IUBMB Life, 58(5 6): 363 369, May June 2006
Critical Review
Protein Turnover
Yoshinori Ohsumi
National Institute for Basic Biology, Okazaki, Japan
Summary
The mechanisms and physiological meanings of protein turnover
are crucial subjects in the understanding of life. However, for a long
time, protein degradation was neglected by most biologists, and was
thought of as a rather passive cellular process.
IUBMB Life, 58: 363 369, 2006
Keywords Protein degradation; lysosomes; autophagy; ubiquitin/
proteasome.
INTRODUCTION
Every cellular activity is maintained by a balance between
synthesis and degradation of cellular proteins. Nutritionists
tell us that we need to eat 60 80 grams of protein every day,
as a source of building blocks of protein, amino acids. We
synthesize about 300 500 grams of protein every day. But we
are able to survive for long time without any ingestion of
protein, which does not mean a cessation of protein synthesis
during starvation. These facts indicate that most proteins are
synthesized from amino acids derived from degradation of
pre-existing proteins, in other words by organisms capable of
ecient recycling of proteins.
LYSOSOMES
More than 55 years ago C. de Duve discovered lysosomes
by his cell fractionation procedures as an organelle contain-
ing various hydrolytic enzymes (1). It must be a reasonable
strategy for cells to segregate the dangerous degradation
process within the membrane of an organelle. Therefore, a
basic problem of lysosomal degradation is how the subs-
trates to be degraded become accessible to the lysosomal
enzymes. Electron microscopic studies revealed the membrane
dynamism of lysosomes and main delivery routes to the
Received 12 April 2006; accepted 12 April 2006
Address correspondence to: Prof. Yoshinori Ohsumi, Division of
Molecular Cell Biology, National Institute for Basic Biology,
Myodaiji, Okazaki 444-8585, Japan. E-mail: yohsumi@nibb.ac.jp
ISSN 1521-6543 print/ISSN 1521-6551 online 2006 IUBMB
DOI: 10.1080/15216540600758539
lysosome. One is the endocytic pathway (heterophagy), which
degrades extracellular proteins, even bacteria and also plasma
membrane proteins, via endocytic vesicles and endosomes (2).
The other was called autophagy, which means self eating, the
process of degradation of cytoplasmic proteins and organelles.
A portion of cytoplasm including organelle is enclosed by a
membrane sac, so called isolation membrane, and results in
a double membrane organelle, autophagosome (3). Subse-
quently it fuses with lysosome, and the contents are digested
and reutilized. Autophagy in mammals seems to be diverse in
dierent organs and also on dierent physiological demands,
and many schematic depictions of autophagy were proposed.
The lysosome is so dynamic and heterogeneous that biochem-
ical analyses on its dynamics have been limited. No good
marker proteins for the autophagic degradation, and no genes
involved in this process have been elucidated. Therefore,
detection of autophagosomes and autolysosomes (autophago-
somes fused with lysosome) in electron micrography was the
only way to analyze autophagy. Not much progress has been
made to dissect the mechanism of autophagy.
TWO MAJOR SYSTEMS OF PROTEIN DEGRADATION
For a long time it was assumed that most intracellular
proteins are degraded in the lysosome. But about 20 years ago,
researchers discovered that a small number of groups revealed
an energy-dependent proteolysis in the cytosol. It opened up a
novel vast eld of the ubiquitin/proteasome system. It was
proved that many critical cellular events are regulated by this
system. Most people started to realize that protein degradation
is as important as synthesis, and now it becomes clear that
signicant percentages of the genome are related to protein
degradation, and proteolysis has become a hot subject in
biology.
Each protein has its own lifetime within a broad range from
a few minutes to more than one month. It is obvious that cells
require dierent pathways of protein turnover for dierent
purposes. It is generally accepted that short-lived proteins,
which consist of most regulatory proteins and damaged or
misfolded proteins, are degraded by the ubiquitin/proteasome
364 OHSUMI
system (4), while long-lived proteins are degraded in the
lysosome via autophagy. Since our body is made up mostly of
long-lived proteins, from the point of view of nutrition
autophagy is crucial for life. The degradation by proteasome
is held in the cytosol, the site of biosynthesis, therefore,
requires a strict recognition of target proteins with consump-
tion of energy of ATP hydrolysis. In contrast autophagy plays
roles in a bulk and non-selective protein turnover.
AUTOPHAGY IN YEAST
In order to make a breakthrough in autophagy, a good
model system was necessary. Budding yeast, S. cerevisiae,
turned out to be the most useful organism for this purpose.
Yeast cells have relatively large compartments, vacuoles,
which are acidied by V-type ATPase and contain various
kinds of hydrolytic enzymes. So it had been postulated that the
vacuole is analogous to the lysosome in mammals. However,
nothing was known about what kind of proteins and how
proteins were degraded within this organelle. Many mutants
of vacuolar functions show a sporulation-negative phenotype.
The sporulation, meiosis of yeast, is triggered by depletion
of nitrogen from the medium. For this cell dierentiation,
bulk protein degradation in the vacuole must be essential.
I observed the vacuolar proteinase-decient mutant cells under
nitrogen starvation, expecting to identify the structures in-
volved in delivery of proteins to the vacuoles, and found an
obvious morphological change of the vacuoles. After 30 min
of starvation spherical bodies appeared in the vacuoles, and
moved around vigorously by Brownian motion. For up to
10 h these bodies gradually accumulated in the vacuoles and
nally lled inside the vacuoles (5).
Electron microscopy of the mutant cells under starvation
revealed that these bodies in the vacuole, named autophagic
bodies, are mostly single membrane-bound vesicles, containing
ribosomes and other cellular structures including mitochon-
dria and rER (5). Then double membrane structures of the
same size to autophagic bodies, yeast autophagosomes, were
found in the cytoplasm of the starved cells. Fusion images
between the outer membrane of autophagosome and the
vacuolar membrane were obtained by rapid freezing or freeze-
fracture electron microscopy (6, 7). Autophagic bodies were
300 900 nm in diameter, about 500 nm on average, which
delivers about 0.2% of the cytoplasm in a quantized manner.
Membrane dynamics of yeast autophagy is topologically
the same as macroautophagy in mammals, though the vacuole
is much larger than the lysosomes. A schematic drawing of
autophagy in yeast is shown in Fig. 1.
INDUCTION OF AUTOPHAGY
In yeast, the extent of autophagy is negligibly small when
growing in a rich medium. Similar membrane phenomena were
induced by carbon, sulfate, phosphate and single auxotrophic
amino acid starvation (5). These observations strongly indi-
cate that yeast cells take up a portion of the cytoplasm to the
lytic compartment via autophagosomes in adverse condi-
tions for growth. So far there is no mutant unable to respond
to a specic starvation signal. Autophagy should be primarily
a physiological response to nutrient limitation and may be
under the control of common but unknown starvation
signals. High cAMP levesl blocked autophagy and activated
A-kinase mutants do not induce autophagy (8), indicating that
autophagy is regulated in an opposite manner to cell growth.
When rapamycin, a specic inhibitor of the Tor kinase, is
added to a nutrient-rich medium, yeast cells induce autophagy
just like during nitrogen starvation (8). Thus the Tor kinase
negatively regulates autophagy during growing conditions as a
master regulator. At present a regulator of Tor and down-
stream pathway toward autophagy are not fully understood.
The mechanism of signal transduction of autophagy is a still
unanswered question.
MEMBRANE DYNAMICS DURING AUTOPHAGY
The most critical event in autophagy is not the proteolytic
step, but the sequestration of a portion of cytoplasm to
be degraded. This autophagosome formation is a unique
membrane dynamic, dierent from the conventional mem-
brane trac. For a long time the origin of the autophagosome
membrane was proposed to be the ER (9). We also showed
that membrane ow from the ER is necessary for autophagy
(10). Another group reported post-Golgi and other organelle
transport are involved in autophagsoome formation. The
autophagosomal membrane in yeast has a distinct morphol-
ogy; thinner than other organelle membranes and the outer
and inner membranes stick together without lumenal space
(5, 6). In freeze fracture images the inner membrane
completely lacked intra-membrane particles, while the outer
membrane contained sparse but signicant particles (7), which
may participate in targeting and fusion of the autophagosome
to the vacuole. The inner and outer membranes are somehow
dierentiated and specialized for delivery of cytoplasm to the
vacuole, though they must be derived from both sides of the
isolation membrane. By electron microscopy we could detect a
cup-shape intermediate membrane structure at low frequency,
but so far nobody has shown membrane vesicles involved in
the membrane elongation step of the isolation membrane. We
proposed that autophagosome formation is not simple an
enwrapping process by a pre-existing large membrane
structure such as the ER, but rather de novo assembly of a
new membrane from its constituents (10, 11). Many questions
still remain to be answered.: Where do the constituents of the
membrane originate? How is the isolation membrane orga-
nized? What factors are involved in the morphogenesis of
the isolation membrane? How does it seal to form a closed
double-membrane-bound compartment? What is the fusion
machinery of autophagosome to the vacuole?
 PROTEIN TURNOVER
Figure 1. A schematic drawing of autophagy in yeast.
GENETIC APPROACH TO THE AUTOPHAGY
To elucidate the molecular mechanism of autophagy, we
applied a genetic approach. The most characteristic feature
of yeast autophagy is that we are able to monitor the progress
of autophagy under a light microscope as the accumulation of
autophagic bodies. Taking advantage of this, we attempted to
obtain autophagy-defective mutants. Mutagenized vacuolar
proteinase-decient cells that failed to accumulate autophagic
bodies during starvation were selected under a light micro-
scope and a mutant, apg1 was obtained (12). The apg1 mutant
did not induce bulk protein degradation under starvation, and
homozygous apg1/apg1 diploid cells did not sporulate. The
apg1 mutant grew normally in a rich medium, but could not
maintain viability under long nitrogen starvation. To obtain
more apg mutants, this loss of viability on starvation was used
for the rst screening, assuming that it is due to the defect
in autophagy. About 100 autophagy-defective mutants were
isolated, and divided into 14 groups (apg2-apg15) by
complementation analysis (12). The original strategy for
365
isolation of autophagy-defective mutants was quite ecient,
so since then only two genes are added as typical autophagy
mutants using two hybrid screens with known Apg proteins.
Another approach taken by Thumm and his colleagues was
immuno-screening of cells that retain a cytosolic enzyme, fatty
acid synthase, after starvation. By this method originally 6 aut
mutants were obtained (13). Klionskys group has been
studying the pathway of maturation of aminopeptidase I
(API), one of the vacuolar enzymes, and isolated defective
mutants in the process (14). Unlike other vacuolar enzymes,
the proform of API is transported from the cytosol to the
vacuole via the Cvt pathway (15). The cvt mutants defective in
the Cvt pathway signicantly overlapped with autophagy
defective apg and aut mutants (16, 17), though the two
pathways are apparently dierent; one is degradative and
starvation-induced, and the other is biosynthetic and consti-
tutive. EM analyses of the Cvt pathway clearly showed
that the Cvt pathway is mediated by quite similar membrane
dynamics to autophagy (18, 19). Later, several groups
366 OHSUMI
identied autophagy-related genes in S. cerevisiae and other
yeast species, and named them dierently. To avoid confusion,
recently all groups involved agreed to use a novel nomen-
clature for the autophagy-related gene, ATG. The original
APGx is now renamed as ATGx (20).
Among ATG genes, the original APG genes and ATG18,
and ATG29 are involved in the autophagosome formation, so
here, APG will be used for these genes collectively.
CHARACTERIZATION OF AUTOPHAGY-DEFECTIVE
MUTANTS
The apg mutants fail to induce bulk protein degradation
under nutrient-depleted conditions, indicating that auto-
phagy is the major pathway of bulk protein degradation.
They grow normally and show no defects in stress responses,
vacuolar functions, secretion, and endocytosis, indicating that
autophagy is not essential for vegetative growth in yeast (12).
Autophagy-defective mutants start to die after 2 days of
starvation and almost completely lose viability after 1 week
(12). Homozygous diploids with any apg mutation cannot
sporulate, indicating that cell remodeling must require bulk
protein degradation via autophagy (12).
Recently we examined the amino acid pool during nitrogen
starvation. In wild type cells, amino acid levels drop dramati-
cally for the rst 3 h of starvation, but recover and maintain a
certain level; in contrast, in atg mutants most amino acid levels
decrease further and some become very low (21). The amount
of nitrogen-starvation-inducible proteins was severely reduced
in atg mutants; probably protein synthesis is limited by the
shortage of amino acids. Recycling of protein via autophagy
must be essential for survival during starvation.
All apg mutants did not accumulate autophagosomes in the
cytoplasm during starvation, indicating that thess genes have
functions at or before the formation step of the autopha-
gosome, and further studies on the Apg proteins conrmed
that all these proteins function at the autophagosome for-
mation step.
FURTHER GENES REQUIRED FOR AUTOPHAGY
Typical autophagy mutants like the original apg mutants
seem to be nearly saturated. However, the strategies of screen
eliminated mutants of aberrant vacuole morphology, partially
defective mutants, and of genes sharing with other essential
functions. Now it is obvious that normal autophagy requires
more both known and unknown genes. Most Gcn proteins are
necessary for the normal extent of autophagy. Several early
secretory genes such as SEC12 an SEC24 were shown to be
required for autophagy (10). Several mutants such as vam5
and ypt7 show accumulation of autophagosomes in the
cytoplasm under starvation, suggesting the fusion machinery
of autophagosome with the vacuole shares SNARE molecules
with that for vacuolar homotypic fusion. In wild type cells
autophagic bodies eectively disappear within a minute. Atg15
and Atg22 were shown to be involved in this process (22, 23).
Atg15 contains a putative lipase domain, but its activity has
not yet been proved. Acidication of the vacuole is a requisite
for eective digestion of autophagic bodies, since a defect in
every subunit of V-ATPase (Vma) causes an accumulation of
autophagic bodies in the vacuole (24). It is still an open
question why the autophagic body membrane is immediately
disintegrated in less than a minute in the vacuoles.
FUNCTION OF ATG PROTEINS
Almost all ATG genes were unknown genes, except Atg6/
Vps30, which is required for vacuolar protein sorting (25). Even
in yeast, autophagy genes had been overlooked for a long time
because they exhibit phenotypes only under starvation conditions.
Recent analyses revealed that Atg proteins are classied into
several functional units. One of the most remarkable ndings is
the discovery of two ubiquitin-like conjugation systems in Atg
proteins (26, 27). Actually half of the APG genes are involved
in these novel conjugation systems. A brief summary of our
present knowledge of the Atg protein system follows.
(A) Atg12 Protein Conjugation System
Apg12 is a novel ubiquitin-like protein (UBL) (26). Atg12 is
activated by an activating enzyme (E1), Atg7, and then
transferred to a conjugating enzyme (E2), Atg10, by forming
thioester conjugates, and nally an isopeptide conjugates with
Atg5. The Atg12 conjugation reaction is similar to ubiquiti-
nation, but has distinct features. Atg12 is synthesized as an
active form with a single glycine at the C-terminus. Recently
the crystal structure of Arabidpsis Atg12 was solved in
Dr Inagakis lab, showing a ubiquitin-fold at the C-terminal
region. A C-terminal ubiquitin fold is necessary and sucient
for conjugation and also for autophagy. Atg5 is the only target
molecule of the Atg12 modication. Atg12 and Atg5 form a
conjugate immediately after their synthesis and free forms are
hardly detectable in cell lysates. This conjugation reaction is
irreversible, since deconjugating enzymes have not been found.
Conjugate formation is not aected by autophagy-inducing
conditions and the conjugate behaves just like a single poly-
peptide and functions as a part of the machinery of auto-
phagosome formation. The conjugate further forms a complex
with Atg16. Apg16 binds to Atg5 at the N-terminal region and
forms an homo-oligomer through a coiled-coil region. Atg12-
Atg5-Atg16 forms a large complex of about 350 kDa, which
is essential for autophagy (28). In mammals this complex
is shown to reside on the outer surface of the isolation mem-
brane, but dissociates from the membrane when autopha-
gosome has been completed.
(B) Atg8 Lipidation System
Atg8 is another UBL and has many homologues in
eukaryotes. Immunoelectron microscopy showed that Atg8
 PROTEIN TURNOVER
is localized on the isolation membrane, autophagosome and
autophagoc body during autophagosome formation (29).
The C-terminal arginine is processed by Atg4 (Atg8DR), a
novel and well conserved cysteine proteinase family protein
(30). Then Atg8 is activated by Atg7, and then is transferred to
Atg3, an E2 enzyme. Atg7 is a unique E1 enzyme which
activates two dierent UBLs, Atg12 and Atg8, and transfers
them to each E2 molecule, Atg10 and Atg3, respectively (27).
By SDS-PAGE in the presence of 6 M urea two forms of Atg8
were separable (30). Mass spectrography of the modied form
of Atg8 showed a covalent conjugate of Atg8 to phosphati-
dylethanolamine (PE). Importantly Atg8-PE formation was
reversible and the processing enzyme, Atg4, functions also for
this deconjugation (27, 30). The expression of Atg8 is induced
by nitrogen starvation, and the Atg8-PE level is also elevated
(29). The cycle of Atg8-lipidation reaction is necessary for
normal autophagy (30).
Atg12 and Atg8 conjugation systems work concertedly;
they not only share the same E1 enzyme, Atg7, but also
functionally, because the Atg8-PE level is severely reduced in
Atg12 system components, Atg5, Atg10, and Atg12 in mutants
(31). Recently we succeeded to reconstitute the in vitro
lipidation reaction using puried Atg8DR, Atg7, Atg3, and
PE-containing liposome (32). This system will elucidate the
molecular details of this interesting reaction.
(C) Atg1 Kinase Complex
The third protein complex required for autophagy is the
Atg1 kinase complex. Atg1 possesses a serine/threonine kinase
domain at the N-terminus region. Kinase-negative Atg1 mut-
ant could not induce autophagy, implying that kinase activity
is essential for function (33, 34). Atg1 kinase activity is en-
hanced during induction of autophagy, which is important for
the regulation of autophagosome formation (35).
Atg1 physically interacts with Atg13, Atg17 and Atg11/
Cvt9. Atg13 is a highly phosphorylated protein under
nutrient-rich condition. On starvation or addition of rapamy-
cin, it is immediately dephosphorylated by an as yet unknown
phosphatase (34). Reversely, on addition of nutrients to
starved cells, Atg13 is rapidly hyperphosphorylated. The
phosphorylation state of Atg13 is controlled by the nutrient
conditions through the Tor signaling pathway. Under starva-
tion, Atg13 is tightly associated with Atg1, while under
nutrient rich conditions the anity is lowered (34). Atg17
binds to Atg13 and forms a ternary complex with Atg1, which
causes activation of Atg1 kinase activity. Atg17 exists as a
large complex.
(D) Autophagy Specific PI3 Kinase Complex
The fourth complex is an autophagy-specic PI3 kinase
complex. ATG6 is allelic to VPS30 (25). Vps30/Atg6 has a dual
function for vacuolar protein sorting and autophagy. There are
at least two PI3 kinase complexes in yeast, Complex I and
Complex II (36). Vps34, Vsp15 and Vps6 are common, but
367
Atg14 and Vps38 dene the specicity of the complex. Atg14,
specically required for autophagy, binds to Vps30 and Vps34
at the coiled-coil region. Vps34 is the sole phosphatidyl inositol
3-kinase in yeast and Vps15 is a regulatory protein kinase of
Vps30. Atg14 determines the precise localization of PI3 kinase
at the site of autophagosome formation, PAS (37).
(E) Other Atg Proteins
The remaining three Atg proteins, Atg2, Atg18 (Aut10)
form a complex and weakly with Atg9. Atg9 is a putative
multi-membrane spanning protein, but its localization does
not t to any known organelle marker (38). Their precise roles
in autophagy are not known yet at the molecular level.
However, they may play important roles in linkage between
the above four reactions.
SITE OF ATG PROTEIN FUNCTIONING
PREAUTOPHAGOSOMAL STRUCTURE
All Atg proteins function spatiotemporally in a very close
manner during the autophagosome formation step. Recently
we found that the autophagosome marker, Atg8, is localized
in a small area close to the vacuole, and showed that almost all
Atg proteins are localized also at the structure, the preauto-
phagosome structure (PAS) (35). Temperature sensitive Atg1ts
mutant cells expressing GFP-Atg8 were completely defective
in autophagosome formation; instead they showed a bright
PAS at a restrictive temperature. On shiftdown to the
permissive temperature, immediately, less brightly uorescent
structures, presumably autophagosomes, were generated from
the PAS, and fused to the vacuole; consequently the vacuo-
lar lumen stained brightly. This structure seems to be an
organizing center of the autophagosome (35). Recently we
performed systematic analyses of the PAS localization of every
Atg protein in all atg disruptants (K. Suzuki, Y. Kubota,
Y. Ohsumi, manuscript submitted). From this analysis we
obtained a hierarchy map of Atg proteins. There are epistatic
relations between functional units of Atg genes with PAS
organization. The Atg12 system and lipidation are necessary
for Atg8 to associate to the PAS. The Atg1 kinase complex is
not required for the recruitment of Atg12 and Atg8. The PI3
kinase and Atg9 are necessary for the association of two
conjugates to the PAS. Atg17 and Atg11 may function as
scaold proteins for all Atg protein localization to the PAS.
Biochemical and ne structural investigation of the PAS will
elucidate the molecular details of autophagsosme formation.
ATG PROTEINS IN HIGHER EUKARYOTES
Most of the ATG genes are conserved from yeast to
mammals and plants, indicating that eukaryotic cells acquired
the autophagic system at an early stage of evolution. Espe-
cially, two conjugation reactions are well conserved. Still
several Atg proteins have not been identied in mammals or
368 OHSUMI
plants. A requirement of PI3 kinase activity for autophagy is
also reported in mammals, but the autophagy specic
component, Atg14, has not been found. Possibly, as in the
case of Atg16, sequence homology may not be sucient to nd
the counterparts, or they may be yeast-specic factors.
Just as in yeast, two conjugation systems provided a good
marker for the membrane structures involved in autophagy in
yeast. LC3, an Atg8 homologue, is also modied to LC3II, in
a tightly membrane bound form after processing by Atg4
homologues. LC3 stains isolation membrane and autophago-
somes. The Atg12 system is also well conserved, and Atg5 is
localized in the isolation membrane, but dissociates when the
autophagosome is completed, providing a good marker for the
intermediate membrane.
In multicellular organisms, autophagy should be diverse
and regulated in a dierent manner. Our recent studies have
made several marker proteins for autophagosomes available.
To understand where and when autophagy occurs in vivo,
transgenic mice systemically expressing GFP fused to LC3 were
established. Cryosections of various organs were prepared and
the occurrence of autophagy was examined by uorescence
microscopy. Active autophagy was observed in various tissues,
such as the skeletal muscle, liver, heart, exocrine glands, thymic
epithelial cells, lens epithelial cells and podocytes. The patterns
of induction of autophagy in dierent tissues are clearly
distinct. In some tissues, autophagy even occurs constitutively.
This transgenic mouse is a useful tool to study mammalian
autophagy (39).
Elucidation of genes essential for autophagy in yeast
facilitated the study of autophagy in various organisms
Dictiosteirum discoidum, Caenorhabditis elegans, Drosophila,
Arabidopsis and mouse and human. Knockout of Atg genes
showed clearly the physiological signicance of autophagy.
Since protein turnover is a basic function of the cell, auto-
phagy must be relevant to many physiological functions.
Researchers in many elds now pay attention to autophagy,
but it is still a rather early and developing eld of biology.
Further studies on the function of Atg proteins will not only
unveil the mystery of autophagosome formation but also may
provide new insights on membrane dynamics within the cell.
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