|autophagy related protein 8|
|The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8.|
|Alt. symbols||Apg8, Aut7, Cvt5|
|Chromosome||VII: 0.16 - 0.16 Mb|
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.
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 GABARAP domain. Even though Atg8 does not show a clear sequence homology to ubiquitin, its crystal structure reveals a conserved ubiquitin-like fold.
Atg8 is one of the key molecular components involved in autophagy, 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 transcriptional 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.
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 phosphatidylethanolamine (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.
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), 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.
Mammalian LC3 isoforms contain a conserved Ser/Thr12, which is phosphorylated by protein kinase A to suppress participation in autophagy/mitophagy.
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.
- PDB 1UGM; Sugawara K, Suzuki NN, Fujioka Y, Mizushima N, Ohsumi Y, Inagaki F (July 2004). "The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8". Genes Cells 9 (7): 611–8. doi:10.1111/j.1356-9597.2004.00750.x. PMID 15265004.
- Geng J, Klionsky DJ (September 2008). "The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein modifications: beyond the usual suspects' review series". EMBO Rep. 9 (9): 859–64. doi:10.1038/embor.2008.163. PMC 2529362. PMID 18704115.
- Sugawara K, Suzuki NN, Fujioka Y, Mizushima N, Ohsumi Y, Inagaki F. (2004). "The crystal structure of microtubule-associated protein light chain 3, a mammalian homologue of Saccharomyces cerevisiae Atg8". Genes to Cells 9 (7): 611–618. doi:10.1111/j.1356-9597.2004.00750.x. PMID 15265004.
- Suzuki NN, Yoshimoto K, Fujioka Y, Ohsumi Y, Inagaki F. (2005). "The crystal structure of plant ATG12 and its biological implication in autophagy". Autophagy 1 (2): 119–126. doi:10.4161/auto.1.2.1859. PMID 16874047.
- Ohsumi, Y. (2001). "Molecular dissection of autophagy: two ubiquitin-like systems". Nature Reviews Molecular Cell Biology 2 (3): 211–216. doi:10.1038/35056522. PMID 11265251.
- Kamada Y, Sekito T, Ohsumi Y. (2004). "Autophagy in yeast: a TOR-mediated response to nutrient starvation". Current Topics in Microbiology and Immunology. Current Topics in Microbiology and Immunology 279: 73–84. doi:10.1007/978-3-642-18930-2_5. ISBN 978-3-540-00534-6. PMID 14560952.
- JEave-Liisa Eskelinen (2008). "New insights into the mechanisms of macroautophagy in mammalian cells". International Review of Cell and Molecular Biology. International Review of Cell and Molecular Biology 266: 207–247. doi:10.1016/S1937-6448(07)66005-5. ISBN 978-0-12-374372-5. PMID 18544495.
- Mizushima N, Yamamoto A, Hatano M, Kobayashi Y, Kabeya Y, Suzuki K, Tokuhisa T, Ohsumi Y, Yoshimori T. (2001). "Dissection of Autophagosome Formation Using Apg5-Deficient Mouse Embryonic Stem Cells". J Cell Biol 152 (4): 657–68. doi:10.1083/jcb.152.4.657. PMC 2195787. PMID 11266458.
- Xie Z, Klionsky DJ. (2007). "Autophagosome formation: core machinery and adaptations". Nat Cell Biol. 9 (10): 1102–9. doi:10.1038/ncb1007-1102. PMID 17909521.
- Huang WP, Scott SV, Kim J, Klionsky DJ. (2000). "The itinerary of a vesicle component, Aut7p/Cvt5p, terminates in the yeast vacuole via the autophagy/Cvt pathways". J Biol Chem. 275 (8): 5845–51. doi:10.1074/jbc.275.8.5845. PMID 10681575.
- Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T. (2000). "LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing". EMBO J. 19 (21): 5720–5728. doi:10.1093/emboj/19.21.5720. PMC 305793. PMID 11060023.
- Kirisako T, Ichimura Y, Okada H, Kabeya Y, Mizushima N, Yoshimori T, Ohsumi M, Takao T, Noda T, Ohsumi Y. (2000). "The Reversible Modification Regulates the Membrane-Binding State of Apg8/Aut7 Essential for Autophagy and the Cytoplasm to Vacuole Targeting Pathway". J Cell Biol. Oct 16; 151(2) (2): 263–76. doi:10.1083/jcb.151.2.263. PMC 2192639. PMID 11038174.
- Xie Z, Nair U, Klionsky DJ. (2008). "Atg8 Controls Phagophore Expansion during Autophagosome Formation". Mol Biol Cell. 19 (8): 3290–8. doi:10.1091/mbc.E07-12-1292. PMC 2488302. PMID 18508918.
- Kim J, Klionsky DJ (2000). "Autophagy, cytoplasm-to-vacuole targeting pathway, and pexophagy in yeast and mammalian cells". Annual Review of Biochemistry 69: 303–342. doi:10.1146/annurev.biochem.69.1.303. PMID 10966461.
- Ichimura Y, Kirisako T, Takao T, Satomi Y, Shimonishi Y, Ishihara N, Mizushima N, Tanida I, Kominami E, Ohsumi M, Noda T, Ohsumi Y. (2000). "A ubiquitin-like system mediates protein lipidation". Nature. Nov 23; 408(6811) (6811): 488–92. doi:10.1038/35044114. PMID 11100732.
- Geng J, Klionsky D (2008). "The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. 'Protein Modifications: Beyond the Usual Suspects' Review Series". EMBO Reports 9 (9): 859–864. doi:10.1038/embor.2008.163. PMC 2529362. PMID 18704115.
- Tanida I, Ueno T, Kominami E. (Dec 2004). "LC3 conjugation system in mammalian autophagy". Int J Biochem Cell Biol. 36 (12): 2503–18. doi:10.1016/j.biocel.2004.05.009. PMID 15325588.
- Cherra SJ, Kulich SM, Uechi G, Balasubramani M, Mountzouris J, Day BW, Chu CT (August 2010). "Regulation of the autophagy protein LC3 by phosphorylation". J. Cell Biol. 190 (4): 533–9. doi:10.1083/jcb.201002108. PMC 2928022. PMID 20713600.
- Sagiv Y, Legesse-Miller A, Porat A, Elazar Z (2000). "GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28". EMBO J. 19 (7): 1494–1504. doi:10.1093/emboj/19.7.1494. PMC 310219. PMID 10747018.
- Chen ZW, Chang CS, Leil TA, Olsen RW. (2007). "C-terminal modification is required for GABARAP-mediated GABA(A) receptor trafficking". J Neurosci. 27 (25): 6655–63. doi:10.1523/JNEUROSCI.0919-07.2007. PMID 17581952.
- Wang H, Bedford FK, Brandon NJ, Moss SJ, Olsen RW. (1999). "GABA(A)-receptor-associated protein links GABA(A) receptors and the cytoskeleton". Nature 397 (6714): 69–72. doi:10.1038/16264. PMID 9892355.
- Tanida I, Sou YS, Minematsu-Ikeguchi N, Ueno T, Kominami E. (2006). "Atg8L/Apg8L is the fourth mammalian modifier of mammalian Atg8 conjugation mediated by human Atg4B, Atg7 and Atg3". FEBS J. 273 (11): 2553–62. doi:10.1111/j.1742-4658.2006.05260.x. PMID 16704426.