stable complex in vivo. Wildtype cells (BY4741) were grown and treated with rapamycin for 1 h.
After cells were converted to spheroplasts, cell lysates were prepared byosmotic lysis. Cytosolic fractions (100,000 x g supernatant, HSS) wereseparated by size exclusion chromatography on a Superdex 200 column.
Each fraction was analyzed by immunoblotting using anti-Atg1, antiAtg17, anti-Atg29, and anti-Atg31antibodies. Positions of molecularmass standards (in kDa) are shown. Open and closed circles indicate
dephosphorylated and phosphorylated Atg31, respectively.

V. S t r u c t u r a l s t u d i e s o f A t g p ro t e i nconjugation systems

Atg proteins contain two ubiquitin-like protein conjugation
systems. In collaboration with Dr. Inagaki’s group at
Hokkaido University, understanding of these systems has
proceeded from the viewpoint of structural biology. The
ubiquitin-like protein Atg8 is synthesized as a precursor form
with an arginine at the C terminus, which is immediately
removed by the cysteine protease Atg4 to expose a glycine
residue at the new C terminus. Then, this glycine of Atg8 isconjugated to the lipid PE (phosphatidylethanolamine) via

12Figure 4. Model of the Atg12-Atg5-Atg16 complex.

the mechanisms of the C-terminal processing anddelipidation of Atg8 by Atg4, especially for molecular
recognition and regulation in these cleavage reactions.
We previously showed that the conjugation reaction of Atg8
is accelerated by a conjugate formed by the other ubiquitinlike protein Atg12 and its target protein Atg5. The Atg12Atg5 conjugate further forms a complex with Atg16, whichis required for the localization of this complex to the PAS.
The crystal structure of Atg16 has been solved, which
revealed that Atg16 forms a parallel coiled-coil dimer.
Together with the previously-reported structures of Atg12and Atg5, the entire architecture of the Atg12-Atg5-Atg16
complex was modeled (Figure 4). This provided an important
structural basis to further elucidate the function of thecomplex and the mechanism of autophagosome formation.

〔Original papers (E-publication ahead of print)〕

Y., Noda, N.N., Nakatogawa, H., Ohsumi, Y., and Inagaki, F.
• Fujioka,
The dimeric coiled-coil structure of Saccharomyces cerevisiae Atg16
and its functional significance in autophagy. J. Biol. Chem. 2009 Nov 4.

Y., Yoshino, K., Kondo, C., Kawamata, T., Oshiro, N.,• Kamada,
Yonezawa, K., and Ohsumi, Y. Tor directly controls the Atg1 kinase
complex to regulate autophagy. Mol. Cell Biol. 2009 Dec 7.

〔Review articles〕

H., Suzuki, K., Kamada, Y., and Ohsumi, Y. (2009).
• Nakatogawa,Dynamics and diversity in autophagy mechanisms: lessons from yeast.
Nat. Rev. Mol. Cell Biol., 10, 458-467.

N.N., Ohsumi, Y., and Inagaki, F. (2009). ATG systems from the• Noda,protein structural point of view. Chem. Rev., 109, 1587-1598.
K., Kondo-Okamoto, N., and Ohsumi, Y. (2009). A landmark• Okamoto,protein essential for mitophagy. Autophagy, 5, 1203-1206.

Publication List

〔Original papers〕

T., Satomi, Y., Takao, T., and Ohsumi, Y. (2009). The amino• Hanada,terminal region of Atg3 is essential for association with•••••••••

phosphatidylethanolamine in Atg8 lipidation. FEBS Lett., 583, 10781083.
Kabeya, Y., Noda, N.N., Fujioka, Y., Suzuki, K., Inagaki, F., andOhsumi, Y. (2009). Characterization of the Atg17-Atg29-Atg31
complex specifically required for starvation-induced autophagy in
Saccharomyces cerevisiae. Biochem. Biophys. Res. Comm., 389, 612615.
Kageyama, T., Suzuki, K., and Ohsumi, Y. (2009). Lap3 is a selectivetarget of autophagy in yeast, Saccharomyces cerevisiae. Biochem.
Biophys. Res. Comm., 378, 551-557.
Okamoto, K., Kondo-Okamoto, N., and Ohsumi, Y. (2009).
Mitochondria-anchored receptor Atg32 mediates degradation ofmitochondria via selective autophagy. Dev. Cell, 17, 87-97.
Satoo, K., Noda, N.N., Kumeta, H., Fujioka, Y., Mizushima, N.,
Ohsumi, Y., and Inagaki, F. (2009). The structure of Atg4B-LC3complex reveals the mechanism of LC3-processing and delipidation
during autophagy. EMBO J., 28, 1341-1350.
Sekito, T., Kawamata, T., Ichikawa, R., Suzuki, K., and Ohsumi, Y.
(2009). Atg17 recruits Atg9 to organize the pre-autophagosomalstructure. Genes Cells, 14, 525-538.
Shin, J.H., Yoshimoto, K., Ohsumi, Y., Jeon, J.S., and An, G. (2009).
OsATG10b, an autophagosome component, is needed for cell survival
against oxidative stresses in rice. Mol. Cells., 27, 67-74.
Wada, S., Ishida, H., Izumi, M., Yoshimoto, K., Ohsumi, Y., Mae, T.,
and Makino, A. (2009). Autophagy plays a role in chloroplastdegradation during senescence in individually darkened leaves. Plant
Physiol., 149, 885-893.
Watanabe, Y., Noda, N.N., Honbou, K., Suzuki, K., Sakai, Y., Ohsumi,Y., and Inagaki, F. (2009). Crystallization of Saccharomyces cerevisiaealpha-mannosidase, a cargo protein of the Cvt pathway. Acta
Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 65, 571-573.
Yoshimoto, K., Jikumaru, Y., Kamiya, Y., Kusano, M., Consonni, C.,
Panstruga, R., Ohsumi, Y., and Shirasu, K. (2009). Autophagy
negatively regulates cell death by controlling NPR1-dependent salicylicacid signaling during senescence and the innate immune response inArabidopsis. Plant Cell, 21, 2914-2927.

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