DIVISION OF MOLECULAR CELL BIOLOGY

Professor

OHSUMI, Yoshinori
Assistant Professors

Technical Staff
NIBB Research Fellow
Postdoctoral Fellows

Graduate Students
Technical Assistants

Secretaries

KAMADA, Yoshiaki
SUZUKI, Kuninori
NAKATOGAWA, Hitoshi
KABEYA, Yukiko
OBARA, Keisuke
HANADA, Takao
FUJIKI, Yuki
HARASHIMA, Toshiaki
OKU, Masahide
YAMAMOTO, Hayashi
OKAMOTO, Koji
HAMASAKI, Maho
SEKITO, Takayuki
YOSHIMOTO, Kohki
KAWAMATA, Tomoko
KAGEYAMA, Takuya
OOOKA, Kyoko
KONDO, Chika
NIIMI, KaoriISHII, Junko
OKAMOTO, Noriko
TSUKESHIBA, KumiICHIKAWA, Rie
HARA, Yoko
SUZUKI, Yuko

All cellular activity is maintained by the balance between
the synthesis and degradation of related proteins.
Degradation processes, therefore, play important roles inmany physiological aspects as well as the regulation of gene
expression. Autophagy is a bulk degradation system forcytosolic proteins and organelles in lysosomes/vacuoles thatis highly conserved in eukaryotic cells. This division aims tounderstand the physiological roles and molecularmechanisms of autophagy in yeast and higher eukaryotes.

Ⅰ. BackgroundUpon nutrient starvation, the autophagic process starts as abuilding up of membrane structures called autophagosomesin the cytoplasm. The autophagosome sequesters cytosol andorganelles nonselectively. It is then delivered to thevacuole/lysosome, and the cytoplasmic materials inside aredegraded by vacuolar/lysosomal hydrolases. We discovered
autophagy in a simple model organism, the budding yeast
Saccharomyces cerevisiae, and morphologically andgenetically defined the whole process.

Ⅱ. Identification and characterization of autophagyspecific proteins, Atg17, Atg29, and Atg31In S. cerevisiae, autophagy is induced under nutrientstarvation. In growing conditions another autophagy-relatedtransport, the Cvt pathway, is observed. While most of theAtg proteins are involved in both pathways, only a few Atg
proteins, such as Atg17 and Atg29, are uniquely required for

starvation-induced autophagy. Identification of novel
autophagy-specific Atg protein(s) and their characterization
are urgent issues for the elucidation of the induction
mechanism of autophagy.
We identified Cis1/Atg31 as a third autophagy-uniqueprotein. ATG31 null mutant cells were defective inautophagy and lost viability under nitrogen-depletedconditions.
Atg29 and Atg31 localize to the pre-autophagosomal
structure (PAS; a putative generation site of theautophagosome) in an ATG17-dependent manner (Figure 1),and Atg17-Atg29 and Atg17-Atg31 physical interactions aredetected, suggesting that these three proteins make a ternarycomplex. We further observed that this autophagy-uniqueprotein complex binds to Atg1, and this association isregulated by nutrient conditions. When the Cvt pathway is
disrupted by deletion of ATG11, encoding a Cvt-specificprotein, recruitment of the Atg17 complex to the PAS isstimulated by starvation. In addition, in atg11Δ cells, thetrimeric complex is required for the PAS recruitment ofother Atg proteins under starvation conditions. Thesefindings indicate that the Atg17 complex plays an importantrole in the assembly of other Atg proteins to the autophagyspecific PAS to generate the autophagosome.

Figure 1. Atg31 localizes to the PAS. Yeast cells expressing Atg31-GFP
and mRFP-Ape1 were incubated with rapamycin for 3 h. Fluorescence andDIC images are shown. Ape1 is used as a marker for the PAS. Bar: 2μm.

Ⅲ. Functions of two ubiquitin-like conjugatesin autophagosome formation
Autophagosome formation involves two ubiquitin-likeproteins, Atg8 and Atg12. We showed that Atg12 forms a
conjugate with another Atg protein, Atg5, whereas Atg8 isconjugated to a lipid, phosphatidylethanolamine (PE). We
have reported on the functions of these conjugates using invitro reconstitution systems consisting of purified protein
components and PE-containing liposomes.
First, we showed that Atg8 mediates the tethering andhemifusion of membranes (liposomes), which are evoked bylipidation of the protein and reversibly modulated by Atg4
that catalyzes the deconjugation of Atg8-PE (Figure 2A). In
addition, mutational analyses suggested that these functionsof Atg8 observed in vitro are required for autophagosome
formation in vivo; membrane tethering and hemifusion
represent bona fide functions of Atg8. These results provide
key insights into the unique membrane dynamics ofautophagy, which cannot be explained by the mechanismsthat have been elucidated in conventional vesiculartrafficking systems.
Previous in vivo observations have suggested that twoubiquitin-like conjugation systems have an interrelationship:11

National Institute for Basic Biology

Cell Biology

the Atg12-Atg5 conjugate is required for effective formationof Atg8-PE. We purified the Atg12-Atg5 conjugate formed
in E. coli and found that it dramatically accelerates the Atg8PE conjugation reaction in vitro, in which transfer of Atg8
from the E2 enzyme Atg3 to PE is thought to be activatedthrough the direct interaction between Atg12-Atg5 and Atg3
(Figure 2B). These results indicate that Atg12-Atg5 serves
as a novel E3 enzyme in the ubiquitin-like conjugation
reaction of Atg8. Further studies will shed light onunderstanding how these ubiquitin-like systems cooperate in
autophagosome formation in the cell.

Figure 2. Functions of two ubiquitin-like protein conjugates. (A)Liposomes clustered upon in vitro production of Atg8-PE were stainedwith phosphotungsticacid and analyzed by electron microscopy.
Arrowheads and an arrow, respectively, show junctions between
liposomes and a structure suggested to represent hemifusion ofliposomes. (B) In vitro conjugation reaction of Atg8-PE was performedin the presence or absence of the Atg12-Atg5 conjugate.

condition, which was dependent on the PI 3’-kinase
components necessary for the cell viability. Moreover, theproduct of the PI 3’-kinase, phosphatidylinositol 3’monophosphate (PI3P), was found to be enriched at the siteof vacuole invagination (Figure 3B). Subsequentmorphological studies indicated that the vacuole
invaginations are followed by a transport of lipid bodies into
vacuole, thereby showing that MIA toward lipid bodies isinduced under the stress condition.

Figure 3. Membrane dynamics of MIA. (A) Schematic drawing ofvacuolar membrane morphology undergoing MIA during glycerol
culture at 37ºC. A part of a round vacuole (1) forms an invagination (2)dependent on some components of the PI 3’-kinase complex. Within theinvagination a smaller vesicle structure emerges (3) and buds off into
vacuolar lumen (4). (B) Localization of PI3P at the site of vacuole
invagination. A tandem repeat of FYVE domain known to specificallybind to PI3P is expressed in fusion to monomeric RFP (mRFP).
Fluorescence microscopy of the mRFP-(2x)FYVE was done for thedenoted strains. Along with the mRFP signal (shown as PI3P), Nomarski
images (Nomarski) and their superimposed images (Merge) are also
shown. Bar, 2 μm.

Ⅳ. Identification of another type of autophagy(microautophagy) under a stress condition
Our efforts to elucidate the molecular mechanism ofautophagy have led to the accumulation of enormous
amounts of knowledge regarding macroautophagy(macroautophagy is here simply described as autophagy). Incontrast, little is known at present about the physiological
functions and molecular machinery of another type ofautophagic pathway, termed microautophagy (MIA). In
MIA cargo components are sequestered directly by thevacuolar (lysosomal) membrane and transported into thevacuolar lumen after an inward pinch-off of the sequestering
membrane (Figure 3A). In the yeast S. cerevisiae, MIA has
been observed under some nutrient-starved conditions, but
its biological significance remains unclear.
After phenotypic analyses of 18 ATG-gene disruptants, wefound that disruptions of genes encoding a subset ofphosphatidylinositol (PI) 3’-kinase complex led to severe
viability defects when the cells were cultured on glycerol asa solo carbon source at 37ºC and eventually exposed toextensive oxidative stress. The frequency of the cells
possessing vacuole invaginations increased under the culture

12Ⅴ . Lap3p is a novel cargo of selective
autophagy during glycerol culture
Under growing conditions, a constitutive autophagy-likepathway, termed the Cvt (cytoplasm-to-vacuole-targeting)pathway, utilizes similar molecular machinery and vesicle
structures to those of autophagy for a biosynthetic transportof aminopeptidase I (Ape1) into the vacuole. Recently, wediscovered that in the cells grown on glycerol as a solecarbon source, the Cvt pathway was enhanced. Under thiscondition, we found that Leucyl aminopeptidase Ⅲ (Lap3),
a neutral cysteine protease, was the selective cargo proteinof the Cvt pathway (Figure 4A). Lap3 co-localized with
Ape1 (Figure 4B), and Atg19 was involved in delivering
Lap3 to the vacuole as a receptor protein. Further studies
showed that a portion of Lap3 was degraded inside vacuoles,and thus suggested that the Cvt pathway might function as adegradative pathway and that Lap3 might be a cargo of thepathway.

Publication List
〔Original papers〕● Adachi, W., Suzuki, N.N., Fujioka, Y., Suzuki, K., Ohsumi, Y., andFigure 4. (A) A proteinase A deficient (pep4Δ) and pep4Δ atg19Δ
cells expressing GFP-Lap3 were grown to logarithmic phase in theglycerol medium and examined by differential interference contrast(DIC) and fluorescence microscopy. Lap3 was selectively delivered tovacuole by the Cvt pathway. Arrows indicate the intravacuolar structures
including GFP-Lap3 positive structures. Arrowheads indicate thepunctate structures near vacuole. (B) Cells co-expressing GFP-Lap3 andRFP-Ape1 were examined by microscopy as described in (A). Lap3 colocalized with Ape1 near the vacuole. Bar, 4 μm.

Inagaki, F. (2007). Crystallization of Saccharomyces cerevisiaeaminopeptidase 1, the major cargo protein of the Cvt pathway. Acta
Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 63, 200-203.
● Fujiki, Y., Yoshimoto, K., and Ohsumi, Y. (2007). An Arabidopsis
homolog of Yeast ATG6/VPS30 is essential for pollen germination.
Plant Physiology 143, 1132-1139.
● Hanada, T., Noda, N.N., Satomi, Y., Ichimura, Y., Fujioka, Y., Takao,T., Inagaki, F., and Ohsumi, Y. (2007). The Atg12-Atg5 conjugate
has a novel E3-like activity for protein lipidation in autophagy. J. Biol.
Chem. 282, 37298-37302.
● Kabeya, Y., Kawamata, T., Suzuki, K., and Ohsumi, Y. (2007).
Cis1/Atg31 is required for autophagosome formation in
Saccharomyces cerevisiae. Biochem. Biophys. Res. Comm. 356, 405410.
● Matsushita, M., Suzuki, N.N., Obara, K., Fujioka, Y., Ohsumi, Y., andInagaki, F. (2007). Structure of Atg5-Atg16, a complex essential forautophagy. J. Biol. Chem. 282, 6763-6772.
● Nakatogawa, H., Ichimura, Y., and Ohsumi Y. (2007). Atg8, aubiquitin-like protein required for autophagosome formation, mediates
membrane tethering and hemifusion. Cell 130, 165-178.
● Satoo, K., Suzuki, N.N., Fujioka, Y., Mizushima, N., Ohsumi, Y., andInagaki, F. (2007). Crystallization and preliminary crystallographic
analysis of human Atg4B-LC3 complex. Acta Crystallograph. Sect. FStruct. Biol. Cryst. Commun. 63, 99-102.
● Suzuki, K., Kubota, Y., Sekito, T., and Ohsumi, Y. (2007). Hierarchyof Atg proteins in pre-autophagsomal structure organization. GenesCells 12, 209-218.
● Yamada, Y., Suzuki, N.N., Hanada, T., Ichimura, Y., Kumeta, H.,
Fujioka, Y., Ohsumi, Y., and Inagaki, F. (2007). The crystal structureof Atg3, an autophagy-related ubiquitin carrier protein (E2) enzymethat mediates Atg8 lipidation. J. Biol. Chem. 282, 8036-8043.
● Yamaguti, M., Suzuki, N.N., Fujioka, Y., Ohsumi, Y., and Inagaki, F.
(2007). Crystallization and preliminary X-ray analysis of Atg10. Acta
Crystallograph. Sect. F Struct. Biol. Cryst. Commun. 63, 443-445.

〔Review article〕● Suzuki, K., and Ohsumi, Y. (2007). Molecular machinery ofautophagosome formation in yeast, Saccharomyces cerevisiae. FEBS
Lett. 581, 2156-2161.

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