ATG8
Encyclopedia
Autophagy-related protein 8 (Atg8) is a ubiquitin
-like protein
required for the formation of autophagosomal membranes. The transient conjugation of Atg8 to the autophagosomal membrane through a ubiquitin-like conjugation system is essential for autophagy
in eukaryotes. Even though there are homologues in animals (see for example GABARAP
, GABARAPL1, GABARAPL2
, MAP1LC3A
, MAP1LC3B
, MAP1LC3B2, and MAP1LC3C), this article mainly focuses on its role in lower eukaryotes such as Saccharomyces cerevisiae
.
domain. Even though Atg8 does not show a clear sequence homology to ubiquitin
, its crystal structure reveals a conserved ubiquitin-like fold.
, the cellular process mediating the lysosome
/vacuole
-dependent turnover of macromolecules and organelles. Autophagy is induced upon nutrient depletion or rapamycin treatment and leads to the response of more than 30 autophagy-related (ATG) genes known so far, including ATG8.
How exactly ATG proteins are regulated is still under investigation, but it is clear that all signals reporting on the availability of carbon and nitrogen sources converge on the TOR signalling pathway and that ATG proteins are downstream effectors of this pathway. In case nutrient supplies are sufficient, the TOR signaling pathway hyperphosphorylates certain Atg proteins, thereby inhibiting autophagosome formation. After starvation autophagy is induced through the activation of Atg proteins both on the protein modification and the transciptional level.
Atg8 is especially important in macroautophagy which is one of three distinct types of autophagy characterized by the formation of double-membrane enclosed vesicles
that sequester portions of the cytosol
, the so called autophagosomes. The outer membrane of these autophagosomes subsequently fuses with the lysosome/vacuole to release an inter single membrane (autophagic body) destined for degradation
. During this process, Atg8 is particularly crucial for autophagosome maturation (lipidation).
Like most Atg proteins, Atg8 is localized in the cytoplasm and at the PAS under nutrient-rich conditions, but becomes membrane-associated in case of autophagy induction. It then localizes to the site of autophagosome nucleation, the phagophore-assembly site (PAS). Nucleation of the phagophore requires the accumulation of a set of Atg proteins and of class III phosphoinositide 3-kinase
complexes on the PAS. The subsequent recruitment of Atg8 and other autophagy-related proteins is believed to trigger vesicle expansion in a concerted manner, presumably by providing the driving force for membrane curvature. The transient conjugation of Atg8 to the membrane lipid phosphatidylethanolamine
is essential for phagophore expansion as its mutation
leads to defects in autophagosome formation. It is distributed symmetrically on both sides of the autophagosome and it is assumed that there is a quantitative correlation between the amount of Atg8 and the vesicle size.
After finishing vesicle expansion, the autophagosome is ready for fusion with the lysosome
and Atg8 can either be released from the membrane for recycling (see below) or gets degraded in the autolysosome if left uncleaved.
ATG8 is also required for a different autophagy-related process called the Cytoplasm-to-vacuole-targeting (Cvt) pathway. This yeast-specific process acts constitutively under nutrient-rich conditions and selectively transports hydrolases such as aminopeptidase I to the yeast vacuole. The Cvt pathway also requires Atg8 localised to the PAS for the formation of Cvt vesicles which then fuse with the vacuole to deliver hydrolases necessary for degradation.
(PE) which is a lipid constituent of plasma membranes. This post-translational modification process, called lipidation, is performed by the Atg8 conjugation system comprising the cysteine protease ATG4 (belonging to the caspase family), as well as the proteins ATG7
, ATG3 and the ATG5
-ATG12
complex.
The Atg8 conjugation system (Fig.1) works in analogy to the ubiquitination system. However, it is Atg8 itself that represents the ubiquitin-like protein (Ubl) being transferred to PE, while ATG7 functions like an E1 enyzme, ATG3 like an E2 enyzme and the ATG12-ATG5 complex like an E3 ligase
The lipidation process is initiated by an ATG4 dependent post-translational cleavage of the last C-terminal amino acid residue of Atg8. After the cleavage, Atg8 exposes a C-terminal glycine residue (Gly 116) to which PE can then be coupled during the following steps. In the first step, the Gly116 residue of Atg8 binds to a cysteine residue of ATG7
via a thioester bond in an ATP-dependent manner. During the second step, Atg8 is transferred to Atg3 assuming the same type of thioester bondage. Finally, Atg8 is detached from Atg3 and coupled to the amine head group of PE via an amide bond. This final step was found to be facilitated and stimulated by the ATG5
-ATG12
complex.
Both proteins, Atg5 and Atg12 were originally identified as part of another Ubl conjugating system that promotes conjugation of ATG12
to ATG5
via ATG7
and Atg10. This implies, that the ATG12
and the Atg8 conjugation system are actually interdependent.
One of them is LC3 (MAP1LC3A
), a light chain of the microtubule-associated protein 1 Like Atg8, LC3 needs to be proteolytically cleaved and lipidated to be turned into its active form which can localize to the autophagosomal membrane. Similar to the situation in yeast, the activation process of LC3 is triggered by nutrient depletion, but interestingly also in response to hormones.
Other homologues are the transport factor GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) which plays an important role in intra-golgi vesicular transport by stimulating NSF
(N-ethylmaleimide-sensitive factor) ATPase activity and interacting with the Golgi v-SNARE GOS-28, and GABARAP
(γ-aminobutyric acid type A receptor associated protein) which facilitates clustering of GABAA receptors in combination with microtubules.
All three proteins are characterized by proteolytic activation processes upon which they get lipidated and localized to the plasma membrane. However, for GATE-16 and GABARAP membrane association seems to be possible even for the non-lipidated forms. Apart from LC3, GABARAP and GATE-16
the most recently but less well characterized mammalian homologue is ATGL8. Little is known about its actual activation process except for its interaction with one of the mammalian ATG4 homologues, hATG4A
.
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
-like protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
required for the formation of autophagosomal membranes. The transient conjugation of Atg8 to the autophagosomal membrane through a ubiquitin-like conjugation system is essential for autophagy
Autophagy
In cell biology, autophagy, or autophagocytosis, is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. It is a tightly regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance...
in eukaryotes. Even though there are homologues in animals (see for example GABARAP
GABARAP
Gamma-aminobutyric acid receptor-associated protein is a protein that in humans is encoded by the GABARAP gene.A study has screened 2000 participants representing more than 600 families of smokers and examined different genes showing linkage to nicotine addiction...
, GABARAPL1, GABARAPL2
GABARAPL2
Gamma-aminobutyric acid receptor-associated protein-like 2 is a protein that in humans is encoded by the GABARAPL2 gene.-Interactions:GABARAPL2 has been shown to interact with ULK1 and ATG4B.-Further reading:...
, MAP1LC3A
MAP1LC3A
Microtubule-associated proteins 1A/1B light chain 3A is a protein that in humans is encoded by the MAP1LC3A gene.. MAPLC3A is the mammalian homologue of yeast ATG8, an important marker and effector of autophagy.-Interactions:...
, MAP1LC3B
MAP1LC3B
Microtubule-associated proteins 1A/1B light chain 3B is a protein that in humans is encoded by the MAP1LC3B gene.-Further reading:...
, MAP1LC3B2, and MAP1LC3C), this article mainly focuses on its role in lower eukaryotes such as Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
.
Structure
Atg8 is a monomer of 117 aminoacids and a molecular weight of 13,6kDa. It consists of a 5-stranded β-sheet, which is enclosed by two α-helices at one side and one α-helix at the other side and exhibits a conserved GABARAPGABARAP
Gamma-aminobutyric acid receptor-associated protein is a protein that in humans is encoded by the GABARAP gene.A study has screened 2000 participants representing more than 600 families of smokers and examined different genes showing linkage to nicotine addiction...
domain. Even though Atg8 does not show a clear sequence homology to ubiquitin
Ubiquitin
Ubiquitin is a small regulatory protein that has been found in almost all tissues of eukaryotic organisms. Among other functions, it directs protein recycling.Ubiquitin can be attached to proteins and label them for destruction...
, its crystal structure reveals a conserved ubiquitin-like fold.
In Autophagy
Atg8 is one of the key molecular components involved in autophagyAutophagy
In cell biology, autophagy, or autophagocytosis, is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. It is a tightly regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance...
, the cellular process mediating the lysosome
Lysosome
thumb|350px|Schematic of typical animal cell, showing subcellular components. [[Organelle]]s: [[nucleoli]] [[cell nucleus|nucleus]] [[ribosomes]] [[vesicle |vesicle]] rough [[endoplasmic reticulum]]...
/vacuole
Vacuole
A vacuole is a membrane-bound organelle which is present in all plant and fungal cells and some protist, animal and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic molecules including enzymes in solution, though in certain...
-dependent turnover of macromolecules and organelles. Autophagy is induced upon nutrient depletion or rapamycin treatment and leads to the response of more than 30 autophagy-related (ATG) genes known so far, including ATG8.
How exactly ATG proteins are regulated is still under investigation, but it is clear that all signals reporting on the availability of carbon and nitrogen sources converge on the TOR signalling pathway and that ATG proteins are downstream effectors of this pathway. In case nutrient supplies are sufficient, the TOR signaling pathway hyperphosphorylates certain Atg proteins, thereby inhibiting autophagosome formation. After starvation autophagy is induced through the activation of Atg proteins both on the protein modification and the transciptional level.
Atg8 is especially important in macroautophagy which is one of three distinct types of autophagy characterized by the formation of double-membrane enclosed vesicles
Vesicle (biology)
A vesicle is a bubble of liquid within another liquid, a supramolecular assembly made up of many different molecules. More technically, a vesicle is a small membrane-enclosed sack that can store or transport substances. Vesicles can form naturally because of the properties of lipid membranes , or...
that sequester portions of the cytosol
Cytosol
The cytosol or intracellular fluid is the liquid found inside cells, that is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondrion into compartments....
, the so called autophagosomes. The outer membrane of these autophagosomes subsequently fuses with the lysosome/vacuole to release an inter single membrane (autophagic body) destined for degradation
Chemical decomposition
Chemical decomposition, analysis or breakdown is the separation of a chemical compound into elements or simpler compounds. It is sometimes defined as the exact opposite of a chemical synthesis. Chemical decomposition is often an undesired chemical reaction...
. During this process, Atg8 is particularly crucial for autophagosome maturation (lipidation).
Like most Atg proteins, Atg8 is localized in the cytoplasm and at the PAS under nutrient-rich conditions, but becomes membrane-associated in case of autophagy induction. It then localizes to the site of autophagosome nucleation, the phagophore-assembly site (PAS). Nucleation of the phagophore requires the accumulation of a set of Atg proteins and of class III phosphoinositide 3-kinase
Phosphoinositide 3-kinase
Phosphatidylinositol 3-kinases are a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking, which in turn are involved in cancer. In response to lipopolysaccharide, PI3K phosphorylates p65, inducing...
complexes on the PAS. The subsequent recruitment of Atg8 and other autophagy-related proteins is believed to trigger vesicle expansion in a concerted manner, presumably by providing the driving force for membrane curvature. The transient conjugation of Atg8 to the membrane lipid phosphatidylethanolamine
Phosphatidylethanolamine
Phosphatidylethanolamine is a lipid found in biological membranes. It is synthesized by the addition of CDP-ethanolamine to diglyceride, releasing CMP. S-adenosyl methionine can subsequently methylate the amine of phosphatidyl ethanolamine to yield phosphatidyl choline.Cephalin is a phospholipid,...
is essential for phagophore expansion as its mutation
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
leads to defects in autophagosome formation. It is distributed symmetrically on both sides of the autophagosome and it is assumed that there is a quantitative correlation between the amount of Atg8 and the vesicle size.
After finishing vesicle expansion, the autophagosome is ready for fusion with the lysosome
Lysosome
thumb|350px|Schematic of typical animal cell, showing subcellular components. [[Organelle]]s: [[nucleoli]] [[cell nucleus|nucleus]] [[ribosomes]] [[vesicle |vesicle]] rough [[endoplasmic reticulum]]...
and Atg8 can either be released from the membrane for recycling (see below) or gets degraded in the autolysosome if left uncleaved.
ATG8 is also required for a different autophagy-related process called the Cytoplasm-to-vacuole-targeting (Cvt) pathway. This yeast-specific process acts constitutively under nutrient-rich conditions and selectively transports hydrolases such as aminopeptidase I to the yeast vacuole. The Cvt pathway also requires Atg8 localised to the PAS for the formation of Cvt vesicles which then fuse with the vacuole to deliver hydrolases necessary for degradation.
Post-translational Modification and Regulatory Cycle
Atg8 exists in a cytoplasmic and in a membrane-associated form. Membrane association is achieved by coupling Atg8 to phosphatidylethanolaminePhosphatidylethanolamine
Phosphatidylethanolamine is a lipid found in biological membranes. It is synthesized by the addition of CDP-ethanolamine to diglyceride, releasing CMP. S-adenosyl methionine can subsequently methylate the amine of phosphatidyl ethanolamine to yield phosphatidyl choline.Cephalin is a phospholipid,...
(PE) which is a lipid constituent of plasma membranes. This post-translational modification process, called lipidation, is performed by the Atg8 conjugation system comprising the cysteine protease ATG4 (belonging to the caspase family), as well as the proteins ATG7
ATG7
Autophagy-related protein 7 is a protein that in humans is encoded by the ATG7 gene.-Further reading:...
, ATG3 and the ATG5
ATG5
Autophagy protein 5 is a protein that in humans is encoded by the ATG5 gene.-Further reading:...
-ATG12
ATG12
Autophagy-related protein 12 is a protein that in humans is encoded by the ATG12 gene.-Further reading:...
complex.
The Atg8 conjugation system (Fig.1) works in analogy to the ubiquitination system. However, it is Atg8 itself that represents the ubiquitin-like protein (Ubl) being transferred to PE, while ATG7 functions like an E1 enyzme, ATG3 like an E2 enyzme and the ATG12-ATG5 complex like an E3 ligase
The lipidation process is initiated by an ATG4 dependent post-translational cleavage of the last C-terminal amino acid residue of Atg8. After the cleavage, Atg8 exposes a C-terminal glycine residue (Gly 116) to which PE can then be coupled during the following steps. In the first step, the Gly116 residue of Atg8 binds to a cysteine residue of ATG7
ATG7
Autophagy-related protein 7 is a protein that in humans is encoded by the ATG7 gene.-Further reading:...
via a thioester bond in an ATP-dependent manner. During the second step, Atg8 is transferred to Atg3 assuming the same type of thioester bondage. Finally, Atg8 is detached from Atg3 and coupled to the amine head group of PE via an amide bond. This final step was found to be facilitated and stimulated by the ATG5
ATG5
Autophagy protein 5 is a protein that in humans is encoded by the ATG5 gene.-Further reading:...
-ATG12
ATG12
Autophagy-related protein 12 is a protein that in humans is encoded by the ATG12 gene.-Further reading:...
complex.
Both proteins, Atg5 and Atg12 were originally identified as part of another Ubl conjugating system that promotes conjugation of ATG12
ATG12
Autophagy-related protein 12 is a protein that in humans is encoded by the ATG12 gene.-Further reading:...
to ATG5
ATG5
Autophagy protein 5 is a protein that in humans is encoded by the ATG5 gene.-Further reading:...
via ATG7
ATG7
Autophagy-related protein 7 is a protein that in humans is encoded by the ATG7 gene.-Further reading:...
and Atg10. This implies, that the ATG12
ATG12
Autophagy-related protein 12 is a protein that in humans is encoded by the ATG12 gene.-Further reading:...
and the Atg8 conjugation system are actually interdependent.
Mammalian Homologues
In higher eukaryotes Atg8 is not encoded by a single gene as in yeast, but derived from a multigene family. Four of its homologues have already been identified in mammalian cells.One of them is LC3 (MAP1LC3A
MAP1LC3A
Microtubule-associated proteins 1A/1B light chain 3A is a protein that in humans is encoded by the MAP1LC3A gene.. MAPLC3A is the mammalian homologue of yeast ATG8, an important marker and effector of autophagy.-Interactions:...
), a light chain of the microtubule-associated protein 1 Like Atg8, LC3 needs to be proteolytically cleaved and lipidated to be turned into its active form which can localize to the autophagosomal membrane. Similar to the situation in yeast, the activation process of LC3 is triggered by nutrient depletion, but interestingly also in response to hormones.
Other homologues are the transport factor GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) which plays an important role in intra-golgi vesicular transport by stimulating NSF
NSF
-Politics:* National Service Framework, any of a series of British government policies on medical care* National Socialist Front, a Swedish Nazi party* NS-Frauenschaft, the women's wing of the former German Nazi party...
(N-ethylmaleimide-sensitive factor) ATPase activity and interacting with the Golgi v-SNARE GOS-28, and GABARAP
GABARAP
Gamma-aminobutyric acid receptor-associated protein is a protein that in humans is encoded by the GABARAP gene.A study has screened 2000 participants representing more than 600 families of smokers and examined different genes showing linkage to nicotine addiction...
(γ-aminobutyric acid type A receptor associated protein) which facilitates clustering of GABAA receptors in combination with microtubules.
All three proteins are characterized by proteolytic activation processes upon which they get lipidated and localized to the plasma membrane. However, for GATE-16 and GABARAP membrane association seems to be possible even for the non-lipidated forms. Apart from LC3, GABARAP and GATE-16
GABARAPL2
Gamma-aminobutyric acid receptor-associated protein-like 2 is a protein that in humans is encoded by the GABARAPL2 gene.-Interactions:GABARAPL2 has been shown to interact with ULK1 and ATG4B.-Further reading:...
the most recently but less well characterized mammalian homologue is ATGL8. Little is known about its actual activation process except for its interaction with one of the mammalian ATG4 homologues, hATG4A
ATG4A
Cysteine protease ATG4A is an enzyme that in humans is encoded by the ATG4A gene.-Further reading:...
.
See also
- autophagyAutophagyIn cell biology, autophagy, or autophagocytosis, is a catabolic process involving the degradation of a cell's own components through the lysosomal machinery. It is a tightly regulated process that plays a normal part in cell growth, development, and homeostasis, helping to maintain a balance...
- autophaginAutophaginAutophagin-1 is a unique cysteine protease responsible for the cleavage of the carboxyl terminus of Atg8/Apg8/Aut7, a reaction essential for its lipidation during autophagy...
- ATG5ATG5Autophagy protein 5 is a protein that in humans is encoded by the ATG5 gene.-Further reading:...
- ATG7ATG7Autophagy-related protein 7 is a protein that in humans is encoded by the ATG7 gene.-Further reading:...
- ATG12ATG12Autophagy-related protein 12 is a protein that in humans is encoded by the ATG12 gene.-Further reading:...
- MAP1LC3AMAP1LC3AMicrotubule-associated proteins 1A/1B light chain 3A is a protein that in humans is encoded by the MAP1LC3A gene.. MAPLC3A is the mammalian homologue of yeast ATG8, an important marker and effector of autophagy.-Interactions:...
- GABARAPGABARAPGamma-aminobutyric acid receptor-associated protein is a protein that in humans is encoded by the GABARAP gene.A study has screened 2000 participants representing more than 600 families of smokers and examined different genes showing linkage to nicotine addiction...