10NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGYDIVISION OF MOLECULAR CELL BIOLOGY
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OHSUMI, Yoshinori
KAMADA, Yoshiaki
NODA, Takeshi IJ
SUZUKI, Kuninori
KABEYA , Yukiko
FUJIKI, Yuki
SEKITO, Takayuki
HANADA, Takao
NAKATOGAWA , Hitoshi
JCHIMURA , Yoshinobu
YOSHIMOTO, Kohki
BABA, Misuzu
OITA, Eiko
ONODERA , Jun
OHNEDA , Mamoru
OBARA , Keisuke
KAWAMATA , Tomoko JJ
MATSUI, Makoto 2;
KAGEYAMA, Takuya lJ
IMA MURA, Yuko
ICHJKA WA, Rie
TSUKESHJBA , Kumi
HARA , Yokotargeted to the membrane of nascent autophagosomes,
eventually sequestered into autophagosomes, and fina llytransported to the vacuolar lumen. Our previous
classification was performed under nutrient-richconditions. In order to obtain further insights for themechanism of autophagosome formation, we analyzed theintracellular localization of GFP-Atg8p in every atg
mutant under autophagy-inducing conditions. GFP-Atg8p
showed several distinct localization patterns in atg
mutants (Figure 1). Subsequent analyses revealed
epistatic relationships between Atg proteins during theprocess of autophagosome formation.
GrowingTerminalClass~(LI.atg4)

t:..vps30

IJ Study abroad.fi·om Mar. 20032) Graduate University for Advanced Studies

J) Kobe University

yThis division aims to understand the autophagy inrespects to its molecular mechanism and its physiologicalroles in yeast and higher eukaryotes. Cells executedegradation processes of their constituents, which are wellcoordinated to regulate the biological activities. In otherword, we must shed light on degradation process to fully
tmderstand the cell, because the study on the degradation
has been retarded compared to the biosynthetic process.
Autophagy is a major route for bulk degradation ofcytoplasmic
constituents
andorganelles
111
lysosome/vacuole, and is well conserved in eukaryotes.

I. Background
Upon nutrient starvation, autophagic process stmis asbuilding up membrane structures, autophagosomes, in thecytoplasm. The autophagosome sequesters cytosol andorganelles nonselectively. Then it is delivered to thevacuole/lysosome, and the cytoplasmic materials inside
are degraded by vacuolar/lysosomal hydrolases. We had
discovered autophagy in a simple model organism, S.
cerevisiae and morphologically and genetically defined
the whole process.

II. Interrelationships among Atg proteins during
autophagy in yeast, Saccharomyces cerevisiae
Macroautophagy is a bulk degradation process induced
nutrient starvation in eukaryotic cells. In
Saccharomyces cerevisiae, 16 ATG genes are essential forautophagosome formation. Recently, we demonstrated
that these ATG genes can be classified into three groups
on the basis of localization of GFP-Atg8p and Atg5p-GFP
under nutrient-rich conditions. In starving cells, Atg8p isby(LI.atg9)a(LI. atg1)

Figure I. Localization of GFP-Atg8p in atg mutants before
and after 24-hour rapamycin treatment. Cell s express ing
GFP-Atg8p were grown in SD + casamino ac id med ium
and observed under the fluorescence microscope. Cells
under growing conditions (Growing; A-D) and after
24-hour rapamycin treatment (Terminal; E-H). (A and E)
t':!.atg4 cells showing the class ~ terminal phenotype. (B andF) t':!.vps30/t':!.atg6 cells showing the t':!.vps30 tenninalphenotype. (C and G) t':!.atg9 ce lls showing a typical c lass yterminal phenotype. Arrows represent dots of GFP-Atg8p
close to the vacuole often seen in the class y mutants. (Dand H.) t':!.atgl ce ll s showing the class a. terminal phenotype.
Bar: 5 J.lll1 .

III. Starvation triggers the delivery of theendoplasmic reticulum to the vacuole via
autophagy in yeast
In the course of studying mechanistic aspect ofautophagy, we recently found that an active flow of
COPII vesicular traffic from the ER is required forautophagosome formation. There are many studies tofo llow the changes in expression levels of proteins under
the starvation, but it has not been studied how the protein
h·ansport pathway like early secretory pathway from theER, the production site of cell surface and secretory
proteins, is affected. Here we studied ER and Golgi
resident proteins to see if and how the starvation stress

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

modulates the early secretory pathway, and found anautophagic degradation of ER under starvation conditionsin addition to cytoso lic protein degradation (Figure 2).
Golgi membrane protein was not engulfed by th e
autophagosome under the same conditions, indicating thatthe uptake of ER by autophagosome is the specific event.
Although the ER exists in a network structure that ismu tually connected and resides predominantly around thenucleus and beneath the plasma membrane, most ofautophagosomes engulfed ER. The extent of the ER
uptake by autophagy was nearly identical to that of thesoluble cytosolic proteins. This phenomenon wasexplained by the appearance of fragmented ER membrane
structures in almost all autophagosomes. Fut1hermore, ER
dynamism is required for this process: ER uptake by
autophagosomes occurs in an actin dependent manner.
Visualizing these proteins revealed dynamic changes inthe early secretmy pathway under the starvation condition.
Significant amounts of both ER and Golgi proteins weretransported to the vacuole during starvation .

IIrecombinants and liposomes demonstrated that theefficiency of Atg8-PE conjugation was strongly affectedby lipid composition. Further, the Atg8 was linked tothe PE in liposomes, but not to the PE in the presence ofdetergent, suggesting that PE in a lipid bilayer isnecessmy for the Atg8 conjugation. These results shouldbe very useful for future works to determine w here
Atg8-PE conjugation occurs during autophagy in yeast.
Interestingly, we found that the PE-conjugated Atg8 has ahigher affinity for an antiserum raised against theN-terminal region of the Atg8 protein. Further, Atg8
became highly sensitive to trypsin digestion afterlipidation. Hence, we concluded that PE conjugationinduces a conformational change of Atg8 .

8~r • • • • • • • • • • • •Atg48GG¥
Rt~

t8 8 8G'A8~- -GD - Mrnt

AlPGc(El)

(EZ)

~

G/

Gc~PEB

Figure 3. Atg8-PE conjugation system
DIGFM

GFP-HDEL

MergedFigure 2. DsRed-HDEL is transp01·ted to the vacuole inan autophagy dependent manner with rapamycin
treatment. The fluorescence and the Nomarski images ofwild-type ce ll s (JK9-3da) expressing DsRed-HDEL (A)under growi ng condition and (B) und er rapamycin treatedco ndition ( I 0 hrs). The vacuolar limiting membrane waslabeled w ith FM4-64. The vacuolar lumen (noted "vac" )became tluorescent with starvation treatment.

IV. In vitro reconstitution of Atg8-PE conjugation
systemIn yeast, S. cerevisiae, Atg8 plays an important role
during autophagosome formation. We have previously
revealed that the Atg8 is covalently attached tophosphatidylethanolamine (PE) via a ubiquitin-likeconjugation system (Figure 3). The C-terminal Arg of117
newly synthesized Atg8 (Atg8R ) is removed by Atg4
protease to expose a Gly residue at the C-terminus
11 6(Atg8G 11 6). The Apg8G
is then activated by Atg7 (Elenzyme) and transferred to Atg3 (E2 enzyme).
116
Following these reactions, the Apg8G
conjugates to PE
tlu·ough an amide bond between its C-terminal Gly andthe amino group of PE. The subsequent deconjugation
reaction by Atg4 is necessary for the normal progressionof autophagy. Notably, Atg8 conjugation system, whi lesimilar to ubiqu itination mechanically, utili zes aubiquitous phospholipid, not a protein, as a target. We
further developed in vitro Atg8-PE reconstitution system.
The Atg8-PE was successfully reconstituted simply withAtg8G 11 6 , Atg7 and Atg3 by using in vitro system. These11 6
results confirmed that Atg8G , Atg7, and Atg3 are theminimum components for Atg8-PE conjugation reaction.
The in vitro Atg8-PE reconstitution system using

Carboxy-termi nal arginine (R) of newly synthesized Atg8 isremoved by Atg4 protease, leaving a glyc ine residue (G) at theC terminus (Atg8G 11 6). The Atg8G11 6 is activated by El enzyme,
Atg7 , and then transfetTed to E2 enzyme, Atg3 . Finally,
Atg8G 11 6 cova lently conjugates with phosphatidylethanolamine(PE) through an amide bond between the C-tenninal glycine
and the amino group of PE. Liberation of Atg8 moiety from
Atg8-PE by the act ion of Atg4 (deconjugation) is required forthe normal progression of autophagy.

V.

Autophagy-defective
senescenceplant

exhibitearly

So far, plant autophagy has been described in reports
by morphological studies . In addition, a recent
genome-wide search revealed significant conservation
among ATG genes in yeasts and plants. It has not been
proved, however, that Arabidopsis ATG genes are
involved in plant autophagy. To evaluate this requirement,
we examined the ubiquitination-like Atg8 lipidation
system. We generated transgenic Arabidopsis expressing
GFP-ATG8 fusion proteins and established a systemmonitoring autophagy in whole plants. In wild-type plants,
GFP-ATG8s were observed as ring shapes in thecytoplasm and were delivered to vacuolar lumens under
nitrogen-starvedconditions.
By
contrast,inT-DNA-insertion double mutant of the ATG4s which
encodes proteases required for C-terminal cleavage of
ATG8 (atg4a4b-l) , autophagosomes were not observed
and the GFP-ATG8s were not delivered to the vacuole. In
addition, we detected autophagic bodies in the vacuoles ofwild-type roots (Figure 4 panel A) but not in those of
atg4a4b-1 (Figure 4 panel B) in the presence of
concanamycin A , a V-ATPase inhibitor. The autophagy
defective mutant exhibited early senescence of rosette

NATIONAL INSTITUTE FOR BASIC BIOLOGYCELL BIOLOGY

12leaves even under nutri ent-rich conditions (Figure 4 panelD) and fewe r seeds under nutri ent-limiting conditions;howeve r, these plants were neverthe less able to undergo acom plete life cycle. In addition, atg mutant di splayed areduction in
the growth rate of roots undernutri ent-limiting conditions . Autophagy is required forthe m ai ntenance of cellular viability regardless of thenutrient conditions and contributes to the development ofa root system under co nditions of nutrient limitation.

form-ll formation. J. Cell Sci. 117, 2805-12 (2004)
Ichimura T, Kubota H, Goma T , Mizushima N , Ohsumi Y,lwago M, Kakiuchi K, Shekhar HU , Shinkawa T, Taoka
M, Ito T, Isobe T. Transcriptomic and proteom ic analys isof a 14-3-3 ge ne-deficient yeast.
Biochemist1y 43,
6 149-58.
Okazaki H, Ono B, Ohsumi Y, Ohsumi M. , apg 15-l , a UGA
mutant allele in the Saccharomyces cerevisiae APG 16
gene, and its suppressio n by a cytoplas mic factor. BiosciBiotechnol Biochem. 68, 1541-8 (2004)
Sugawara K, Suzuk i NN , Fujioka Y, Mi zushima N, Ohsumi
Y,lnagakiF.
Thecrysta lstructureofm icrotub ule-associated protein li g ht chain 3, a mamma li an
ho mologue of Saccharomyces cerevisiae Atg8. Genes.
Cells 9, 6 11-8 (2004)
Ichimura Y, Imamura Y, E moto K, Umeda M , Noda T,Ohsumi Y. In vivo and in vitro reconstitution of Atg8
conjugation essential for autophagy . J. Bioi. Chem. 279,40584-92 (2004)
Suzuki K, Noda T, Ohsumi Y., Interre latio nships among Atg
proteins during autoph agy in Saccharomyces cerevisiae.
Yeast 21 , 1057-65 (2004)
Yoshimoto K, H anaoka H, Sato S, Kato T, Tabata S, Noda T,Ohsumi Y., Processi ng of ATG8s, ubiquitin-li ke prote ins,and their deconjugation by ATG4s are essentia l for plant
autophagy. Plant Cel/ 16, 2967-83 (2 004)
Kuma A, Hatano M , Matsui M, Yamamoto A, Nakaya H,
Figure 4 . atg mutant is defective in accum ul ation ofau tophagic bodies and exhibited early senescencephenotype(A) Electron micrographs of wild-type roots treated withconca namycin A.
(B) Electron mi crographs of atg4a4b-l mutant roots treatedwith concanamycin A.
Roots of one-week-old wild-type and atg4a4b-l seedlings
were treated with concanamyc in A ( 1 ~lM ) under
nitrogen-starved conditions for 6 h. The same reg ions of
the root tip were compared. Bars = 5 f.ll11(C) 7-week-old wi ld-type plant(D) 7-week-old atg4a4b-l plant

Publication List:
Mizushima, N., Methods for monitorin g autophagy. Int. J.
Biochem. Cell Bioi. 36 , 2491 -2502 (2004)

Yoshimori T, Ohsumi Y, Tokuh isa T , Mi zushima N. The
ro le of autophagy during the earl y neonata l starvati on
period. Natu re in press (2004)
Ano Y, Hattori T, Oku M, Mukaiyama H, Baba M , Ohsumi
Y, KatoN, Sakai Y ., A Sorting Nexin PpAtg24 Regul ates
Vacuo lar Membrane Dynami cs during Pexophagy via
Binding to Phosphatidylinosito l-3-Phosphate. Mol Bioi.

Cell in press (2004)
Shimazu M, Sekito T, Akiyama K, Ohsumi Y, Kakinuma Y.
A fa mil y of basic amino acid transporters of the vacuo larmembrane from Saccharomyces cerevisiae. J Bioi. Chem. ,in press (2004)
Hamasak i M, Noda T, Baba M, Ohsumi Y ., Starvation
Triggers the D eli ve1y of the Endoplasmic Reticulum to theVacuole via Autophagy in Yeast. Traffic

6, 56-65 (2005)

Mukaiyama H, Baba M, Osumi M, Aoyag i S, Kato N ,Ohswni Y, Sakai Y., Modification of a ubiquitin-like

Reviewprotein Paz2 conducted micropexophagy throughfo rmation of a nove l membrane stru cture. Mol. Bioi. Cell15, 58-70 (2 004)

Ohs umi Y., Cellul ar recycling system- molec ular m echani smof autophagy. In Cell Growth, Cold Spring Harbor Press,
pp 412-429 (2004)

Mizushima N, Yamamoto A, Matsui M, Yoshimori T,
Ohsumi Y., In vivo ana lysis of autophagy in response tonutri ent starvation using tran sgeni c mice expressi ng afluorescent autophagosome marker. Mol. Bioi. Cell 15,
11 01-11 (2004)

Ohsumi Y. , Mizuhs ima N. , Two ubiquitin-like conjugationsystems essenti al for autophagy. Semin. Cell Dev. Bioi. IS,231 -6 (2004)
Kamada, Y., Sekito,T., Ohsumi Y., Autophgy in yeast: aTOR-med iated respo nse to nutri ent starvation . Curr Top
Microbial !mmunol. 279, 73-84 (2004)

Onodera J, Ohsumi Y. , A ld6p is a prefened target forautophagy in yeast, Saccharomyces cerevisiae. J. Bioi.
Chem. 279, 1607 1-6 (2004)
Kabeya Y, Mizushima N, Yamamoto A, Os hitani-Okamoto S,O hsum i Y, Yoshimori T., LC3, GABARAP and GATE I6locali ze to autophagosoma l membrane depend ing on

Ohsumi , Y. and Mi zushima, N. T wo ubiquitin-li ke
conjugation systems essen ti al for auto phagy. Sem. Cell
Dev. Bioi., in press (2004)
Suzuki K. , Ohsumi Y. , A utophagy of yeast as a syste m forselective protein transpo rt to the vac uole. Jikken Jgaku 22,
150-4 . (2004)