FOR BASIC BIOLOGY13

DIVISION OF BIOENERGETICS
Professor:
Associate Profes sor:
Research Associates:
Technical Staff:
Postdoctoral Fellow:

Graduate Students:

Visiting Scientists:

OHSUMI, Yoshinori
YOSHIMORJ, Tamotsu
KAMADA, Yoshiaki
NODA, Takeshi
KABEYA, Yukiko
MIZUSHIMA, Noboru n
KIRISAKO, Takayoshi 21
SEKITO,Takayuki 131
YOSHIMOTO, Kohki 4!
ICHIMURA, Yoshinobu 51
SUZUKI, Kuninori 51
KOBAYASHI, Yoshinori 5•71
NARA, Atsuki 51
HANAOKA, Hideki 51
KUMA. Akiko 51
HAMASAKI, Mah o 51
SHOJI, Shisako 51
ONODERA,Jun ,5·61
OKAMOTO, Satsukiacterized of the gene product. We drew a rough functional map of each Apg proteins this year. This is basedon the finding of novel structure called preautophagosomal structure (PAS). Many Apg proteins (Apgl ,Apg2, Apg5 , Apg8 , Apgl6) are colocalized on preautphagosomal structure. Autophagy-specific phosphatidylinositol 3-kanise complex (Apg6, Apgl4, Vps34 andVpsl5) are essential for organization of PAS. Apg8,ubiqitin-like protein, gathers to PAS depending on lipidation reaction and the Apg5-Apg 12 conjugates. Apg5
is recruited to PAS depending on Apgl6 but independent on Apgl2-conjugation. Apg2 is localized on
PAS via Apgl. Apgl protein kinase function in transition from PAS to autophagosome. Thus, PAS is theorganizing center of autophagosome.

n PRESTO, JST, Oct 1999-.
21

fromApr2001-.
from Feb2001-.
41
from Sep2001-.
51
Graduate University for Advanced Studies.
61
from Apr2001 .
71
til Oct 2001
31

This division aims to understand the autophagy in respects to its molecular mechanism and its physiologicalrole in higher eukaryotes. Cells execute degradation
processes of their constituents together with biosynthetic processes. These two processes are well coordinated to regulate the biological activities. In other word,
we must shed light on degradation process to fully understand the cell, because the study on the degradation
has been retarded compared to the bio-synthetic process.
Autophagy is well conserved in eukaryotes and is amajor route for bulk degradation of cytoplasmic constituents and organelles in a lytic compartment,
lysosome/vacuole.

I. Background
Upon nutrient starvation, autophagic process starts asbuilding up a membrane structure, an autophagosome,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/lysosomal proteases.
We had discovered autophagy in a simple eukaryotic
model organism, Saccharomyces cerevisiae · and morphologically defined the whole process. We have isolated a set of autophagy-deficient mutants (apg ), and havecloned most of the APG genes essential for autophagy.
We are now characterizing these gene products andtheir homologues in mammals and higher plants.

2. Discovery of novel preautophagosomal structure
We have cloned the last APG genes, APG2, and char-Fig.! Discovery of preautophagosomal structure
Most Apg proteins are gathered on preautophagosmal structure. Based on this finding, function al role of each Apg protein
can be discussed in detail.

3. Autophagosome formation depends on specificsets of coat proteinsWe revealed that autophagosme fuses with the vacuole by NSF/SNARE meditated mechanism, which iscommon through endmembrane system, such as ER andGolgi. However, autophagosome formation is not dependent of NSF. Instead, the involvement the proteinsof early secretory pathway are revealed. Secl2, Secl6,
Sec23, Sec24 are required for autophagosome formation .
Interestingly, they are not prerequisite for Cvt pathway,
the similar pathway to autophagy. The findings may
imply that early phase of autophagosome formation isrelated to secretory pathway in some way .

4. Molecular dissection of autophagosome formation
Apgl2 and Apg5 are covalently attached in a manner
similar to the ubiquitin conjugation system. The Apg12Apg5 conjugation system is well conserved in mammalian cells. Part of the mammalian Apg12-Apg5 conjugate localized to the isolation membranes when autophagy proceeds, whereas most of it existed in the cytoplasm. Using GFP-tagged Apg5 , we revealed that the14

NATIONAL INSTITUTE

cup-shaped isolation membrane is developed from asmall crescent-shaped compartment. Apg5 localized on
the isolation membrane throughout its elongation process. Apg5 was preferentially distributed in the outer sideof the membrane and detaches from it immediately
before or after autophagosome formation is completed.
In contrast, LC3, the mammalian homologue of Apg8,
was associated with both sides of isolation membranes
and autophagosomes as well as their precursors. To
examine the role of Apg5, we generated Apg5-deficient
ES cells. APG5 + cells are viable but bulk protein degradation was significantly reduced. Autophagosome
formation was impaired in these cells. The mutant
Apg5K 130R, which is unable to be conjugated with Apgl2,could bind to the autophagosome precursors but could
not start elongation of the membranes. Thus, the covalent modification of Apg5 with Apg12 was not requiredfor its membrane targeting but is essential for involvement of Apg5 in elongation of the isolation membranes.
Intriguingly, Apg12-Apg5 was required for processingof LC3 and its targeting to the membranes. Therefore,
the Apg12-Apg5 conjugate plays essential roles in isolation membrane development in co-operation with LC3.
In addition, our studies provided good molecular markers, LC3 and Apg12-Apg5, for autophagic membrane at
all stages and isolation membranes, respectively, which
so far have been defined only by morphology.

5. Production of phosphatidylinositol 3-phosphate(PI3P) at the trans-Golgi network (TGN) is required for autophagy
This year, we reported that, in yeast, Vps34 PI3kinase and Apg6 form a protein complex that plays aessential role in autophagy by producing PI3P. Then,
we found in mammalian cells that the complex of the
mammalian Apg6 homologue, beclin and PI3-kinase
was distributed in the TGN rather than autophagosomes(Figure 2A). We propose that beclin functions as aregulatory-subunit of the PI3-kinase complex at theTGN, which supplies PI3P for autophagosome formation (Figure 2B).
A

8Fig.2A. Beclin localizes in the TGN.
B. Model for beclin function. The beclin-PI3-kinase complex
localized at the TGN supplies PBP to the membranes in theautophagic pathway.

Original papers :Ishihara, T., Hamasaki, M., Yokota, S., Suzuki, K.,
Kamada, Y., Kihara, A., Yoshimori, T., Noda, T.,
and Ohsumi, Y. (2001) Autophagosome requires
specific early Sec proteins for its formation andNSF/SNARE for its fusion to the vacuole. Mol. Bioi.
Cell12, 3690-3702.
Kim, J., Kamada, Y., Stromhaug, P. E. Guan, J., Hefner-Gravinnk, A., Bevan, A., Scott, S. V., Ohsumi, Y.,Dunn, Jr., W. A., Klionsky, D. J. (2001) Cvt9/Gsa9function in sequestering selective cytosolic cargo
destined for the vacuole. J. Cell. Bioi. 153, 381-396.
Kihara, A., Noda, T., Ishihara, N. and Ohsumi, Y.
Two distinct Vps34 Ptdlns 3-kinase complex functionin autophagy and CPY. (2001) sorting in Saccharomyces cevisiae. J. Cell. Biol. 152, 519-530.
Kihara, A., Kabeya, Y., Ohsumi, Y., and Yoshimori, T.
(2001) Beclin-phosphatidylinositol 3-kinase complex
functions at the trans-Golgi Network. EMBO Report,2, 330-335.
Komtatsu, M., Tanida, I., Ueno, T., Ohsumi, M., Ohsurni, Y., and Kominami, E. (2001) The C-terminal
region of an Apg7p/Ctv2p is required for homodimerization and is essential for its E1-activity and E1E2 complex-formation. E. J. Biol. Chern. 276, 98469854.
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi,Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y
and Yoshimori, T. (2001) Dissection of autophagosome formation using Apg 5-deficient mouseembryonic stem cells. J. Cell. Bioi. 152, 657-667.
Shintani, T., Suzuki,K., Kamada, Y., Noda, T., Ohsumi,Y. (2001)Apg2p functions in autophagosome formation on the perivacuolar structure. J. Bioi. Chern. 276,30452-30460.
Suzuki, K., Kirisako, T., Kamada, Y. , Mizushima, N.,
Noda, T. and Ohsumi, Y. (2001) The preautophagosomal structure organized by concertedfunctions of APG genes is essential for autophagosome formation. EMBO. J. 20,5971-5981.

Review:
Ohsumi, Y. (2001) Molecular dissection of autophagy:
Two ubiquitin-lke systems. Nature Reviews,
Molecular Cell Bioi, 2, 211-216.