13FOR BASIC BIOLOGY

to the lytic compartment (Fig.l ). Since cellular lyticcompartment, lysosome, was identified, biogenesis oflysosomal enzymes and their enzymatic characterization have been studied thoroughly. However, themechanisms of delivery of proteins to the lysosomes
are not known at a molecular level. Biochemical
analysis of lysosome/vacuole system is quite difficult
because of its complexity and dynamic feature. Autophagic process has been studied mostly by electron
microscopy, and molecular mech 1ism of autophagy
remains to be elucidated.

DIVISION OF BIOENERGETICSYoshinori Ohsumi
Tamotsu Yoshimori
Takeshi Noda
Yoshiaki Kamada
Post Doctral Fellow: Tomoko Funakoshi 1
Noboru Mizushima 2
Satoshi Kametaka (Univ. of Tokyo)
Graduate Students:
Takayoshi Kirisako (Univ. ofTokyo)
Yukiko Kabeya
Technical Staff:
Fumi Yamagata (Teikyo Univ. ofVisiting Fellow:
Sci. and Tech.)

Professor:
Associate Professor:
Research Associate:

Yeast Induces Autophagy as Mammalian Cells
Recently we discovered yeast, Saccharomyces
cerevisiae, induces bulk protein turnover in the vacuoles under starvation conditions. This whole process
corresponds to that of macroautophagy in higher eukaryotic cells. By electron microscopic analyses wesucceeded in detecting double membrane structures in
the cytoplasm enclosing a portion of cytosol. These
yeast autophagosomes immediately fuse with vacuoles, delivering single inner membrane structures, autophagic bodies in the vacuole. When vacuolar
proteinase activities are blocked genetically or by specific inhibitor such as PMSF autophagic bodies areaccumulated in the vacuoles. They moves around vigorously in the vacuoles by Brownian motion, and areeasily detectable by light microscope. Thus we canfollow the progression of autophagy as the accumula-This division aims to elucidate the mechanism andits regulation of intracellular protein degradation in alytic compartment. Recently it was realized that degradation process plays essential roles for various
physiological function. In the cytoplasm selectiveprotein degradation takes place by ubiquitin/proteasome system. Short-lived or abnormal proteins
are selectively eliminated by this pathway. Whilevacuole/lysosome contributes to the bulk turnover ofcytosolic and organelles proteins.
Little is known about the mechanism of protein
degradation in contrast with that of biosynthesis.
Bulk protein degradation is induced by various nutrient starvation condition, which is obligatory to celldifferentiation and maintenance of cell viability. Autophagy is a major route for sequestration of proteins

(a)

(b)

Fig. l. (a) Freeze-Fracture Image of Yeast under Nitrogen Starvation.
When vacuolar proteinases are blocked, spherical membrane structures, autophagic bodies accumulate in the vacuole.
(b) Fusion of Autophagosome to the Vacuolar Membrane.
Double membrane structure, autophagosome (AP), encloses a portion of the cytosol, then fuses with the vacuole to deliver an
autophagic body (AB) in the vacuole. These membrane structures show very few intramembrane particles.

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NATIONAL INSTITUTE

tion of autophagic bodies in real time. Biochemicaland immunoelectron microscopic analyses of vacuoles
containing autophagic bodies showed that starvationinduced sequestration is non-selective, that is, any
kind of cytosolic enzymes and organelles are sequestered randomly to the 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 single amino aciddepletion. Signal transduction of starvation for induction of autophagy is a key question to be solved.
Recently we found rapamycin induces autophagy inthe cell growing in a rich medium and that Tor, phosphatidyl inositol kinase homologue plays essential
role for the regulation of autophagy (Fig.2).
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.

Starvation

Genetical dissection of autophagic process
Yeast, S. cerevisiae, has made great contribution tosolve many fundamental problems in cell biology
because of tractability of genetic and molecular biological techniques . In order to dissect the complex
process of autophagy to its elementary steps westarted to isolate mutants in the process of autophagy,taking advantage of morphological selection underlight microscope. Total 14 autophagy defective mutants, agp, were isolated. They cannot induce protein
degradation upon shift from growth medium to thestarvation medium. They grow normally in a richmedium, but start to die after 2 days in the starvation
medium. This suggests that the autophagy is necessary for long time maintenance of cell viability. Homozygous diploid of each apg mutant is sporulationdefective as expected.
Analyses of APG genes
Now we are focusing on characterization of these
APG genes, essential for autophagy. So far we havefinished cloning and identification of twelve APGgenes. APGJ codes a novel Ser/Thr protein kinaseessential for the induction of autophagy. This pro-GrowingTorinactive

Autophagosome

0//Ap~

Cvt Vesicle®

Aminopeptidase I

ril

Tor
active\Vacuole\\o~ oFig. 2 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.

FOR BASIC BIOLOGY

vides the first direct evidence for involvement of protein phosphorylation in the process of autophagy.
Apg13p interacts with Apg1p and might function as aregulator of its kinase activity. So far most APG
genes analyzed are novel and non-essential for vegetative growth. Using the assay system of autophagy wedeveloped, we are studying genetic interaction among
APG genes. Some APG gene products are inducible orchange phosphorylation state by nutrient starvation.
Analyses of intracellular localization of these Apgps
may provide us specific marker to elucidate the membrane dynamics during autophagy.
Diverse pathways of autophagy
Autophagy is ubiquitous and fundamental physiological response of every eukaryotic cell. We foundyeast cells induce macroautophagy under starvation,
which is a main route for bulk and non-selective protein degradation. While it is known that excessive
amount of organelles are also degraded by autophagic
process. In methylotrophic yeast, Pichia pastoris degrades peroxisomes by invagination of vacuolar membrane, microautophagic process. Further studies will
uncover more sophisticated pathways for the degradation in the lysosome/vacuoles.
Recently it was found that all APG genes are required for targeting of one of vacuolar enzyme, API,
from cytosol to the vacuole. Electron microscopic
analyses of API transport process revealed that API
first forms a complex in the cytosol and delivered to
the vacuole by the distinct but topologically similar
mechanism to the macroautophagy (Fig.2). However,
API transport is selective and constitutive process. It
is interesting that biosynthetic pathway shares themachinery with degradative process. Some selective
degradation may also be mediated by autophagy-related mechanism.
Perspective
Autophagy is essential for maintenance of cell viability during starvation. Degradation products may
provide essential nutrients necessary for minimal protein synthesis, or reduction of certain critical activities
in the cytosol may be essential for the maintenance ofviability. Molecules involved in the membrane dynamics are well conserved from yeast to higher eukaryotes. We realized some APG genes in yeast show
homologues in mammals or higher plants. Knowledge in yeast must give us key to uncover the mechanism of autophagy in higher eukaryotic organisms.
We are now developing systems for studying autophagy in mammalian cell culture.
Publication List:Nakamura, N., Matsuura, A., Wada, Y., and Ohsumi,
Y. (1997) Acidification of vacuoles is required forautophagic degradation in the yeast, Saccharomyces
cerevisiae. J. Biochem., 121, 338-344.
Wada, Y., Nakamura, N., Ohsumi, Y, and Hirata, A.
(1997) Vam3p, a new member of syntaxin related

15protein, is required for vacuolar assembly in theyeast Saccharomyces cerevisiae. J. Cell Sci, 110,1299-1306.
Funakoshi, T., Matsuura, A., Noda, T., and Ohsumi, Y.
(1997) Analyses of APG13 gene involved in theautophagy in yeast, Saccharomyces cerevisiae.
Gene, 192, 207-213.
Matsuura, A., Wada, Y., and Ohsumi, Y. (1997)
Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae.
Gene, 192, 245-250.
Scott, S. V., Baba, M., Ohsumi, Y., and Klionsky, D.
J. (1997) Aminopeptidase I is targeted to the vacuole by a nonclassical vescicular mechanism. J. Cell
Bioi. 138, 37-44.
Nakamura, N., Hirata, A., Ohsumi, Y., and Wada, Y.
(1997) Vam2/Vps41p and Vam6/Vps39p are components of a protein complex on the vacuolar membranes and involved in the vacuolar assembly in theyeast, Saccharomyces cerevisiae. J. Bioi. Chern.,
272,11644-11349.
Baba, M., Osumi, M., Scott, S. V., Klionsky, D. J., andOhsumi, Y. (1997) Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome. J. Cell Bioi. 139: 1687-1695.
Shirahama, K., Noda, T., and Ohsumi, Y. (1997)
Mutational analysis of Cscl!Vps2p-involvement ofendosome in regulation of the autophagy in yeast,
Cell Struc. Func. 22, 501-509.
Noda, T., and Ohsumi, Y. (1998) Tor, a phosphatidyl
inositol kinase homologue, controls autophagy inyeast. J. Bioi. Chern. 273, 3963-3966.