FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS

Professor:
Research Associates:

OHSUMI, Yoshinori
KAMADA, Yoshiaki
NODA, Takeshi 11
MIZUSHIMA, Noboru
SUZUKI, Kuninori
Technical Staff:
KABEYA, Yukiko
NIBB Research Fellows: YOSHIMOTO, Kohki
FUJIK/, Yuki
JSPS Postdoctoral Fellows: SEKITO, Takayuki
HANADA, Takao
HAMASAKI, Maho
Postdoctoral Fellows:
ICHIMURA, Yoshinobu
KUMA, Akiko (~May 2003)
ICHIKAWA, Hisashi
KANAZAWA, Takumi
BABA, Misuzu
ONODERA, Jun 2)
Graduate Students:
KAWAMATA, Tomoko 3!
MATSUI, Makoto 2 )Technical Assistants:
MIWA, Mas ami
IMAMURA Yuko
TAKEUCHI, Yuka
TSUKESHIBA Kumi
HARA, Yoko
Secretary:
Study abroad from Mar. 2003
Graduate University for Advanced Studies
J) KOBE Univ.
/J2!

This division aims to understand the autophagy inrespects to its molecular mechanism and its
physiological role in yeast and higher eukaryotes. Cells
execute degradation processes of their constituents
together with biosynthetic processes. These twoprocesses are well coordinated to regulate the biologicalactivities. In other word, we must shed light ondegradation process to fully understand the cell,because the study on the degradation has been retarded
compared to the biosynthetic process. Autophagy is amajor route for bulk degradation of cytoplasmic
constituents and organelles in lysosome/vacuole, and iswell conserved in eukaryotes.

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 .i'sdelivered to the vacuole/lysosome, and the cytoplasmic
materials inside are degraded by vacuolar/lysosomalhydrolases. We had discovered autophagy in a simple
model organism, S. cerevisiae and morphologically andgenetically defined the whole process.

U. In vitro reconstitution of Atg8-PE conjugation
systemIn yeast, S. cerevisiae, Atg8 plays an important role
during autophagosome formation. We have previously
reported that the Atg8 is covalently attached tophosphatidylethanolamine (PE) via a ubiquitin-likeconjugation system. The C-terminal Arg of newly

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synthesized Atg8 (Atg8R" 7 ) is removed by Atg4
protease to expose a Gly residue at the C-terminus(Atg8° 116). The Apg8°" 6 is then activated by Atg7 (Elenzyme) and transferred to Atg3 (E2 enzyme).
Following these reactions, the Apg8° 116 conjugates to PE
through an amide bond between its C-terminal Gly andthe amino group of PE. The subsequent deconjugation
reaction by Atg4 is necessary for the normal
progression of autophagy. We developed in vitro Atg8PE reconstitution system. The Atg8-PE wassuccessfully reconstituted simply with Atg8° 11 ", Atg7and Atg3 by using in vivo in E. coli and in vitro system.
These results suggested that Atg7 and Atg3 arenecessary and sufficient for the Atg8-PE conjugation
reaction. The in vitro Atg8-PE reconstitution systemusing recombinants and liposomes demonstrated thatthe efficiency of Atg8-PE conjugation was strongly
affected by lipid composition. Further, the Atg8 waslinked to the PE in liposomes, but not to the PE in thepresence of detergent, suggesting that the lipid bilayerof membrane is essential for the Atg8-PE conjugation.

III. Molecular dissection of Atg12
Atgl2 is activated by Atg7, transferred to AtglO andattached to Atg5 in a manner similar to theubiquitination. Although Atgl2 has scarcely sequencesimilarity with ubiquitin, its secondary structure waspredicted to have ubiquitin-like domain in the Cterminus region. We prepared N-terminally truncatedAtgl2 mutant of the yeastS. cerevisiae according to thepredicted secondary structure. Truncated form of Atgl2
having only a predicted ubiquitin-like domain
conjugated with Atg5 was still active in autophagy.
While a truncated Atg12 mutant lacking the first beta
strand in the ubiquitin-like domain didn't conjugatewith Atg5. These results showed that the ubiquitin-likedomain of Atgl2 is necessary and sufficient for
conjugation and autophagy. Furthermore, we altered
several hydrophobic amino acid residues in theubiqutin-like domain of Atg8 and found a certain aminoacid residue is critical for autophagy, even though still
active for conjugation with Atg5.

IV. Preferential degradation by autophagy
In contrast to the ubiqutin/proteasome pathway,
autophagy is thought to be non-selective protein
degradation process. We surveyed the changes ofprotein profiles after nitrogen starvation of wild-type
and !1atg7 cells using 2D-PAGE, and we found thatcytosolic acetaldehyde dehydrogenase (Ald6p; FigureI) was degraded more rapidly than other cytosolic
proteins in an autophagy dependent manner. We
observed that Ald6p was enclosed in theautophagosome and delivered to the vacuole
preferentially. The Ald6p may be harmful to cells
during nitrogen starvation, since disruption of ALD6
improved the loss of viability of the !1atg7 mutant. Thisis the first report of the preferential degradation of a
detrimental protein via autophagy.

NATIONAL INSTITUTE

12Nitrogen starvation 24 hWild-typet:.atgl•Ald6pFigure I. Ald6p was degraded by autophagy. Protein
spots from 2D-PAGE using soluble lysates .of nitrogenstarved cells were shown. Ald6p, cytosolic acetaldehydedeh)'drogenase; Adhlp, alcohol dehydrogenase (cmmol).

V. Involvement of early secretory pathway
In addition to Atg proteins , we have previously
identified the involvement of secretory proteins inautophagy. Autophagosome formation is completely
blocked when some early Sec proteins such as Sec24p, aredefective , which are involved in the formation of theCOPII coated vesicles from ER to Golgi. The autophagic
defect in sec24 deleted mutant cells was, however ,suppressed upon the recovery of ER-Golgi secretory flowby the overexpression of its homologue, Sfb2p. We foundthat the autophagic defect is not observed in sec13 andsec31 mutants, a phenomenon that can be explained by thefact that starvation stress suppresses the secretory defect ofthese mutants . These observations indicate that the active
flow in the early secretory pathway plays an important rolein autophagy. Both autophagy and its closely related
cytoplasm to vacuole-targeting (Cvt) pathway occur
through a pre-autophagosomal structure (PAS), and since
the PAS and the functional Cvt pathway exist in all sec
mutants, the early secretory pathway must be involved
specifically in autophagy, subsequent to PAS formation .
23 oc +Rap

37 oc +Rap

ubiquitin-like system, is essential for autophagosome
formation. In yeast, the Atgl2-Atg5 conjugate interacts
with a small coiled-coil protein , Atgl6, to form a ~350kDa multimeric complex . We demonstrate that the mouse
Atgl2-Atg5 conjugate forms a ~800-kDa protein complex
containing a novel WD repeat protein (Fig. 3). As the Nterminal region of this novel protein shows homology withyeast Atgl6, we have designated it mouse Atgl6-likeprotein (Atgl6L). Atgl6L, however , has a large Cterminal domain containing seven WD repeats , absent
from yeast Atgl6. Atg16L interacts with both AtgS andadditional Atgl6L monomers ; neither interaction ,however, depends on the WD-repeat domain. In
conjunction with Atgl2-Atg5 , Atgl6L associates with theautophagic isolation membrane for the duration ofautophagosome formation. As these features are similar toyeast Atgl6 , we concluded Atgl6L is the functionalcounterpart of the yeast Atgl6 . We also found that
membrane targeting of Atgl6L requires Atg5 , but not
Atgl2. As WD repeat proteins provide a platform forprotein-protein interactions , the ~800-kDa complex is
expected to function in autophagosome formation, furtherinteracting with other proteins in mammalian cells.
800 kD complex~•(Apg12-Apg5 · Apg16)i•

3/LC ~lg$ -·C -·0 ~isolation membrane

autophagosome

autolysosome

Figgure 3. Model of autophagosome formation in
mammalian cells.
The Atg12-Atg5 conjugate and Atgl6L localize to theisolation membrane throughout its elongation process.
LC3 is recruited to the membrane in the Atg5-dependent
manner. Atg12-Atg5 and Atgl6L dissociate from themembrane upon completion of autophagosome
formation , while LC3 remains on the autophagosome
membrane. Atg5 and its modification by Atg 12 arerequired for elongation of the isolation membrane.

VII. Mice with a fluorescent marker forautophagy

Figure 2. Localization of GFP-Atg8p in sec24 mutant
cells. Wild-type (KVY55) and sec24 (MHY24) cells
haboring pRS316 GFP-Atg8p were used. GFP-Atg8p
was visualized under growing condition (a, d), starvation
condition at permissive temperature (b, e) and starvation
condition at non-permissive temperature (c, f).

VI. Mammalian Atg16-like protein: a novel WDrepeat protein
We have shown that, in yeast and mammalian cells, theAtg 12-AtgS protein conjugate, which is formed by aIn yeast, autophagy is required for cell survival during
starvation and is necessary for spore formation. In contrast,
the role of autophagy in mammals is still poorly
understood. Although the possible involvement ofautophagy in development, cell death and pathogenesis has
been repeatedly pointed out, systematic analysis has notbeen petformed, mainly due to a limitation of monitoring
methods. Our recent studies have made available several
marker proteins for autophagosomes. To understand whereand when autophagy occurs in vivo, we have generated·transgenic mice systemically expressing GFP fused toLC3 , which is a mammalian homologue of yeast
Aut7/Atg8. Cryosections of various organs were preparedand the occurrence of autophagy was examined byfluorescence microscopy (Fig. 4) . Active autophagy wasobserved in various tissues, such as the skeletal muscle,
liver, heart, exocrine glands, thymic epithelial cells, lensFOR BASIC BIOLOGY

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epithelial cells and podocytes. Autophagy is differently
induced by nutrient starvation in most tis sues. In so meti ssues, autophagy even occurs spontaneo usly. Our results
s ugges t that the reg ulation of a utoph agy is orga ndependent and the role of autophagy is not restricted to thestarvation response. This transgenic mouse is a usefu l tool
to study mammalian autophagy .

Therefore, Atg8 is a us eful molecular marker formonitorin g autophagic process . To establish a systemmonitoring autophagy in a whole p lant, we generated
tran sge ni c Arabidopsis ex press in g GFP-AtATG8 fusion
protein. In wild-type pl ants, GFP-AtATGSs were observed
as rin g structures in the cytoplasm (Figure 5 panel A), andd e liv e red to the lumen s of vacuo le under nitrogenstarvation condition (Figure 5 pane l B). On the basis ofanalogy of yeast, we rega rded the se ring structures as
autophagosomes. In contrast, in double disruptant of
AtATG4 s which are required for C-terminal cleavage ofAt g8, GFP-AtATGS s were not locali ze d on
autophagosomes (Figure 5 panel C) and were not delivered
to the vacuole under nitrogen-starvation condition (Figure5 pan e l D) . These re s ults indicate that AtATGS is asuitable marker for mon itoring autophagy in plant.

Publication List:
Fi g ure 4 . Muscle autophagy in res pon se to foodwithdrawal. GFP images of gastrocneniius muscles at 0 h
and 24 h starvation. Small dots indicate autophagosomes.
Bar, 10 !-lm.

VIII. Monitoring of autophagic process in a wholeplant
So far, autophagy in plant h as been de sc ribed bymorphological studies. Recent genome-wide searc h
revea led significant conservation in a utophagy ge nes
between yeast and plant. But little is known about thephysiolog ical roles and mol ecul ar mechanisms underlying
autophagy in higher pl a nts. To elucidate the plant
autophagy, we focused ubiquitination-like Atg8 lipidation
syste m. In yeast, Atg8 binds to autophagosomes and isde livered to the vacuol e through autophagic proces s .

MS

MS-NFigure 5. Localization of GFP-AtA TG8a fusion protein
in Arabidopsis roots. The roots of !-week-old transgenic
seedlin gs were grown on MS and MS -N medium. For
sta r va tion , see dlin gs g rown on MS medium we retransferred to MS-N medium for additional 7 days. (A,B) Wild-type Arabidopsis roots stably expressing GFPAtATGSa. (C, D) atatg4ab -J di sruption mutant rootsstabl y expressing GFP-AtA TG8a. Bar: 20 Jtm.

Mi zushima, N. , Kuma , A., Kobayas hi , Y., Yamamoto, A.,
Takao, T., Natsume, T. , Ohsumi , Y., and Yoshimori , T.,
Mouse Apgl6L, a novel WD repeat protein, targets to theautoph ag ic isolation membrane with the Apg 12-Apg5conjugate. J. Cell Sci. , 116, 1679- 1688 (2003)
Hamasak i, M., Noda, T ., and Ohsumi Y. The early secretory
pathway contributes to autoph agoso me formation. CellStruc. Func., 28, 49-54 (2003)
Su gawa ra, K. , Suzuki , N ., Fujioka, Y. , Mizu shima, N .,Ohsumi , Y. , Inagaki , F. Crystalization and preliminary Xray ana lyses of LC3-I, Acta Crys. , 059, 1464- 1465 , (2003)
Mukaiyama, H. , Baba, M., Osumi , M., Aoyag i, S., Kato, N. ,Ohsumi, Y. , Sakai, Y. Modification of a Ubiquitin-likepro te in Pa z2 condu c ted mi cro pexo phagy throughformation of a membrane structure. Mol. Bioi. Cell. , inpress (2003)
Qu X, Yu J, Bhagat G, Furuya N, Hibshoosh H, Troxel A,Ro se n J , E ske linen EL , Mizushima N , Oh sumi Y ,Cattoretti G, Levine B. , Promotion of tumorigenesis byheterozygous disruption of the beclin 1 autophagy gene. J.
Clin.lnvest. , 112, 1809-1820 (2003)
Klionsk y OJ, Cregg JM , Dunn WA Jr, Emr SO, Sakai Y ,Sandoval IV , Sibirny A , Subramani S , Thumm M ,Veenhuis M, Ohsumi Y. Dev. Cell. , 5, 539-45 (2003)
Mizushima, N., Yamamoto, A. , Matsui , M. , Yoshimori, T.
and Ohsumi , Y. In vivo ana lysis of autophagy in response
to nutri ent starvation using transge ni c mice expressi ng afluorescent autophagosome marker. Mol . Bioi. Cell, inpress (2004)
Ohas hi, M., Mi zushima, N., Kabeya, Y. and Yoshimori, T.
Locali za tion of mamm a lian NAD (P) H steroid
dehydrogenase-like protein on lipid droplets J. Bioi. Chem.
278, 36819-36829 (2003)
Mi zushima, N. , Yamamoto, A. , Matsui , M. , Yoshimori , T.
and Ohsumi , Y. In vivo analysis of autophagy in response
to nutrient starvation usin g tran sgenic mice expressing afluorescent autophagosome marker. Mol . Bioi. Cell inpress
Mizushima, N., Methods for monitoring autophagy. Int. J.
Biochem. Cell Bioi. In press (2004)
Mizushima, N, Yoshimori, T. , Ohsumi, Y., Role of the Apg 12
co nju ga tion sys tem in mamma lian autophagy. Int . J.
Biochem. Cell Biol. , 35, 553-561 (2003)
Ohsumi, Y. Cellular recycling system- molecular mechanismof autophagy. In Cell Growth, Cold Spring Harbor Press,in press (2003)
Kamada, Y., Sekito,T. , Ohsumi Y. , Autophgy in yeast: aTOR-mediated response to nutrient starvation. Curr Top
Microbiollmmunol., 279, 73-84 (2004)
Oh s umi , Y. and Mi z us him a, N . Two ubiquitin-likeconju gation sys tems essenti al for autophagy. Sem. CellDev. Bioi. , in press (2004)