NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGYautophagy. We also found that two hydrophobic residues
within the ubiquitin-fold region are important forautophagy: mutations at Y149 affected conjugate
formation catalyzed by Atg10, an E2-like enzyme, while
mutations at F154 had no effect on Atg12-Atg5 conjugate
formation but its hydrophobic nature was essential forautophagy. In the cells expressing F154 mutants, Atg8-PE
conjugation, the other ubiquitin-like conjugation inautophagy, was severely reduced and autophagosome
formation failed. A gel filtration analysis suggested that
F154 plays a critical role in the assembly of a functional
Atg12-Atg5·Atg16 complex requisite for autophagosome
formation.
Crystal structure of Arabidopsis thaliana Atg12
homolog (AtATG12) has shown that it adopts theubiquitin-like fold (Figure 1). Residues Y57 and F62,
corresponding to Y149 and F154 in yeast, respectively,
were found to be located adjacent to each other but form
different hydrophobic patches, suggesting Atg12 utilizes
two different hydrophobic patches for autophagy: one for
conjugation reaction and the other for autophagosomeformation.

DIVISION OF MOLECULAR CELL BIOLOGY
Professor:
Research Associates:

Technical Staff:
NIBB Research Fellow:
Postdoctoral Fellows:

Graduate Students:
Visiting Scientists:
Technical Assistants:
Secretary:

OHSUMI, Yoshinori
KAMADA, Yoshiaki
NODA, Takeshi
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
KABEYA, Yukiko
OBARA, Keisuke
ICHIMURA, Yoshinobu
SEKITO, Takayuki
YOSHIMOTO, Kohki
HANADA, Takao
FUJIKI, Yuki
OHNEDA, Mamoru
OITA, Eiko
ONODERA, Jun
OKU, Masahide
HARASHIMA, Toshiaki
MATSUI, Makoto
KAGEYAMA, Takuya
BABA, Misuzu
KAWAMATA, Tomoko
TSUKESHIBA, Kumi
ICHIKAWA, Rie
KONDO, Chika
HARA, Yoko

This division aims to understand the physiologicalroles and molecular mechanism of autophagy in yeast andhigher eukaryotes. All cellular activity is maintained by
the balance between the synthesis and degradation ofrelated proteins. It is now well known that the degradation
process plays important roles in many physiologicalaspects. Autophagy is a bulk degradation system ofcytosolic proteins and organelles in lysosome/vacuoles.
Membrane dynamics during autophagy remain to bediscovered.

I. Background
Upon nutrient starvation, the autophagic process starts
as a building up of membrane structures(autophagosomes) in the cytoplasm. The autophagosome
sequesters cytosol and organelles nonselectively. Then it
is delivered to the vacuole/lysosome, and the cytoplasmic
materials inside are degraded by vacuolar/lysosomalhydrolases. We discovered autophagy in a simple modelorganism, Saccharomyces cerevisiae and morphologicallyand genetically defined the whole process.

Figure 1. Two hydrophobic residues important for
autophagy are located in close vicinity but form differenthydrophobic patches.

III. Atg17 plays a pivotal role in autophagosome
formation directed by Atg1 kinase activity
In the yeast S. cerevisiae, most of ATG genes areinvolved in not only the process of degradative autophagy,
but also a biosynthetic process of vacuolar enzymes. In
contrast, the ATG17 gene is required specifically inautophagy. We found that the atg17∆ mutant understarvation condition was severely impaired in
autophagosome formation. Autophagosomes with asmaller number and size were observed in the atg17∆
cells (Figure 2). We showed that Atg17 physicallyassociated with an Atg1-Atg13 complex, and that it wasenhanced under starvation conditions. The complex
formation resulted in upregulation of an Atg1 kinase

II. Hydrophobic residues in a ubiquitin-fold ofAtg12 are important for autophagy in yeast
Atg12, a post-translational modifier, is activated andconjugated to Atg5 by a ubiquitin-like conjugation system,
though it has no obvious sequence homology to ubiquitin.
The carboxyl-terminal region of Atg12 is predicted to foldinto a ubiquitin-like structure. We constructedamino-terminally truncated Atg12 mutants of the yeast S.
cerevisiae according to the predicted secondary structureand showed that the ubiquitin-fold region of Atg12 isnecessary and sufficient for both conjugation and9NATIONAL INSTITUTE FOR BASIC BIOLOGCELL BIOLOGY

activity, suggesting that Atg17 regulates the Atg1 kinase
activity in concert with Atg13. Taken together, theseresults indicated that Atg17-Atg13 complex formation
plays an important role in normal autophagosome
formation via binding to and activating the Atg1 kinase.

V. Autophagy is essential for protein synthesis
under nitrogen starvation
Autophagy is involved in degradation of cytoplasmic
components including a significant amount of proteins,rRNA and phospholipids of organelle membranes. So far,the precise fate and the physiological importance of eachdegradation products generated by autophagy had been
unclear. Recently, we examined changes in protein
synthesis after long term nitrogen starvation and foundthat a heat shock protein (Hsp26p) and an enzyme
involved in an amino acid biosynthesis (Arg1p) wereup-regulated by nitrogen starvation in wild-type cells, butthese changes did not occur in atg mutant cells (Figure4A). The mRNA levels in the atg mutant cells were ashigh as those in the wild-type cells under nitrogenstarvation (Figure 4B). Thus, it was suggested thatsynthesis of these proteins was inhibited at thetranslational step. We also found that bulk protein
synthesis was substantially reduced in the atg mutant cells
under nitrogen starvation compared with the wild-typecells. The wild-type cells maintained amino acid levels tosurvive under starvation conditions. In contrast, the total
intracellular amino acid pool was reduced in the atg
mutant cells, and the levels of several amino acids fell
below critical values. These results indicate that
autophagy is required to maintain physiological aminoacid levels under nitrogen starvation conditions, andinability to synthesize proteins newly may explain mostphenotypes in autophagy-defective mutants (e.g. inabilityof spore formation and so on).

WT

atg17∆VV

Figure 2. ATG17 is essential for normal autophagosome
formation. In a nitrogen-starvation medium, theatg17∆ mutantgeneratesfewer
andsmaller
autophagosomes (arrowheads) than those in the wild-type.
Bar: 200 nm. V, vacuole.

IV. Identification and characterization of a novel
autophagy-specific gene, ATG29
At present, at least 16 autophagy-related genes (ATG)
have been identified. To search for novel genes involvedin autophagy, we performed a genome-wide screen using
a collection of ~4,500 yeast single-gene deletion mutants
and identified a novel ORF, ATG29/YPL166w. atg29∆
cells were sensitive to starvation, and induction ofautophagy was severely retarded. However, the Cvt(Cytoplasm to vacuole transport) pathway, which sharesmechanistic components with autophagy, operated
normally. Therefore, Atg29 is the second protein
specifically required for autophagy. Furthermore weobserved that an Atg29-GFP fusion protein was localized
to the PAS, at which most Atg proteins are colocalized(Figure 3). From these results, we propose that Atg29
functions in autophagosome formation at the PAS incollaboration with other Atg proteins.

Figure 4. Autophagy is required for the expression ofnitrogen starvation-induced proteins at the translational
level. (A) Immuno-blot analysis of starvation-inducedproteins, Arg1p and Hsp26p. (B) Northern-blot analysisof starvation-induced gene, ARG1 and HSP26. ADH andrRNA were used as loading controls.

Figure 3. Localization of Atg29 at the PAS. Growing (0 h)and rapamycin treated (3 h) cells were observed byfluorescence microscopy. Atg8 and API were used as
markers for the PAS.

10

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

death. J. Biol. Chem. 280, 20722-20729.
Sugawara, K., Suzuki, N.N., Fujioka, Y., Mizushima, N.,
Ohsumi, Y., and Inagaki, F. (2005). Structural basis forthe specificity and catalysis of human Atg4B responsible
for mammalian autophagy. J. Biol. Chem., in press.
Suzuki, N.N., Yoshimoto, K., Fujioka, Y., Ohsumi, Y., andInagaki, F. (2005). The crystal structure of plant ATG12and its biological implication in autophagy. Autophagy 1,119-126.

VI. Autophagy plays important roles in various
aspects of plant life
By establishing an autophagy-monitoring system in awhole plant we were able show that atatg mutants weredefective in autophagy. Our recent studies indicated thatthe autophagy defective mutants exhibited a reduction in
the growth rate of roots under nitrogen-starved conditions
and early senescence phenotype even in nutrient-richconditions. In addition, hypersensitive response cell
death was accelerated in the mutants during the plant
innate immune response. These results indicate that,regardless of nutrient conditions, autophagy plays
important roles in various aspects in plant life.

Review article
Bassham, D.C., Laporte, M., Marty, F., Moriyasu, Y.,
Ohsumi, Y., Olsen, L.J., and Yoshimoto, K. (2005).
Autophagy in development and stress responses of plants.
Autophagy, 2, in press.

Publication List:

Original papers
Hamasaki, M., Noda, T., Baba, M., and Ohsumi, Y. (2005).
Starvation triggers the delivery of the endoplasmic
reticulum to the vacuole via autophagy in yeast. Traffic
6, 56-65.
Hanada, T., and Ohsumi, Y. (2005). Structure-Function
relationship of Atg12, a ubiquitin-like modifier essential
for autophagy. Autophagy 1, 110-118.
Kabeya, Y., Kamada, Y., Baba, M., Takikawa, H., Sasaki, M.,and Ohsumi, Y. (2005). Atg17 functions in cooperation
with Atg1 and Atg13 in yeast autophagy. Mol. Biol. Cell16, 2544-2553.
Kamada, Y., Fujioka, Y., Suzuki, N.N., Inagaki, F.,
Wullschleger, S., Loewith, R., Hall, M.N., and Ohsumi, Y.
(2005). Tor2 directly phosphorylates the AGC kinase
Ypk2 to regulate actin polarization. Mol. Cell Biol. 25,7239-7248.
Kawamata, T., Kamada, Y., Suzuki, K., Kuboshima, N.,Akimatsu, H., Oota, S., Ohsumi, M., and Ohsumi, Y.
(2005). Characterization of a novel autophagy-specificgene, ATG29. Biochem. Biophys. Res. Comm., in press.
Komatsu, M., Waguri, S., Ueno, T., Iwata, J., Murata, S.,Tanida, I., Ezaki, J., Mizushima, N., Ohsumi, Y.,
Uchiyama, Y., Kominami, E., Tanaka, K., and Chiba, T.
(2005). Impairment of starvation-induced and constitutiveautophagy in Atg7-deficient mice. J. Cell Biol. 169,425-434.
Matsui, M., Yamamoto, A., Kuma, A., Ohsumi, Y., andMizushima, N. (2005). Organelle degradation during thelens and erythroid differentiation is independent ofautophagy. Biochem. Biophys. Res. Comm., in press.
Onodera, J., and Ohsumi, Y. (2005). Autophagy is required
for maintenance of amino acids levels and protein
synthesis under nitrogen starvation. J. Biol. Chem. 280,31582-31586.
Pyo, J.O., Jang, M.H., Kwon, Y.K., Lee, H.J., Jun, J.I., Woo,H.N., Cho, D.H., Choi, B., Lee, H., Kim, J.H., Mizushima,N., Ohsumi, Y., and Jung, Y.K. (2005). Essential roles ofAtg5 and FADD in autophagic cell death: dissection ofautophagic cell death into vacuole formation and cell11