11

FOR BASIC BIOLOGYDIVISION OF BIOENERGETICS
Graduate University for Advanced Studies Professor:
Yoshinori Ohsumi
Tamotsu Yoshimori
Associate Professor:
Research Associates: Yoshiaki Kamada
Takeshi Noda
Technical Staff:
Yukiko Kabeya
Post Doctoral Fellows: Noboru Mizushima 1!
Takahiro Shintani
Naotada lshihara 2!
Akio Kihara 3!
Graduate Students:
Takayoshi Kirisako4!
Yoshinobu lchimura4!
Kuninori Suzukt><~!
Yoshinori Kobayasht><~!
Atsuki Nara4!
Hideki Hanaoka 4!
Akiko Kuma4·5!
Maho Hamasakt-4. 5!
Shisako Shojt><~. 6!
Visiting Scientists:
Satsuki Okamoto
I! PRESTO, JST, Oct 1999-.
! til March 2000
JJ til Dec 2000
4
! Graduate University for Advanced Studies.
5!from April 2000.
6! from Oct.2000
2

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.

duction of autophagy during growing condition, since
we observed that rapamycin, a Tor-specific inhibitor,
induced autophagy even under such a condition. Next
question is how inhibition of Tor function leads autophagy induction. Among 15 Apg proteins Apglp andApgl3p show close relations with Tor. Apglp is a protein kinase, and Apgl kinase activity is enhanced bynutrient starvation or rapamycin-treatment. This activation requires APGJ3. Apgl3p is highly phosphorylated
in a Tor-dependent manner. In starved or rapamycintreated cell, Apgl3p is immediately dephosphorylated.
And defect of autophagy by deletion of APGJ3 is rescued by overexpression of APG1. These suggest that
Apgl3p plays a key role in signal transduction from Tor
to Apglp. We found that only dephosphorylated form
(but not hyper-phosphorylated form) of Apgl3p associates to Apglp under starved condition. And we demonstrated that this association confers Apgl activation
which is required for the induction of autophagy. We
also identified a couple of Apgl-binding proteins
Apg17p and Cvt9p suggesting that Apgl makes a large
protein complex.

III. Two Distinct Phosphatidylinositol 3-Kinase
Complexes Function in Autophagy and Carboxypeptidase Y Sorting
Vps30p/Apg6p is required for both autophagy andsorting of carboxypeptidase Y (CPY). Although Vps30p
is known to interact with Apgl4p, its precise role hasremained unclear. We found that two proteins copurify
with Vps30p. By mass spectrometry they were identified to be Vps38p and Vps34p, a phosphatidylinositol(Ptdins) 3-kinase. Vps34p, Vps38p, Apgl4p, andVps15p, an activator of Vps34p, were coimmunoprecipitated with Vps30p. These results indicate that
Vps30p functions as a subunit of a Vps34 Ptdins 3kinase complex. Phenotypic analyses indicated thatcomplex I<<

complex II

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.

II. Apgl-Apg13 protein kinase CQJllPlex mediates
induction of autophagy in response to starvation
signalWe previously reported that Tor protein represses in-

Autophagy

CPY sorting

Fig. I PI3 kinase complex is required for autophagy.
PI3 kinase( vps34) and its activator (vps 15) form at least twocomplex. Complex I (Vps34, Vps15, Apg14, and Vps30) is
essential for autophagy, and Complex II (Vps34, Vps15,
Vps38, and Vps30) functions for vacuolar enzyme CPY
sorting.

12
Apg14p and Vps38p are required for autophagy and
CPY sorting, respectively, whereas Vps30p, Vps34p,
and Vps15p are required for both processes. Coimmunoprecipitation using anti-Apg14p and anti-Vps38p
antibodies and pull-down experiments showed that two
distinct Vps34 Ptdlns 3-kinase complexes exist: one,
containing Vps15p, Vps30p, and Apg14p, functions in
autophagy and the other containing Vps15p, Vps30p,
and Vps38p functions in CPY sorting. The vps34 andvpsl5 mutants displayed additional phenotypes such as
defects in transport of proteinase A and proteinase B,
implying the existence of another Ptdlns 3-kinase complex(es). We propose that multiple Vps34p-Vps15p
complexes associated with specific regulatory proteinsmight fulfil their membrane trafficking events at different sites (Fig. I).

VI. Discovery of novel lipidation reaction mediated by ubiquitin-like system
Apg8/Aut7, which plays an important role in theformation of autophagosome, tends to bind to membranes in spite of its. hydrophilic nature. We showedthat the mode of the association of Apg8 with membranes changes depending on a series of modificationsof the protein itself. First, the carboxy-terminal Arg of
newly synthesized Apg8 is removed by Apg4/ Aut2, anovel cysteine protease, and a Gly residue becomes the
carboxy-terminal residue of the protein that is now
designated Apg8FG. Subsequently, Apg8FG forms a
conjugate with an unidentified molecule "X" and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and
Apg7 , a ubiquitin El-like enzyme. Finally, the adduct
Apg8FG-X is reversed to soluble or loosely membranebound Apg8FG by cleavage by Apg4. The mode ofaction of Apg4, which cleaves both newly synthesized
Apg8 and Apg8FG-X, resembles that of deubiquitinating enzymes.
We then succeeded to identify molecule X and discovred a novel mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine (PE)through an amide bond between the C-terminal glycine
and the amino group of PE. This lipidation is mediatedby a ubiquitination-like system (Fig. 2). Apg8 is a ubiquitin-like protein that is activated by an El protein,Apg7, and is transferred subsequently to an E2 enzyme
Apg3/Autl. Apg7 activates two different ubiquitin-Iikeproteins, Apgl2 and Apg8, and assigns them to specificE2 enzymes, ApglO and Apg3, respectively. This reversible lipidation of Apg8 appears to be coupled to themembrane dynamics of autophagy and the Cvt pathway.

V. Mammalian Apg proteins; localization andfunction
Autophagy was fust described in mammalian ceiJs in
the 1960s and its morphology and regulation have been
extensively investigated. However, molecular mechanism underlying its process is poorly understood. Recent studies on yeast Apg proteins revealed that

NATIONAL INSTITUTE

Apg7

El

Apg4 .-.

Apg3 ApglO

.. E2

E2 .

Fig.2 Two Ubiquitin-like systems essential for autophagy
Apgl2 and Apg8 are activated by common El enzyme,
Apg7. Then they are assigned to different conjugating
enzymes, ApglO and Apg3 , respectively. Finally Apgl2
forms a conjugate with a single target molecule, Apg5 .
Apg8 conjugate with a membrane phospholipid, phosphatidylethanolamine (PE). Apg8-PE is reversed to Apg8 by
Apg4.

molecular machinery of autophagy seems highly conserved among eukaryotes. We are analyzing the mammalian orthologues of Apg proteins.
Apg8 has at least three mammalian homologues, oneof which, LC3, was identified to be involved in autophagy. Newly synthesized rat LC3, a 142 amino acidprotein, was immediately processed to remove the Cterminal 22 amino acids to generate the LC3-I form ,which was further converted to the LC3-II form . While
LC3-I was cytosolic, LC3-II was membrane bound andenriched in the autophagic vacuole fraction (Figure 3A
and B). LC3-II may correspond to yeast Apg8-PE (thePE-conjugated form of Apg8). Imrnunoelectron microscopy revealed LC3 was present both inside and outsideof autophagosomes in addition to cytoplasm. LC3 wasless detected on autolysosome, suggesting that someLC3 dissociated from the membrane after autophagosome formation (Figure 3B). Consistent with such
localization, the amount of LC3-II was correlated withthe extent of the autophagosome formation . Although
LC3 was originally identified as microtubule-associatedprotein 1 light chain 3, involvement of microtuble in
LC3 function and autophagy has remained to be clarified . LC3-II is the first mammalian protein identified
that specifically associates with the autophagosome
membranes.
The Apg 12-Apg5 protein conjugation system is another well conserved machinery. Most mammalian
Apg5 was present in the Apgl2-conjugated form in thecytoplasm. However, a small fraction of the Apgl2Apg5 conjugate localized to the isolation membranes
when autophagy proceeds (Figure 3A and B). Using
GFP-tagged Apg5 , we revealed that the cup-shaped
isolation membrane is developed from a small crescentshaped compartment. These small structures have neverbeen recognized unless labeled with Apg5, but they areFOR BASIC BIOLOGY

13

Fig.3.
A. Distribution of ApgS and LC3 in mouse embryonic stem cells.
YFP-LC3 (middle panel) localizes to spherical autophagosomes and semi-spherical isolation membranes, whereas CFP-ApgS (leftpanel) localizes to only isolation membranes. Bar, 2 J.!m.
B. Model of dynamic association of Apg 12-ApgS and LC3 with autophagic membranes.
Apgl2-Apg5 conjugate localizes to the crescent-shaped autophagosome precursors. While these structures elongate and maturate into
cup-shaped isolation membranes, cytosolic LC3-I is recruited ·to the membrane in the Apgl2-Apg5-dependent manner and accumulated
as the LC3-II form. ApgS biases its localization to the outer side of the membrane. ApgS plays an essential role in this membrane development. Immediately before or after the completion of autophagosome formation, ApgS detaches from the membrane. Some LC3
also dissociate from the autophagosomal membrane thereafter.

likely to be direct precursors of the autophagosome.
Apg5 localized on the isolation membrane throughout
its elongation process. Apg5 was preferentially distributed in the outer side of the membrane and detaches
from it immediately before or after autophagosome
formation is completed (Figure 3B). To examine therole of Apg5, we generated Apg5-deficient ES cells.
APG5 ·f· cells are viable but bulk protein degradation

was significantly reduced. Morphological analysis revealed that autophagosome formation was impaired in
APG5 .f. cells. We also showed that the covalent modification of Apg5 with Apg 12 was not required for its
membrane targeting but is essential for involvement of
Apg5 in elongation of the isolation membranes. Intriguingly, Apgl2-Apg5 was required for targeting of
LC3 to the isolation membranes and LC3-II generation.

14
Therefore, the Apg12-Apg5 conjugate plays essential
roles in isolation membrane development in cooperation with LC3. In addition, our studies provided
good molecular markers, LC3 and Apg12-Apg5, forautophagic membrane at all stages and isolation membranes, respectively, which so far have been defined
only by morphology.

Original papers :Yoshimori, T., Yamagata, F., Yamamoto, A., Mizushima, N., Kabeya, Y., Nara, A., Ishido, M., Ohashi,M., Ohsumi, M., and Ohsumi, Y. SKDl, an AAA
ATPase related to the yeast Vps4p, regulates morphology and transport function of early endosomes.
Mol. Biol. Cell., 11, 747-763 (2000)
George, M, D., Baba, M., Scott, S. V., Mizushima, N.,
Garrison, B., Ohsumi, Y. and Klionsky, D. J. Apg5p
functions in the sequestration step in the cytoplasm tovacuole targeting and macroautophagy pathways.
Mol. Biol. Cell, 11, 969-982 (2000)
Noda, T., Kim. J., Huang, W-P., Baba, M., Tokunaga,C., Ohsumi, Y., Klionsky, D. J. Apg9/Cvt7p is an
integral membrane protein required for transport
vesicle formation in the Cvt and autophagy pathways.
J. Cell Biol. 148,465-479 (2000)
Furukawa, K., Mizushima, N., Noda, T. and Ohsumi, Y.
A protein conjugation system in yeast with homology
to biosyntheic enzyme reaction of prokaryotes. J.
Bioi. Chem. 275, 11,747-763, (2000)·
Scott, S. V., Nice, ill, D. C., Nau, J. J., Weisman, L. S.,
Kamada, Y., Keizer-Gunnink, I., Funakoshi, T.,
Veenhuis, M., Ohsumi, Y., and Klionsky, D. J.
Apg13p and Vac8p are part of a complex of phosphoproteins that are required for cytoplasm to vacuole targeting. J. Biol. Chem. 275, 25840-25849(2000)
Kamada, Y., Funakoshi, T, Shintani, T, Nagano, T,
Ohsumi, M, Ohsumi, Y. Tor-mediated induction of
autophagy via an Apgl protein kinase complex. J.
Cell Biol. 150, 1507-1513 (2000)
Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mi-

NATIONAL INSTITUTE

zushima, N., Yoshimori, T., Ohsumi, M., Noda, T.,
Ohsumi, Y. Membrane association cycle of Apg8 isessential for authophagy and the Cvt pathway. J. Cell
Biol. 151, 263-275 (2000)
Kabeya, Y., Mizushima, N., Ueno, T., Yamamoto, A.,
Kirisako, T., Noda, T., Kominami, E., Ohsumi, Y.,
and Yoshimori, T. LC3, a mammalian homologue of
yeast Apg8p is processed and localized in autophagosome membranes, EMBO. J. 19, 5720-5728,(2000)
Grote, E., Baba, M., Osumi, M. Ohsumi, Y., Novick,P. J. Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion. J. Cell Biol. 151, 453466 (2000).
Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi
Y, Ishihara N, Mizushima N, Tanida I, Kominami E,
Ohsumi M, Noda T, Ohsumi Y. A ubiquitin-likesystem mediates protein lipidation. Nature, 408, 488492 (2000)
Kihara, A., Noda, T., Ishihara, N., and Ohsumi, Y. Two
distinct Vps34 Ptdlns 3-kinase Complexes function
in autophagy and CPY sorting in Saccharomyces
cerevisiae. J. Cell Biol., 152, 519-530. (2001)
Kihara, A., Yamamoto, A., Ohsumi, Y., and Yoshimori
T., Beclin-Phsophatidylinositol 3-kinase complex
functions at the trans-Golgi Network. EMBO Report,
in press (2001)
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi,Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y.,
and Yoshimori, T. Dissection of autophagosome
formation using Apg5-deficient mouse embryonicstem cells. J. Cell Biol. in press (2001)
Komatsu,M., Tanida, 1., Ueno, T., Ohsumi, M., Ohsumi,
Y., and Kominami, E. The C-terminal region of an
Apg7p/Cvt2p is required for homodimerization andis essential for its E1-activity and E1-E2 complexformation. J. Biol. Chem. in press (2001)

Review:
Noda, T. Ohsumi, Y., Klionsky, D. J. The yeast vacuole: a paradigm for plant cell biologists? Annual
Plant Reviews, 5, 1-19 (2000)