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What is Autophagy?

The term "autophagy" means self-eating and is derived from the Greek words, "auto" for "self" and "phagy" for "eating." Although an octopus eating its own arms is sometimes given as an example of autophagy, the octopus behavior seems to be caused exclusively by stress.


An intracellular decomposition mechanism equal in importance to the proteasome system

The role of autophagy is generally understood to be securing nutrient sources through autodigestion in order to survive starvation conditions. However, in recent years, it has been found to work along with the proteasome system in the metabolism of cell components even in normal environments.

Compared to proteasomes, which selectively degrade ubiquitinated proteins as their targets, in autophagy the space within the cell is entirely digested; therefore it is called a bulk degradation system.

An intracellular decomposition mechanism equal in importance to the proteasome system

When a cell is placed under starvation conditions, a flat vesicle called an isolate membrane appears in the cytoplasm (1). Subsequently, the membrane extends while taking in the cytoplasm (2), its edges fusing to compose an autophagosome (AP) (3). Mitochondria and other large organelles are also contained within the AP. When the AP fuses with a lysosome (4), its contents are degraded (5). The amino acids gained through autodigestion are reused as nutrient sources. It is not known at this time how the isolate membrane appears or how its components are obtained.

The control mechanism of autophagy

The phrase “bulk degradation system” may suggest a degradation reaction without specificity, but the large-scale autodigestion of the cytoplasm is a dangerous business for the cell. Therefore, the progress of autophagy is sure to be severely controlled.

What factors, then, are present in autophagy, and in what way does each work?


The discovery of APG/ATG genes

Autophagy has been attracting attention in recent years, but it was originally observed with an electron microscope more than forty years before. However, because the factors of its process were for a long time unclear, the functional analysis of autophagy made little progress.

Professor Ohsumi and his colleagues created a yeast strain in which the autophagosome could not digest its contents, and succeeded in the cloning of autophagy-related APG/ATG genes (Tsukada and Ohsumi, 1993). At present, there are 36 forms of the ATG genes known in budding yeast (2012), and most of them are also retained in mammals and plants (the homology of amino acid sequences between species appears to be low, but the conformation is similar).

Spurred by the discovery of these APG/ATG genes, the functional analysis of various proteins has proceeded rapidly, and the details of the mechanism and roles of autophagy are becoming better and better known.

Atg proteins and their functions

:The 17 factors necessary for autophagosome formation
Yeast Mammalian Functions
Atg1 ULK1, 2 Atg1/Atg13/Atg17/Atg29 kinase complex
Atg2 Atg2? Atg2/Atg18/Atg9 complex (autophagosome formation)
Atg3 Atg3 E2-like enzyme specific for Atg8 (autophagy induction)
Atg4 Atg4A, 4B, Autophagin3, 4 Cysteine protease: Cleavage of C-terminal of Atg8 and de-PE
Atg5 Atg5 Atg12/Atg5/Atg16 complex (autophagosome formation)
Atg6 Beclin-1 A subunit of Vps34 PI3K complex (autophagosome formation)
Atg7 Atg7 E1-like enzyme: Both Atg8 and Atg12 are Atg7 substrates
Atg8 LC3, GABARAP, GATE-16 Ubiquitin-like protein: Atg8-PE formation (phosphatidylethanolamine)
Atg9 Atg9L1, L2 Atg2/Atg18/Atg9 complex (autophagosome formation)
Atg10 Atg10 E2-like enzyme specific for Atg12 (autophagy induction)
Atg11   Adaptor molecule: Incorporation of API into Cvt vesicle in yeast
Atg12 Atg12 Ubiquitin-like protein: Atg12/Atg5/Atg16 complex
Atg13 Atg13 A subunit of Atg1 complex (autophagy induction)
Atg14 Atg14L A subunit of Vps34 PI3K complex (autophagy induction?)
Atg15 Lipase-like protein: degradation of autophagic body in yeast
Atg16 Atg16L Atg12/Atg5/Atg16 complex (autophagosome formation)
Atg17 A subunit of Atg1 kinase complex (autophagosome formation)
Atg18 WIPI-1, 2, 3, 4 PIP3 binding protein: Atg9/Atg2/Atg18 complex
Atg19 The receptor of API in Cvt pathway (yeast)
Atg20 PI3P binding protein in Cvt pathway (yeast)
Atg21 PI3P binding protein in Cvt pathway (yeast)
Atg22 A membrane protein of vacuoles in yeast
Atg23 Cvt vesicle formation in yeast
Atg24 Cvt pathway and degradation of peroxisome (yeast)
Atg25 Degradation of peroxisome in yeast
Atg26 Degradation of peroxisome in yeast
Atg27 PI3P binding protein in Cvt pathway (yeast)
Atg28 Degradation of peroxisome in yeast
Atg29 A subunit of Atg1 kinase complex (autophagy induction?)
Atg30 Peroxisome degradation (Pexophagy)
Atg31 Atg17-Atg29-Atg31 complex in starvation induced autophagy
Atg32 Mitochondria degradation (Mitophagy)
Atg33 Mitochondria degradation (Mitophagy)
Atg34 Alpha-mannosidase transport
Atg35 Peroxisome degradation (Pexophagy)
Atg36 Peroxisome degradation (Pexophagy)

5 complexes which are involved in autophagosome formation


Atg8-conjugation system

Atg8-conjugation system

After the ubiquitin-like protein Atg8 has had its C-terminal arginine residue cleaved by the cysteine protease Atg4, it is passed on to E1 (Atg7) and E2 (Atg3) and transferred into the head group of its substrate phosphatidylethanolamine (PE). This Atg8-PE conjugate functions as part of the membrane component of the autophagosome. When Atg8-PE is once again deconjugated PE by Atg4, the Atg8 is recycled. Incidentally, an E3-like protein in autophagy has not yet been found.


Atg12-conjugation system

Atg12-conjugation system

Unlike other ubiquitin-like proteins, the ubiquitin-like protein Atg12 has a C-terminal glycine, which protects it from processing. Atg12 is conjugated to the substrate Atg5 by Atg7 (an E1-like protein, and as seen in the conjugation of Atg8 to PE) and Atg10 (an E2-like protein). The Atg12-Atg5 conjugate forms a complex with Atg16. Self-oligomerization of Atg16 results in a multimer of the Atg12-Atg5-Atg16 complex. Although its functions remain unknown, this complex is shown to accumulate on the pre-autophagosomal structure (PAS) in yeast and to play an essential role in autophagosome formation.


Atg1 protein kinase complex

Atg1 protein kinase complex

Induction of autophagy is mediated by inactivation of the serine/threonine protein kinase known as target of rapamycin (TOR). Among other molecules, Atg13 is highly phosphorylated by the TOR under nutrient-rich conditions, being unable to bind to Atg1. In such conditions, the kinase activity of Atg1 is kept low as well.

In contrast, under starvation conditions, TOR is inactivated and the phosphorylation levels of Atg13 decrease. Then, Atg13 and Atg17 bind to Atg1, and kinase activity of Atg1 also increases. Induction of autophagy is known to be dependent on this kinase activity of Atg1, but further researches are necessary to fully understand its detail. Rapamycin, a TOR inhibitor, also induces the same phenomenon. It has been shown that Atg29 binds to Atg17 in the budding yeast.


The Vps34 PI3 kinase complex

The Vps34 PI3 kinase complex

Phosphatidylinositol 3-kinase (PI3K) phosphorylates the phosphatidylinositol (PI) in the membrane lipid to create phosphatidylinositol 3-phosphate (PI3P). Class III PI3K activity, called Vps34, is necessary for the progress of autophagy.

Vps34 forms a complex with Vps15 (protein kinase), Vps30/Atg6 (Beclin-1 in mammals), and Atg14. If even one of these subunits is rendered defective, autophagy will not be induced. As well, because lipid kinase activity in Vps34 is necessary for autophagy to take place, it is thought that the PI3P produced thereby plays some role in autophagy. Beclin 1/hVps34/p150/Rubicon/UVRAG complex involves in autophagosome maturation. On the other hand, Beclin 1 has been reported to be defective in various forms of cancer (breast, ovarian, prostate), and it is known that cancer is frequent in Beclin 1 hetero-defective mice. As well, DNA defects related to cancer have been found in the Beclin 1 binding protein UVRAG.


Atg9 and Atg2-Atg18 complex

Atg9 and Atg2-Atg18 complex

It has not yet known where the initiation of autophagy takes place. In the yeast, however, autophagosome formation is suggested to start at the pre-autophagosomal structure (PAS). Atg9 is thought to be involved in the early phase of PAS formation, since many Atg proteins are not localized in the PAS in the budding yeast lacking ATG9.

Atg2-Atg18 complex binds to Atg9 on the surface of the PAS and Atg18 is known to interact with PI3P. But, the function of Atg2-Atg18 complex is still not known. In the yeast with mutated ATG18 and ATG2, Atg9 is highly accumulated at the PAS. Meanwhile, Atg9 is not localized at the surface of autophagosome. These findings suggest there must be mechanisms for Atg9 to detach from the PAS surface. Atg2-Atg18 complex may be involved in this process.

Factors related to autophagy continue to increase

As we have seen so far, it is now understood that the seventeen varieties of Atg proteins necessary for the formation of an autophagosome act as at least five functional aggregates. There are many issues yet to be understood regarding the role of each complex and the interactions among complexes, but in recent years more and more factors related to autophagy, beginning with Atg conjugated proteins, continue to be identified.

UVRAG, related to UV resistivity, is receiving renewed attention as a Beclin 1 conjugated protein, and it is now clear that it is involved in the formation of the autophagosome through the activation of PI3K.
p62/a170/SQSTM1 (p62), which interacts with Atg8 (LC3), is a scaffolding protein for signal transmission through ERK or PKC; because it has a ubiquitin binding domain at its C-terminal, it suggests crosstalk between ubiquitin proteasome degradation through p62 and autophagy. As well, regarding pathogen elimination through autophagy, it is known that LRGM/LRG-47, a variety of GTP conjugated protein with the toll-like receptor and IFN-γ inductor important for pathogen recognition, is used in innate immunity.

In this way, spurred by research into autophagy, new and diverse interactions among biological phenomena have become visible.

In the future, expectations will rest on approaches using Atg-factor defective yeast strains and Tg/KO mice, as well as development of factor monitoring tools such as antibodies to Atg proteins and conjugate factors, and fluorescent protein fusion proteins.


Localization of Atg proteins

Localization of Atg proteins

In budding yeast, almost all the Atg group necessary for autophagosome formation aggregate in the PAS. A structure equivalent to the PAS in the mammals has not yet been confirmed. Proteins in Atg8- and Atg12-conjugation system exist in the isolate membrane, but when the autophagosome formation is complete the proteins in Atg-12-conjugation system detach from the membrane. Subsequently, the autophagosome which has fused with the lysosome (the autolysosome) digests its contents, but the Atg8 remains on the outer membrane. When looked at this way, it is clear that Atg8 is effective as a monitoring marker of autophagy.

LC3 & the LC3-conjugation system

APマーカー:Atg8/LC3図

A yeast autophagy-related gene product (Atg8) has three mammalian homologues: LC3, GABAA receptor-associated protein (GABARAP), and Golgi-associated ATPase enhancer (GATE-16).

Among them, LC3 is most actively studied and frequently used as a mammalian autophagy marker. Shortly after translation (proLC3), LC3 is processed at the C-terminus by Atg4B or Atg4A into LC3-I. Upon induction of autophagy, LC3-I is conjugated to the substrate phosphatidylethanolamine (PE) via E1 (Atg7) and E2 (Atg3). The PE-LC3-I conjugate is referred to as LC3-II. Despite having a higher molecular weight than LC3-I, LC3-II is more hydrophobic and shows a higher mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE). GABARAP and GATE-16 are also demonstrated to undergo the same process of conjugation with PE.

Anti-LC3 Antibodies: useful tools for monitoring autophagy

With the collaboration of Professor Yoshimori Tamotsu of Osaka University and Professor Mizushima Noboru of The University of Tokyo, MBL handles a large number of antibodies useful for autophagy monitoring.

Anti-LC3 Antibodies: useful tools for monitoring autophagy

When serum-starvation treatment is applied to cultured cells, autophagy becomes active. At this time, LC3 puncta increase in cytoplasm. Correlating with this, in western blotting the quantity of LC3-II increases. As well, because LC3 is converted into LC3-I immediately after translation, it is impossible to confirm proLC3 as a band.


Selective degradation of p62/SQSTM1 via interaction with LC3

Immunohistochemistry

p62/SQSTM1 is considered to be the substrate that binds directly to LC3 causing its selective degradation by autophagy. Anti-p62/SQSTM1 antibody, which has a variety of applications including Western blotting (WB), immunoprecipitation (IP), immunocytochemistry (IC), and immunohistochemistry (IHC), is useful for observation of p62-caused pathogenesis and dynamics at the molecular level. The ubiquitin-conjugating protein p62/SQSTM1 is thought to be a scaffold protein because it interacts with a variety of molecules involved in toll-like receptor (TLR) signaling, such as TRAF6, ERK, and aPKC. Recent evidence shows that p62 binds directly to the autophagosome marker LC3 and then is selectively degraded by autophagy. Actually, in liver- or brain-specific autophagy-defective mice, excessive p62 accumulation leads to formation of ubiquitin- and p62-positive inclusions. Importantly, ubiquitin- and p62-positive inclusions have been detected in tissues from patients with neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis), alcoholic hepatitis, fatty liver, or liver cancer. Currently, the association of autophagy-induced p62 metabolic failure with the pathogenesis of these diseases is an important focus of attention.



Development of autophagy research

Development of autophagy research

Autophagy was first discovered in yeast as a form of cellular self-digestion for the purpose of providing nutrients to the cell in order to survive starvation. Research in autophagy has been expanding from its initial discovery in yeast, as a form of cellular self digestion, to having implications in major drug discovery pipelines. Recent researches and advances have shown an association of mammalian autophagy with diseases such as neurodegenerative disease, infection disease, cardiac disease as well as cancers.


The literatures below were used as references for the text and figures in this page. We'd like to take this opportunity to express our gratitude.

Tanaka Keiji, Osumi Yoshinori (eds.), Protein, Nucleic Acid and Enzyme "The ubiquitn-proteasome sytstem and Autophagy", Kyoritsu Shuppan Co., Ltd.

BostonBiochem Product Catalogue, 2006