DIVISION OF MOLECULAR CELL BIOLOGY

Professor

OHSUMI, Yoshinori
KAMADA, Yoshiaki
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
Technical Staff
KABEYA, Yukiko
NIBB Research Fellows OBARA, Keisuke*
HANADA, Takao
Postdoctoral Fellows
OBARA, Keisuke
HANADA, Takao*
FUJIKI, Yuki
OKU, Masahide
HARASHIMA, Toshiaki
YAMAMOTO, Hayashi
OKAMOTO, Koji
KAKUTA, Soichiro
KOBAYASHI, Takafumi
Graduate Students
KAGEYAMA, Takuya
OH-OKA, Kyoko
Technical Assistants
KONDO, Chika
NIIMI, KaoriISHII, Junko
OKAMOTO, Noriko
ICHIKAWA, RieSecretaries
HARA, Yoko
SUZUKI, Yuko
Assistant Professors

Most cellular activity is maintained by the balance between
the synthesis and degradation of related proteins. Therefore,degradation processes as well as the regulation of gene
expression play important roles in many physiologicalaspeets. 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.

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 byvacuolar/lysosomal hydrolases. We discovered autophagy ina simple model organism, the budding yeast Saccharomyces
cerevisiae, and have morphologically and geneticallydefined the whole process.

Ⅱ. Lap3 is a selective target of autophagy inyeast
Although autophagy is, in principle, a non-selectivedegradation process, several proteins and organelles werefound to be selectively incorporated into theautophagosome. For instance, Ald6, a soluble cytoplasmic
enzyme in yeast, is preferentially eliminated from thecytoplasm via autophagy; Little is known, however, about
the mechanisms of Ald6 targeting to autophagosomes.
Autophagy is also involved in the selective transport ofApe1, a vacuolar hydrolase, as a biosynthetic route.
Recently, we discovered that leucine aminopeptidase III(Lap3), a soluble cytosolic cysteine protease, was spatiallyassociated with Ape1 and selectively transported to thevacuole during nitrogen starvation (Figure 1). The rate of
Lap3 transport was much higher than that of Ald6 and was
similar to that of Ape1. Moreover, Atg11 and Atg19,
essential factors for Ape1 transport, were important for Lap3transport. Most of the Lap3 was degraded within a couple ofhours in the vacuole, in contrast to Ape1; therefore, weconcluded that the machinery required for Ape1 biosynthesis
is used for the selective degradation of Lap3.

Figure 1. Microscopic analyses of Lap3. (A) Localization of GFP-Lap3 in
pep4Δ atg7Δ (A) and pep4Δ (B) cells. Cells were grown in YPD media andthen incubated in nitrogen starvation media (SD-N) for 3 h. The GFP andmRFP signals were observed simultaneously. Arrowheads indicate the colocalization of Lap3 and Ape1. Arrows point to intravacuolar structures.
Insets are high-magnification images of dots marked with asterisks. GFPLap3 and mRFP-Ape1 dots do not merge, but are localized very closely toeach other. Bars, 5 μm.

Ⅲ. Structural basis of cargo recognition by
Atg8 homologs in selective autophagy
Atg8 homologs are ubiquitin-like proteins that localize on
autophagosomal membranes and play crucial roles in theformation of the membranes. These proteins are also
involved in the selective incorporation of specific cargomolecules into autophagosomes, in which Atg8 and its
mammalian homolog, LC3, interact with Atg19 and p62,
receptor proteins for a number of vacuolar enzymes
including Ape1 and disease-related, ubiquitin-positive
protein inclusions, respectively. In collaboration with Dr.
Inagaki's group at Hokkaido University, we determined thestructures of Atg8 and LC3 in complex with Atg19 and p62
peptides, respectively, which contain amino acid residues
necessary and sufficient for complex formation.
Remarkably, Atg8 and LC3 proved to interact with Atg19
and p62 in a quite similar manner, even though the entire
sequences of Atg19 and p62 are unrelated to each other.
Hydrophobic pockets conserved among Atg8 homologs
recognized the side-chains of Trp and Leu in the WXXL
motif in Atg19 and p62 (Figure 2). Mutational analyses ofAtg8 and Atg19 showed that this interaction is indeed

Note: Those members appearing in the above list twice under different titles are members whose title changed during 2008. The former title isindicated by an asterisk (*)

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National Institute for Basic Biology

Cell Biology

required for the vacuolar transport of Ape1. Thus, werevealed the fundamental interaction between receptors andAtg8 homologs by which specific cargo molecules areefficiently captured by autophagosomes.

not observed for Atg8–PS at all. Therefore, it was suggested
that intracellular milieus, the pH of the cytosol and acidic
phospholipids in membranes, contribute to the selective
formation of Atg8–PE in the cell. We also showed thatacidic phospholipids facilitate the binding of Atg8-Atg7 andAtg8-Atg3 thioester intermediates to the membrane(Figure 3).

Ⅴ. Amino-terminal region of Atg3 is essential
for the association with PE in Atg8
lipidation

Figure 2. The structure of the interaction site of Atg8 with Atg19. The
evolutionally conserved, hydrophobic binding pocket of Atg8 (surfacemodel) accommodating a peptide corresponding to the Atg8-binding regionof Atg19 (stick model) is shown.

Ⅳ. The determinants of substrate specificity inlipidation of Atg8
A ubiquitin-like system consisting of the E1 enzyme Atg7
and the E2 enzyme Atg3 mediates the conjugation of Atg8
with the lipid phosphatidylethanolamine (PE). The Cterminal carboxyl group of Atg8 is activated by Atg7,
sequentially forms thioester intermediates with active site
cysteine residues of Atg7 and Atg3, and eventually forms anamide bond with the amino group of PE. Probably as thislipid-modified form (Atg8–PE), Atg8 localizes toautophagosomal membranes and participates in theirformation. The conjugation reaction of Atg8 can bereconstituted in vitro using purified proteins and liposomes
containing PE. Whereas PE was identified as the sole in vivo
target of Atg8, in vitro studies showed that the same system
can mediate the conjugation of Atg8 with phosphatidylserine(PS) as efficiently as with PE. We found that, in contrast toPE conjugation, the PS conjugation of Atg8 is markedly
suppressed at physiological pH (Figure 3). Furthermore,while the addition of acidic phospholipids to liposomes
significantly promotes the production of Atg8–PE, this was

We previously showed using the in vitro system that theconjugate of the ubiquitin-like protein Atg12 with Atg5
(Atg12–Atg5) directly interacts with Atg3 and drasticallystimulates its conjugating activity (transfer of Atg8 to PE).
When PE-containing liposomes were absent from thereaction in the presence of Atg12–Atg5, Atg8 was mistransferred to the serine residues in the N-terminal region ofAtg3 from its active site cysteine, suggesting theinvolvement of the region in Atg8 lipidation. To examine
the role of the N-terminal region of Atg3 in the formation ofAtg8–PE, we prepared Atg3 mutants having deletions ormutations in this region. We found that the conjugating
activities of the Atg3 mutants lacking N-terminal 7 aminoacid residues or having the Leu-to-Asp mutation at position
6 were severely impaired both in vivo and in vitro.
Furthermore, we revealed that the N-terminal region of Atg3
is responsible for the proper interaction with PE-containing
membranes (Figure 4).

Figure 4. Floatation assay of Atg3 mutants. Purified Atg3 mutant proteins
were incubated with liposomes containing PE, and then subjected tofloatation assay. Fractions (fraction 1 represents the top fraction) wereanalyzed by western blotting with anti-Atg3 antibodies.

Ⅵ. The yeast Tor signaling pathway is involvedin G2/M transition via Polo-kinase

Figure 3. The neutral pH and the presence of acidic phospholipids result in
the preferential formation of Atg8–PE.

12The target of rapamycin (Tor) protein plays a central rolein cell growth. Rapamycin inhibits cell growth and promotes
cell cycle arrest at G1 (G0). Little is known, however, about
whether or not Tor is involved in other stages of the celldivision cycle. Unexpectedly, we found that the rapamycinsensitive Tor complex 1 (TORC1) is involved in G2/M
transition in S. cerevisiae. Strains carrying a temperaturesensitive allele of KOG1 (kog1-105) encoding an essential
component of TORC1, as well as yeast cells treated withrapamycin, show mitotic delay with prolonged G2 (Figure5A). Overexpression of Cdc5, the yeast polo-like kinase,rescues the growth defect of kog1-105, and in turn, Cdc5
activity is attenuated in kog1-105 cells. The TORC1Type2A phosphatase pathway mediates nucleocytoplasmictransport of Cdc5, which is a prerequisite for its proper
localization and function (Figure 5B). The C-terminal polobox domain of Cdc5 has an inhibitory role in nuclear
translocation. Taken together, our results indicate a novel
function of Tor in the regulation of cell cycle andproliferation.

● Nakashima, A., Maruki, Y., Imamura, Y., Kondo, C., Kawamata, T.,

Kawanishi, I., Takata, H., Matsuura, A., Lee, K.S., Kikkawa, U.,
Ohsumi, Y., Yonezawa, K., and Kamada, Y. (2008). The yeast Tor
signaling pathway is involved in G2/M transition via Polo-kinase.
PLoS ONE 3, e2223.
● Noda, N.N., Fujioka, Y., Ohsumi, Y., and Inagaki, F. (2008).
Crystallization of the Atg12-Atg5 conjugate bound to Atg16 by thefree-interface diffusion method. J. Synchrotron Radiat. 15, 266-268.
● Noda, N.N., Kumeta, H., Nakatogawa, H., Satoo, K., Adachi, W.,Ishii, J., Fujioka, Y., Ohsumi, Y., and Inagaki, F. (2008). Structural
basis of target recognition by Atg8/LC3 during selective autophagy.
Genes Cells 12, 1211-1218.
● Obara, K., Noda, T., Niimi, K., and Ohsumi, Y. (2008). Transport ofphosphatidylinositol 3-phosphate into the vacuole via autophagic
membranes in S. cerevisiae. Genes Cells 13, 537-547.
● Obara, K., Sekito, T., Niimi, K., and Ohsumi, Y. (2008). The Atg18Atg2 complex is recruited to autophagic membranes via PtdIns(3)Pand exerts an essential function. J. Biol. Chem. 283, 23972-23980.
● Oh-oka, K., Nakatogawa, H., and Ohsumi, Y. (2008). PhysiologicalpH and acidic phospholipids contribute to substrate specificity inlipidation of Atg8. J. Biol. Chem. 283, 21847-21852.

〔Review articles〕● Nakatogawa, H., Oh-oka, K., and Ohsumi, Y. (2008). Lipidation of

Atg8: How is substrate specificity determined without a canonical E3
enzyme? Autophagy 4, 911-913.
● Nakatogawa, H., and Ohsumi, Y. (2008). Starved cells eat ribosomes.
Nat. Cell Biol. 10, 505-507.
● Obara, K., and Ohsumi, Y. (2008). Dynamics and function ofPtdIns(3)P in autophagy. Autophagy 4, 952-954.
● Sekito, T., Fujiki, Y., Ohsumi, Y., and Kakinuma, Y. (2008). Novelfamilies of vacuolar amino acid transporters. IUBMB Life 60, 519525.

Figure 5. Tor regulates mitotic entry. (A) FACS analysis of wild type andtsts
kog1-105 strains. After temperature shift to 37°C, kog1-105 cells stop cell
cycle progression with 2C DNA content, suggesting mitotic delay. (B)Localization of GFP-tagged Cdc5, the yeast polo-kinase. In wild type cells,Cdc5 localizes at the spindle pole body and the nucleus. In contrast,ts
localization of Cdc5 is disrupted in the kog1-105 mutant.

Publication List〔Original papers〕● Fujioka, Y., Noda, N.N., Fujii, K., Yoshimoto, K., Ohsumi, Y., andInagaki, F. (2008). In vitro reconstitution of plant ATG8 and ATG12
conjugation systems essential for autophagy. J. Biol. Chem. 283,1921-1928.
● Fujioka, Y., Noda, N.N., Matsushita, M., Ohsumi, Y., and Inagaki, F.
(2008). Crystallization of the coiled-coil domain of Atg16 essential forautophagy. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 64,
1046-1048.
● Hu, G., Hacham, M., Waterman, S.R., Panepinto, J., Shin, S., Liu, X.,
Gibbons, J., Valyi-Nagy, T., Obara, K., Jaffe, H.A., Ohsumi, Y., andWilliamson, P.R. (2008). PI3K signaling of autophagy is required forstarvation tolerance and virulence of Cryptococcus neoformans. J.
Clin. Invest. 118, 1186-1197.
● Ishida, H., Yoshimoto, K., Izumi, M., Reisen, D., Yano, Y., Makino,A., Ohsumi, Y., Hanson, M.R., and Mae, T. (2008). Mobilization ofRubisco and stromal-localized fluorescent proteins of chloroplasts tothe vacuole by an ATG gene-dependent autophagic process. Plant
Physiol. 148, 142-155.
● Kawamata, T., Kamada, Y., Kabeya, Y., Sekito, T., and Ohsumi, Y.
(2008). Organization of the pre-autophagosomal structure responsible
for autophagosome formation. Mol. Biol. Cell 19, 2039-2050.

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