FOR BASIC BIOLOGY

15Professor: Yoshinori Ohswni
Associate Professor: Tamolsu Yoshimuri
Research Associates: Takeshi Noda
Yoshiaki Kamada
Graduate Students: Kanae Shirahama (Un iv. of Tokyo)
Tomoko Funakoshi (Univ. of Tokyo)
Satoshi Kame taka ( Univ. of Tokyo)
Chikara Tokunaga (Univ. of Tokyo)
Takayoshi Kirisako (Univ. of Tokyo)
Technical Staff: Yukiko Kabeya
Visiting Professor: Michael Filosa

nance of cell viability. Autophagy is a major route forsequestration of proteins to the lytic compartment.
Vacuole/lysosome is also the destination for endocytic
pathway. Since in 1952 du Duve identified cellular
lytic compartment, lysosome, enzymatic characterization and biogenesis of lysosomal enzymes have beenstudied thoroughly. However, the mechanisms of delivery of proteins to the lysosomes are not known at
a molecular level. Dynamism of lysosomal system has
been studied mostly with electron microscope. Autophagy is one of the most important problems in cellbiology remained to be solved.

This division aims to elucidate the mechanism andregulation of intracellular protein degradation in alytic compartment and its physiological function. Recently it was realized that degradation process plays
essential role for cellular regulation. In the cytoplasm
selective protein 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. However, little isknown about the mechanism of protein degradation incontrast with biosynthesis. Bulk protein degradation isinduced by various nutrient starvation condition,
which is obligatory to cell differentiation and mainte-

Yeast Induces Autophagy as Mammalian Cells
Recently we discovered yeast, Saccharomyces
cerevisiae, induces bulk protein turnover in the vacuoles under various starvation conditions. This wholeprocess corresponds to the process of macroautophagy
in higher eukaryotic cells. By electron microscopic
analyses we succeeded to detect double membrane
structure enclosing a portion of cytosol in the cytoplasm. These yeast autophagosomes immediately fusewith vacuolar membrane, resulting to deliver a single
membrane bound vesicles, autophagic bodies in thevacuoles. When vacuolar proteinase activities areblocked genetically or by specific inhibitor such as
PMSF autophagic bodies are accumulated in the vacu-DIVISION OF BIOENERGETICS

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

16

NATIONAL INSTITUTEoles. They moves around vigorously in the vacuoles
by Brownian motion, and are easily observed by lightmicroscope. Thus we can follow the progression ofautophagy by the accumulation of autophagic bodies
in real time. Biochemical and immunoelectron microscopic analyses of vacuoles containing autophagic
bodies showed that starvation-induced sequestration is
non-selective, that is, any kind of cytosolic enzymes
and organelles are sequestered to the vacuoles to be
degraded. Freeze-fracture electron microscopy
showed that autophagosomal membrane has characteristic feature and density of intramembrane particles
is quite few. The origin of the autophagosomal membrane and mechanism of formation are key problems
remained to be solved.
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 molecularbiological 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 agp mutants, defective in
the induction of autophagy, were isolated. They
cannot induce protein degradation upon shift fromgrowth. medium to the starvation medium. But theygrow normally in a rich medium, but start to die after
2 days in the starvation medium. This suggests thatthe autophagy is necessary for long time maintenanceof cell viability. Homozygous diploid of each apg
mutants is sporulation defective as expected.
Analyses of APG genesNow we are focusing on characterization of these
APG genes, essential for autophagy. This year wehave finished cloning of twelve APG genes. So far ten
APG genes have been identified. APG 1 codes a novel
Serffhr kinase essential for the induction of autophagy. This provides the first direct evidence for involvement of protein phosphorylation in the processof autophagy. So far all genes analyzed are novel andnon-essential for vegetative growth. Using the assay
system of autophagy we developed, we are now analyzing genetic interaction among APG genes. Furthermolecular biological and biochemical studies on these
APG gene products might provide important clues tounderstand the mechanism of autophagy at a molecular level. Now we obtained antibodies against to these
APG gene products. Intracellular localization of them
may give us clues to elucidate the function of theseproteins.
Diverse pathways of autophagy
Autophagy is one of the fundamental function of all
eukaryotic cells. We found yeast cells induce macro-Aminopeptidase IAPG5APG14 APG7APG13APG9 APG4APG6fAPG12APGS... . ..
.
• ••....
• •. .• .
..
,#•••• • •• ••• •,•'•• •••••,,ProteinaseFig. 2. Scheme of Autophagy in Yeast
All APG genes identified are novel and required at or before the formation of autophagosome.

FOR BASIC BIOLOGY

autophagy under starvation, which is a main route forbulk and non-selective protein degradation. However,
it is also reported that some enzymes are selectively
taken up to vacuoles according to the physiologicaldemands. 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 lysome/vacuoles. Recently it was foundthat all APG genes are reuired for targeting of API
from cytosol to the vacuole. It is interest problem how
this selective and constitutive process shares machinery with the autophagy.
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. Basic molecular devices for the membrane
dynamics are conserved from yeast to higher eukaryotes. We realized some APG genes in yeast show homologues in mammals or higher plants. We are now
developing systems of autophagy in mammalian cultured cell.

Publication List:
Funakoshi, T., Matsuura, A., Noda, T. and Ohsumi, Y.
(1997) Analyses of APG13 gene involved in the autophagy in yeast, Saccharomyces cerevisiae. Gene
179, in press.
Kametaka, S., Matsuura, A., Wada, Y. and Ohsumi, Y.
(1966) Structural and functional analyses of APG5,
a gene involved in autophagy in yeast. Gene 178,139-143.
Matsuura, A., Wada, Y. and Ohsumi, Y. (1977) Apg1p,
a novel protein kinase required for the autophagic
process in Saccharomyces cerevisiae. Gene 179, inpress.
Moriyasu, Y. and Ohsumi, Y. (1996) Autophagy intabbaco suspension-cultured cell (BY-2) in response
to sucrose starvation. Plant Phsiol. 111, 1233-1241.
Nakamura, N., Matsuura, A., Wada, Y. and Ohsumi, Y.
(1997) Acidification of vacuoles is required for autophagic degradation in the yeast, Saccharomyces
cerevisiae. J. Biochem. 193, in press.
Nakamura, N., Hirata, A., Ohsumi, Y. and Wada, Y.
(1997) VAM6 is required for the vacuolar morphogenesis and maturation of vacuolar membrane protein in the yeast Saccharomyces cerevisiae. J. Biol.
Chern. in press.
Scott, S. V., Hefner-Gravink, A., Morano, K. A., Noda,T., Ohsumi, Y. and D. J. Klionsky (1996) Cytoplasmto-vacuole targeting and autophagy employ the samemachinery to deliver proteins to the yeast vacuole.
Proc. Natl. Acad. Sci. USA 93, 12304-12308.
Shirahama, K., Yazaki, Y., Sakano, K., Wada, Y. andOhsumi, Y. (1996) Vacuolar function in the phosphate homeostasis of the yeast Saccharomyces

17cerevisiae. Plant Cell Physiol. 37, 1090-1093.
Wada, Y., Ohsumi, Y., Kawai, E. and Ohsumi, M.,(1996) Mutational <yWysis of Vam4/Ypt7p functionin the vacuolar biogenesis and morphogenesis in theyeast, Saccharomyces cerevisiae. Protoplasma 191,
126-135.