14NATIONAL JNSTITUTEDIVISION OF BIOENERGETICS
Professor:
Associate Professor:
Research Associates:

Yoshinori Ohsumi
Tamotsu Yoshimori
Yoshiaki Kamada
Takes hi Noda
Technical Staff:
Yukiko Kabeya
Post Doctoral Fellows: Noboru Mizushima l)
Takahiro Shintani
Naotada Ishihara
Akio Kihara 2!
Graduate Students:
Takayoshi Kirisako 3!
Yoshinobu /chimura 3J
Kuninori Suzufd 4J
Yoshinori Kobayashi4J
Atsuki Nara 4!
Hideki Hanaoka 4!
Visiting Fellow:
Hisashi Okada 5!
Fumi Yamagata 6!
Visiting Scientists:
Kazuaki Furukawa 7!
Satsuki Okamoto
IJ PRESTO, JST, Oct 1999-.
lJ Apr 1999-.
JJ Graduate University for Advanced Studies4) Apr 1999-, Graduate University fo r Advanced Studies.
s; from Teikyo Univ. ofSci. and Tech.
6) from Teikyo Univ. of Sci. and Tech. -March 1999
7) from Suntory Co. LTD, -July 1999.

This division aims to understand the autophagy in respects to its molecular mechanism and its physiologicalrole in multicellular organisms. Cells execute degradation processes of their constituents together with biosynthetic processes. These two processes are well coordinated to regulate the biological activities. In otherword, we must shed light on degradation process tofully understand the cell, because the study on the degradation has been retarded compared to the biosynthetic process. Autophagy is well conserved in eukaryotes and is a major route for bulk degradation of cytoplasmic constituents and organelles.

Apg5 , another Apg protein, via an isopeptide bond. This
conjugation reaction is distinct from but quite similar toubiquitination and the other related reaction such as
SUMO-I and Rub 1 system. All of these ubiquitinationlike (Ubl) reactions are catalyzed by a series of enzyme
system.
First, C-terminal glycine of Ubi protein is activatedby binding to active center of El -like enzyme via athioester bond. El-like enzymes are ATPase whichshare sequence similarity each other, and the ATPase
activity is prerequisite for the reaction. Collaboratingwith Dr. Kominami's laboratory(Juntendo Med. Univ.),
we have reported that Apg7 is an El-like enzyme involved in Apgl2 conjugation reaction.
Next, C-terminal glycine of Ubi protein is conjugatedto the active center of E2-like enzyme via thioester ·bond. We characterized ApglO, which is required forconjugation reaction of Apg12 . ApglO binds to Apg12
depending on Apg7 function. We identified active center, Cys residue, of ApglO. C-terminal Gly of Apgl2
bind to this Cys via a thioester bond. The active Apg10
is required for Apg12-Apg5 conjugation, and necessaryfor autophagy. Most notably, all previously known E2like enzymes show sequence similarity each other,
however ApglO does not show the similarity to anyknown E2 enzymes. ApglO is a quite unique E2-likeenzyme. These findings prompted us the idea that Ubi
conjugation system is more prevailing than ever expected from sequence similarity.

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, Saccharomy ces cerevisiae and morphologically defined the whole process. We have already isolated a set of autophagy-deficient mutants(apg), and have cloned most of APG genes essential forautophagy. We are now characterizing these geneproducts.

II. ApglO is a unique protein conjugating enzyme
Last year, we reported on novel protein conjugation
system required for autophagy. Apg12, one of Apg
proteins, is terminated with glycine at its C-terminus.
The C-terminal glycine is bound to a lysine residue ofFigure 1 Molecular apparatuses for autophagy in yeast. Upper
Panel: The diagram of Apg12 system. The numerals in the boxes
denotes the corresponding Apg proteins. The number of each
subunits in the final complex is still not determined. Arrowrelated to Apg16 does not necessarily represent the order of theevents, mther do show the concept of the organization. Lower
Panel: Immunofluorescence (Left) and Nomarsky Images(Right) of Apg8 localization. The vacuolar protease deficientmutants were starved and the tagged Apg8 is observed as theFITC signal (the arrow in left panel). Autophagic body is indicated by the arrow in the right panel

FOR BASIC BIOLOGY

15III. Apg16 crosslinks multiple Apg12-Apg5 conjugates
Despite these fmdings of novel Ubi system, themechanism how the system is involved in autophagicmechanism is still to be determined. However, an accessory part related to this system, which will aid us understanding the point, was discovered.
In a two-hybrid screen with Apgl2 as a bait, weidentified a novel protein, Apgl6, which is required forautophagy. Thereafter, we noticed that the Apgl6 is
not bound to Apgl2 but Apgl2-Apg5 conjugate.
Moreover, Apgl6 is bound to Apg5 alone, but more
preferentially to Apgl2-Apg5 conjugate. The binding
occurs at theN-terminal region of Apgl6. On the otherhand, Apgl6 have a coiled coil domain at its C-terminus,
which generally functions for protein interaction. Actually Apgl6 forms an homooligomer via the coiled coildomain. In accordance with this scheme, two or more
independent Apgl2-Apg5 conjugates were shown to be
cross-linked via Apgl6. Hence, this hetero-oligomericlarge protein complex may play a role in autophagic
process.
IV. Apg8 as a tracer of membrane flow in autophagosome formation
Autophagosome is a double membrane structure. The
outer membrane fuses with the vacuolar membrane, andthe inner membrane structure is released into thevacuolar lumen. The released membrane structure,
autophagic body, is disintegrated depending on thevacuolar protease activity, and the cytosolic materials
inside are degraded. Therefore, when vacuolar proteaseactivity is blocked, autophagic bodies are accumulated
inside the vacuole.
Apg8, one of Apg proteins, is transcriptionally upregulated upon starvation via Tor-kinase dependent
signaling pathway, which controls induction of autophagy. Indirect immunofluorescence study showed thatduring a nutrient-rich growing phase, Apg8 is mostly
dispersed throughout the cytoplasm as tiny dot structures. Upon starvation, Apg8-positive structure appears
adjacent to the vacuole, which represents autophagosome or its intermediate. When vacuolar proteaseactivity is blocked, Apg8 was observed to be accumulated inside autophagic bodies. According to immuno
EM analysis in collaboration with Dr. M. Baba (JapanWomen's Univ.), Apg8 is mostly enriched on the membrane of forming intermediate of autophagosome. Uponcompletion of the formation, Apg8 is detached from themembrane and some are released to the cytosol and theothers are entrapped within the autophagosome. Thus,
Apg8 could be a useful tracer for membrane flow in
autophagosome formation. More detailed EM observation clarified that Apg8-positive electron lucent small
structures are gathering around forming autophagosome.
We postulate that these structures are one of the precursors of autophagosomal membrane. If this is the case,formation process of autophagosome is accompaniedwith multiple fusion of small precursor structures to theFigure 2. TfR and the endocytosed dextran are accumulated in thecompartments where the mutant SK.Dl is localized. HeLa cells
transfected with the mutant SKD l fused with GFP were incubatedin the presence of l mg/ml TRITC-dextran at 37 oc for 8 hours.
The cells were then fixed , permeabilized, and subjected to immunofluorescence confocal microscopy for TfR. GFP-mutant SKDI
labeling (a), TfR staining (b), TRITC-dextran labeling (c), and amerged image (d) of a same field are shown. Arrows heads andarrows indicate the untransfected cells and the mutant SKD !positive compartments in the transfected cells, respectively. Bar,20 J.lm.

intermediate structure. This contradicts to the generally
accepted scheme that autophagosome is formed byengulfing of the preexisting large membrane structure,such as ER. This hypothesis will be tested in our ongoing project.
V. SKDl regulates morphology of endosomes andmembrane traffic through them in mammaliancells
We also engage in the study of mammalian to pursue
cell biological and physiological issues of autophagy. In
the course of the study, we encountered an interesting
molecule, which regulates membrane dynamics in endosomal system.
The mouse SKDl is an AAA-type ATPase homologous to the yeast Vps4p/ Csclp implicated in bothautophagy and transport from endosomes to the vacuole.
To elucidate a possible role of SKDl in mammalianendocytosis, we generated a mutant SKD 1, harbouring amutation (E235Q) that is equivalent to the dominantnegative mutation (E233Q) in Vps4p/Csclp. Overexpression of the mutant SKDl in cultured mammaliancells caused defect in uptake of transferrin and lowdensity lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface
transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein
displayed a perinuclear punctate distribution in contrast
to a diffuse pattern of the wild type SKD 1. TfR, thelysosomal protein Iamp-l, endocytosed dextran andepidermal growth factor (EGF), but not markers for thesecretory pathway were accumulated in the mutant

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SKDl-localized compartments. Degradation of EGF
was inhibited. Electron microscopy revealed that thecompartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and an endocytosed horseradish peroxidase.
The early endosome antigen EEAl was also redistributed to these aberrant membranes. Taken together, ourfindings suggest that SKD 1 regulates morphology ofendosomes and membrane traffic through them.

Publication List:
Original Articles
Tanida, 1., Mizushima, N., Kiyooka, M., Ohsumi, M.,Ueno, T., Ohsumi, Y. and Kominami, E. (1999).
Apg7p/Cvt2p: A novel protein-activating enzyme essential for autophagy. Mol Bioi Cell. 10, 1367-1379.
Shintani, T., Mizushima, N., Ogawa, Y., Matsuura, A.,
Noda, T. and Ohsumi, Y. (1999). ApglOp, a novel protein-conjugating enzyme essential for autophagy in yeast.
EMBOJ. 18,5234-5241.
Mizushima, N., Noda, T. and Ohsumi, Y. (1999). Apg16p
is required for the function of the Apg12p-Apg5p conjugate in the yeast autophagy pathway. EMBO J. 18, 38883896.
Kirisako, T., Baba, M., Ishihara, N., Miyazawa, K., Ohsumi, M., Yoshimori, T., Noda, T. and Ohsumi, Y.
(1999). Formation process of autophagosome is traced
with Apg8/Aut7p in yeast. J Cell Bioi. 147,435-446.
Tagawa, Y., Yamamoto, A., Yoshimori, T., Masaki, R.,Omori, K., Himeno, M., Inoue, K. and Tashiro, Y.
(1999). A 60 kDa plasma membrane protein changes its
localization to autophagosome and autolysosome membranes during induction of autophagy in rat hepatoma
cell line, H-4-II-E cells. Cell Struct Funct. 24,59-70.
Yabe, I., Horiuchi, K., Nakahara, K., Hiyama, T., Yama-NATIONAL INSTITUTEnaka, T., Wang, P. C., Toda, K., Hirata, A., Ohsumi, Y.,
Hirata, R., Anraku, Y. and Kusaka, I. (1999). Patchclamp studies on V-type ATPase of vacuolar membraneof haploid Saccharomyces cerevisiae. Preparation andutilization of a giant cell containing a giant vacuole. JBioi Chem. 274, 34903-34910.
Noda T., Kim J., Huang WP., Baba M., Tokunaga C.,
Ohsumi Y., and Klionsky D.J., (2000) Apg9p/Cvt7p is
an integral membrane protein required for transport
vesicle formation in the cvt and autophagy pathways. JCell Biol./148, 465-480
Yoshimori T., Yamagata F., Yamamoto A., Mizushima N.,
Kabeya Y., Nara A., Miwako I., Ohashi M., Ohsumi M.,
Ohsumi Y., (2000) The mouse SKDl, a homologue ofyeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells. Mol Bioi Cell.
11, 747-763
George, M.D., Baba M., Scott, S.V., Mizushima N., Garrison B., Ohsumi Y., Klionsky D.J., (2000) Apg5p functions in the sequestration step in the cytoplasm to vacuole targeting and macroautophagy pathways. Mol Bioi
Cell. in press
Furukawa, K. Mizushima, N., Noda, T., Ohsumi, Y.,(2000) A protein conjugation system in yeast with homology to biosynthtic enzyme reaction ofprokaryotes. J
Bioi Chem. 275, 7462-7465· Review Articles
Klionsky, D. J. and Ohsumi, Y. (1999). Vacuolar import ofproteins and organelles from the cytoplasm. Annu Rev
Cell Dev Bioi. 15, l-32.
Ohsumi, Y. (1999). Molecular mechanism of autophagy inyeast, Saccharomyces cerevisiae. Philos Trans R. Soc.
Lond, B 354, 1577-1580; discussion 1580-1581.
Ohsumi, Y. (1999). Pictures in cell biology. Autophagosomes in yeast. Trends Cell Bioi. 9, 162.