14NATIONAL INSTITUTEDIVISION OF BIOENERGETICS
Professor:
Associate Professor:
Research As$ociate:

Yoshinori Ohsumi
Tamotsu Yoshimori
Takeshi Noda
Yoshiaki Kamada
Post Doctral Fellow: Noboru Mizushima 1
Takahiro Shintani 2 (from April)
Naotada Ishihara 1 (from July)
Graduate Students:
Takayoshi Kirisako(Graduate Univ. of Advanced Studies)
Yoshinobu lchimura(Graduate Univ. of Advanced Studies)Technical Staff:
Yukiko Kabeya
Visiting Fellow:
Fumi Yamagata(Teikyo Univ. of Sci. and Tech.)
Visiting Scientist:
Kazuaki Furukawa(from November)
Satsuki Okamoto

This division aims to elucidate the physiologicalrole and mechanism of intracellular protein degradation in a lytic compartinent. Up to recently, molecularbiologicaf studies on the topic of cellular regulation
have focused on protein synthesis, and (the control of)gene expression. However, it is clear that cellular activity is maintained by a delicate balance between
synthesis and breakdown of individual proteins. For
the last few years, protein degradation has become ahot field in cell biology, because it plays critical roles
in many aspects of cellular functions. Most of the selective protein degradation is believed to occur in thecytosol through the ubiquitin/proteasome system. In
contrast, major bulk protein degradation takes place inthe lysosome/vacuole. Non-specific protein turnover
has essential roles under nutrient stress or for cell differentiation of eukaryotic cells. Until now, the molecular details of protein degradation in the lytic
compartment remained to be uncovered due to thelack of a sensitive monitoring system, and the dynamic features of the lysosomal/vacuolar membranesystem.
Autophagy is defined as the process of bulk degradation of cytoplasmic proteins or organelles in thelytic compartment and seems to be a ubiquitous andbasic cellular activity in all eukaryotic cell types.
Autophagy has been described mostly in higher
eukaryotic cells by using electron microscopy. The
lysosome/vacuole is a single membrane bound compartment containing various hydrolytic enzymes. Segregation of lytic enzymes from the cytosol requires anobvious cell biological process how to deliver proteinsor organelles destined for degradation into the lumenof this compartment. Macroautophagy is the majorroute to the lysosome/vacuole under nutrient starvation conditions. This process involves the formation of
a double membrane structure called an autophagosome (AP). The AP fuses with a primary lysosome and matures to be an autophagolysosome, thenits contents are digested and recycled to be reused.
Autophagy has been studied extensively using various kinds of mammalian tissues and cultured cells. It
is highly regulated by various factors such as aminoacid or metabolites, hormones and growth factors, andsecond messengers. Because the modes of autophagy
are so diverse and the physiological role and regulation of autophagy depend upon the cell type andphysiological demands, it is difficult to present a general model for autophagy in higher eukaryotes. For
this reason, it has been helpful to develop a simple
system to elucidate the fundamental machinery of theautophagic process.
Yeast Induces Autophagy as Mammalian Cells
Recently we discovered yeast, S. cerevisiae, induces
bulk protein turnover in the vacuoles under starvation
conditions. This whole process corresponds to that ofmacroautophagy in higher eukaryotic cells. Byelectron microscopic analyses we succeeded in
detecting double membrane structures in thecytoplasm enclosing a portion of cytosol. This
autophagosome immediately fuses with vacuoles,delivering single inner membrane structures,
autophagic body (AB) in the vacuole. When vacuolar
proteinase activities are blocked genetically or by
specific inhibitor ABs are accumulated in thevacuoles. They move around vigorously in thevacuoles by Brownian motion, and are easily
detectable by light microscope. Thus we can follow
the progression of autophagy as the accumulation of
ABs in real time. Biochemical and immunoelectron
microscopic analyses showed that starvation-induced
sequestration is non-selective, that is, cytosolic
enzymes and organelles are sequestered randomly tothe vacuoles to be degraded.
Autophagy in yeast is a kind of general stress response to adverse environmental condition and is induced by various starvation conditions such as
nitrogen, carbon, sulfate, and even amino acid depletion. Signal transduction of starvation for induction ofautophagy is a key question to be solved. Recently wefound rapamycin induces autophagy in the cellgrowing in a rich medium and that Tor, phosphatidylinositol kinase homologue plays essential role
for the regulation of autophagy.
Another crucial and controversial question is themechanism of formation of autophagosome. Still
nothing is clear about the origin of the membrane.
Freeze-fracture electron microscopy showed that
autophagosomal membrane has quite characteristicfeature and the density of intramembrane particles is
extremely low as compared with other intracellular
membrane.
Genetical dissection of autophagic process
Yeast has made great contribution to solve many
fundamental problems in cell biology because of tractability of genetic and molecular biological techniques. In order to dissect the complex process ofautophagy to its elementary steps we started to isolate
mutants defective in the process of autophagy. Total
14 autophagy defective agp CAutQphagy) mutants

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FOR BASIC BIOLOGY

APG 1 codes a novel Serffhr protein kinase essential
for the induction of autophagy. This provides the first
direct evidence for involvement of protein phosphorylation in the process of autophagy. Apg13p interacts
with Apglp and might function as a regulator of its
kinase activity. So far most APG genes are novel except APG6 and non-essential for vegetative growth.
The APG6 gene is allelic to VPS30 , which is required
for retrieval of the carboxypeptidase, CPY receptor tothe late Golgi from the endosome. APG 14 encodes anovel hydrophilic protein of 40.5kDa. Subcellular
fractionation experiments indicate that both Apg14p
and Apg6p are peripheral membrane proteins andform a stable complex. The apg14 mutant is normalfor CPY sorting. While overexpression of Apg14p
suppresses the autophagy defect of apg6-1, APG14
does not function as a multicopy suppressor of theCPY sorting defect of this mutant. These results suggest that Apg6p has dual functions in autophagy andvacuolar protein sorting, and that Apg14p is specifically required for the autophagic process.
Using the assay system of autophagy we developed,
we are studying genetic interaction among APG
genes. Some APG gene products are starvation-inducible or change phosphorylation state by nutrient starvation. Analyses of intracellular localization of theseApgps may provide us specific marker to elucidate themembrane dynamics during autophagy.

were isolated. They cannot induce protein degradation
upon shift from growth medium to the starvation
medium. They grow normally in a rich medium, but
start to die after 2 days in the starvation medium. Thissuggests that the autophagy is necessary for the maintenance of cell viability under starvation. Homozygous diploid of each apg mutant cannot undergo
sporulation, indicating that this cell differenciation
requires bulk protein degradation via autophagy.

APG genes are essential for Cvt pathway
One of vacuolar enzymes, aminopeptidase I, API issynthesized as a proform and sequestered to the vacuole directly from cytosol to the vacuole. All apg genes
are necessary for this process. Electron microscopic
analyses revealed that API first forms a complex in thecytosol and delivered to the vacuole by the distinct but
topologically similar mechanism to themacroautophagy. Though this Cvt pathway is biosynthetic and constitutive, and kinetically quite different
from the autophagy, but it must share most machinerywith the autophagy.

Analyses of APG genes
Now we are focusing on characterization of APGgenes, essential for autophagy. So far we have finished cloning and identification of twelve APG genes.

StarvationGrowing
TorI'·~Tor

Cvt Vesicle
Aminopeptidase IVacuoleFig. 1 Schema of autophagy in yeast. When yeast cells face to starvation, isolation membrane starts to enclose a portion ofcytosol and form an autophagosome. Autophagosome fuses to the vacuolar membrane, delivering autophagic body in the vacuoles(Autophagic pathway). Under growing condition one of vacuolar enzymes, API is selectively sequestered to the vacuole (Cvtpathway). Two distinct pathways share most molecular machinery.

16NATIONAL INSTITUTE

Novel protein conjugation system essential for autophagy
Recently we found that four APG genes are involved in a novel protein conjugation system essential
for autophagy and the Cvt pathway. Apgl2p is a2lkDa hydrophilic protein. Western blot analysis ofHA-tagged Apgl2p shows an additional band at
70kDa in addition to a band with the expected molecular mass. Sequence analysis of APG5 shows thatit encodes a hydrophilic protein of 34kDa. A western
blot of Apg5p also shows the 70kDa band, suggestingcovalent binding between Apg5p and Apg12p. Elimination of a single Gly at the C-terminus of Apg12p
results in a complete loss of the higher molecularmass band. Similarly, substitution of Lys 149 ofApg5p, among 19 Lys residues, with Arg also abolishes this band. These results strongly suggest that theC-terminal Gly of Apg12p is conjugated to Lysl49 of
Apg5p via an isopeptide bond. Cells expressing thesemutant forms of Apg12p or Apg5p are defective inautophagy -and also Cvt pathway, indicating that theconjugate formation of Apg5p-Apg12p is essential forboth pathways.
Furthermore, among the apg mutants , apg7 andapglO, could not .form the Apg5p-Apg12p conjugate.
These two genes may encode enzymes required forthe conjugate formation . From sequence data, Apg7p
is predicted to be an E1 like activating enzyme for
Apgl2p. Recently, many ubiquitin-related molecules
have been identified, and shown to function in various
regulatory steps. However, Apg12p has no homology
with ubiquitin or ubiquitin-related molecules. Apg12p
is much larger than ubiquitin and seems to have asingle target, Apg5p. The Apg5p-Apg12p conjugation
reaction was reconstituted in vitro and shown to beATP-dependent. Similarly, mutations in the ATP-binding site of Apg7p block conjugate formation . The
function of this conjugate in the Apg and Cvt path-( Apg12p )G~GG

Approach to mammalian autophagy
Then, there is no reason for us hesitating to challenge mammalian autophagy again, by taking advantage of identified genes involved in yeast autophagy.
By searching databases, we have found several mammalian eDNA related to the yeast genes. Homologuesof Apgl2p and Apg5p are widely distributed fromyeast to higher organisms . We cloned recently thehuman homologues of Apg12p and Apg5p and demonstrated a similar conjugation reaction in mammaliancultured cells. This result strongly suggests that thenovel conjugation system is conserved in all eukaryotes. To elucidate roles of autophagy in development,
differentiation, physiology, pathology in a multi-cellular system, we are making Apg 12-deficient mice.
We also found that one of the Apgp homologues is
specifically associated to the autophagosome membrane in human cultured cells. Post-translational processing of the molecule regulates the association. Thisis the first identified protein that is localized in mammalian autophagosomes. Yeast Csc1p is an AAA-type
ATPase implicated in regulation of autophagy. We
showed evidences that mouse SKD1 protein homologous to Csc1p plays a pivotal role on membrane transport via early endosomes in human cultured cells.
This suggests involvement of endosomal system in theautophagic process.
Knowledge in yeast must give us key to uncover themechanism of autophagy in higher eukaryotic organisms such as mammals. We are now developing anassay for monitoring autophagy in mammalian cultured cells to perform systematic evaluation of identified candidates for components in machinery of
mammalian autophagy.

( Apg1 2p )G( Apg12p )GII?

ATP AMP+PPi\. }ways is not known yet but that the conjugation may benecessary for autophagosome or Cvt vesicle formation.

... (ATP AM P+PPicApg7pc) ____. ( Apg10p)( Apg12p )GI... (KApg5p)~GI\___} ... (~1----. ( target protein )Fig. 2 The Apg12p conjugation system and ubiquitin system
Apgl2p is activated by Apg7p in an ATP-dependent manner. After formation of a thioester with Apg7p, and then possibly with
ApglOp, Apg l2p is fin ally conjugated to Apg5p via an isopeptide bond (upper).
Although Apgl2p has no homology to ubiquitin, the Apg12p conjugation system is quite similar to that of ubiquitin (lower) whichis involved in selective proteolysis.

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Publication List:
Kametaka, S., Okano, T., Ohsumi, M., and Ohsumi, Y.
Apg14p and Apg6Nps30p form a protein complex
essential for autophagy in the yeast, Saccharomyces
cerevisiae. J. Biol. Chern. 273, 22284-22291 (1998).
Mizushima, N., Noda, T, Yoshimori, T., Tanaka, Y.,Ishii, T., Geoge, M. D., Klionsky, D. J. Ohsumi, M.,
Ohsumi, Y. A protein conjugation system essentialfor autophagy, Nature 395, 395-398 (1998).
Mizushima, N., Sugita, H., Yoshimori, T., andOhsumi, Y. A New Protein Conjugation System inHuman. The counterpart of the yeast Apg12p conjugation sytem essential for autophagy. J. Biol. Chern.
in press.
Noda, T. and Ohsumi, Y. Tor, a phosphatidylinositol
kinase homologue, controls autophagy in yeast. J.
Biol. Chern. 273, 3963-3966 (1998).
Tagawa Y., Yamamoto A.,Yoshimori T., Masaki R.,
Omori K., Himeno M., Inoue K., Tashiro Y. A 60
kDa plasma membrane protein changes its localization to autophagosome and autolysosome membranes during induction of autophagy in rathepatoma cell line, H-4-11-E cells. Cell Struct.
Funct. in press.
Tanida, 1., Mizushima, N., Kiyooka, M., Ohsumi, M.,
Ueno, T., Ohsumi, Y., and Kominami, E. Apg7p/
Cvt2p: a novel protein activating enzyme essential
for autophagy. Mol. Biol. Cell, in press.
Yamamoto A., Tagawa Y., Yoshimori T., Moriyama Y.,
Masaki R., Tashiro Y. Bafilomycin Al preventsmaturation of autophagic vacuoles by inhibiting
fusion between autophagosomes and lysosomes inrat hepatoma cell line, H-4-11-E cells. Cell Struct.
Funct. 23; 33-42 (1998).

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