13

FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS
OHSUMI, Yoshinori
YOSHIMORI, Tamotsu 11
KAMADA, Yoshiaki
NODA, Takeshi
MIZUSHIMA, Noboru 21
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellow:
YOSHIMOTO, Kohki
JSPS Postdoctral Fellow: SEKITO, Takayuki
SUZUKI, Kuninori
KIRISAKO, Takayoshi n
Postdoctoral Fellow:
ICHIMURA , Yoshinobu
HANADA, Takao
HANAOKA, Hideki 31
Graduate Students:
NARA , Atsuki 11
KUMA, Akiko 41
HAMASAKI, Maho 41
SHOJI, Shisako ' !
ONODERA, fun 41
MJWA , Masami
Technical Assistant:
TSUKESHIBA KumiHARA , Yohko
Secretary
Professor:
Associate Professor:
Research Associates:

11

until Mar 2002
ljrom Apr2002
31
until Jun 2002
41
Graduate University for Advanced Studies
2I. Background
This division aims to understand the autophagy in respectsof its molecular mechanism and its physiological role within
higher eukaryotes. Biosynthetic processes and degradation
processes are well coordinated to regulate the biologicalactivities , however the study on the latter has been retarded
compared to the one on the former and we must shed light on
the degradation process to fully understand the cell . Autophagy is a well conserved degradation process in eukaryotes
and is a major route for bulk degradation of cytoplasmic
constituents in a lytic compartment, lysosome/vacuole.
Upon nutrient starvation, autophagic process starts in thecytoplasm as building up of a membrane structure, an autophagosome, which sequesters a portion of cytosol. Then it isdelivered to the vacuole/lysosome, and the cytoplasmic materials inside are degraded by the vacuolar/lysosomal proteases.
We had discovered autophagy in a simple eukaryotic modelorganism, Saccharomyces cerevisiae and morphologicallydescribed its whole process. We have isolated a set ofautophagy-deficient mutants (apg), and cloned APG genes
essential for autophagy.

plex in the cytosol. Because Apgl6 was suggested to form ahomo-oligomer, we generated an in vivo system that allowed
us to control the oligomerization state of Apg16. With thissystem, we demonstrated that formation of the approximately
350-kDa complex and autophagic activity depended on theoligomerization state of Apgl6. These results suggest that theApg12-Apg5 conjugate and Apg16 form a multimeric complex mediated by the Apg16 homo-oligomer, and formationof the approximately 350-kDa complex is required for autophagy in yeast.

3. Cargo Delivery to the Vacuole Mediated byAutophagosomes
In the yeast Saccharomyces cerevisiae , aminopeptidase I(API) , a vacuolar hydrolase, is selectively transported to thevacuole via the autophagosome. API forms a cytosol to vacuole targeting (Cvt) complex in the cytoplasm. The complex isengulfed by the autophagosome under starvation conditions.
In this study, the Cvt complex is visualized as a dot in thecytoplasm by fluorescence microscopy with API-GFP. TheCvt complex associates with the pre-autophagosomal structure (PAS), which plays a central role in autophagosome
formation. In a cvtl9 mutant, which is specifically defectivein API transport, but not in autophagy, the Cvt complex
forms normally but never associates with the PAS . This indicates that Cvt19p mediates association between the Cvt complex and the PAS.

Figure 1 API-GFP labels the vacuoles and dots close to thevacuole. Wild type cells expressing API-GFP under growing
conditions. (A) API-GFP, (B) vacuoles labeled with FM464, (C) merged image of API-GFP (green) and FM4-64(red), and (D) Nomarski image. Bar: 2 /.1m .

4. Studies of mammalian autophagy
2. Formation of the Apg12-Apg5-Apg16 multimericcomplex is essential for autophagy in yeast.
Autophagy requires a ubiquitin-like protein conjugation
system, in which Apg12 is covalently bound to Apg5. In theyeast Saccharomyces cerevisiae, the Apgl2-Apg5 conjugate
further interacts with a small coiled-coil protein, Apg16. The
Apgl2-Apg5 and Apgl6 are localized in the cytosol and preautophagosomal structures and play an essential role in autophagosome formation. We showed that the Apgl2-Apg5
conjugate and Apgl6 form an approximately 350-kDa com-We cloned mouse ApglO and showed that the Apg12
conjugation system is completely conserved between yeast
and mammals . In addition, we also cloned a novel Apg5interacting protein, Apg16L. Although it was considered tobe a functional counterpart of yeast Apgl6, Apg16L has alarge C-terminal region containing a WD repeat domain thatis absent from yeast Apgl6. This unique feature of Apg16L
will provide new insights into role of Apg12-Apg5 in autophagosome formation.
Once an autophagosome is formed , it matures by fusion

14with endosomes. Using a dominat negative mutant of mouse
SKD1, an AAA ATPase involved in the sorting and transport
from endosomes, we showed that SKD1-dependent endosomal membrane trafficking is required for further fusion withlysosome .
To monitor autophagy in vivo, we generated transgenic
mice expressing a fluorescent marker for autophagosome. It
would be a useful tool for studies on mammalian autophagy.

5. Leaf senescence is accelerated by the disruptionof an Arabi do psis autophagy gene.
The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at thecellular level, but the role that autophagy plays at the organismallevel has yet to be determined. In this study, a genomewide search revealed significant conservation between yeast
and plant autophagy genes. Twenty-five plant genes that arehomologous to 12 yeast genes essential for autophagy werediscovered . We identified an Arabidopsis mutant carrying aT-DNA insertion within AtAPG9, which is the only orthologof yeast Apg9 in Arabidopsis (atapg9 -l). AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9l mutant. Under nitrogen or carbon-starvation conditions,chlorosis was observed earlier in atapg9-l cotyledons androsette leaves compared with wild-type plants. Furthermore,
atapg9-l exhibited a reduction in seed set when nitrogenstarved. Even under nutrient growth conditions, bolting andnatural leaf senescence were accelerated in atapg9-l plants.
Senescence-associated genes SENl and YSL4 were upregulated in atapg9-l before induction of senescence, unlike
in wild type . All of these phenotypes were complemented by
the expression of wild-type AtAPG9 in atapg9-l plants.
These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.

Figure 2 The Arabidopsis during natural leaf senescence.
Wild type, atapg9-J, and atapg9- J plants transformed withwild-type AtAPG9 gene were grown under standard conditions and photographed on 35 day after germination.

NATIONAL INSTITUTE

Original papers :Hamasaki M, Noda T , Ohsumi Y. (2003) The early seretory
pathway contributes to autophagy in yeast. Cell Struct.
Funct. (in press)
Hanaoka H, Noda T, Shirano Y, Kato T , Hayashi H, Shibata
D, Tabata S, Ohsumi Y. (2002) Leaf senescence andstarvation-induced chlorosis are accelerated by thedisruption of an Arabidopsis autophagy gene. Plant
Physiol 129, 1181-1193.
Kuma A, Mizushima N , Ishihara N, Ohsumi Y . (2002) Formation of the approximately 350-kDa Apg12Apg5.Apg16 multimeric complex , mediated by Apg16
oligomerization, is essential for autophagy in yeast. J Bioi
Chem277, 18619-18625.
Mizushima N, Yoshimori T, Ohsumi Y. (2002) Mouse
ApglO as an Apg12-conjugating enzyme: analysis by theconjugation-mediated yeast two-hybrid method. FEESLett. 532,450-454.
Mimura T., Reid R.J., Ohsumi Y., Smith F.A. (2002) Induction of the Na+/Pi co-transport system in the plasma
membrane of Chara corallina requires external Na+ andlow levels of Pi. Plant Cell & Environment 25:1475-1481.
Nara A, Mizushima N, Yamamoto A, Kabeya Y, Ohsumi Y,
Yoshimori T. (2002) SK.Dl AAA ATPase-dependent endosomal transport is involved in autolysosome formation.
Cell Struct Funct. 27, 29-37.
Suzuki K, Kamada Y, Ohsumi Y. (2002) Studies of cargodelivery to the vacuole mediated by autophagosomes in
Saccharomyces cerevisiae. DevCell3, 815-824.
Suzuki T, Nakagawa M, Yoshikawa A, Sasagawa N,Yoshimori T , Ohsumi Y, Nishino I, Ishiura S , Nonaka I.
(2002) The first molecular evidence that autophagy relates
rimmed vacuole formation in chloroquine myopathy J
Biochem (Tokyo). 131 ,647-651.
Reviews:Kamada Y. Sekito T. Ohsumi Y. (2003)Autophgy in yeast- a
Tor-mediated response to nutrient starvation- Curr. Top .
Microbiol.lmmunol. (in press)
Mizushima N, Yoshirnmi T , Ohsumi Y. (2003) Role of theApgl2 conjugation system in mammalian autophagy Int. J.
Biochem. Cell Biol. (in press)
Mizushima N, Ohsumi Y, Yoshimori T. (2002) Autophagosome formation in mammalian cells Cell StructFunct in press
Noda T, Suzuki K, Ohsumi Y. (2002) Yeast autophagosomes: de novo formation of a membrane structure.
Trends Cell Biol. 12,23 1-235
Ohsumi, Y. (2003) Cellular Recycling System- Molecular
Mechanism of Autophagy-.ln Cell Growth Cold Spring
Harbour Press . (in press)