ATG8

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. (1,2)

Evolution
Autophagy-related 8 proteins (Atg8s) are one of the 62 highly conserved eukaryote-specific protein families. Whereas yeast and other fungal species have a single Atg8 gene, multicellular animals, green plants and some protists have several. Animal Atg8 proteins comprise three subfamilies: microtubule-associated protein 1 light chain 3 (MAP1LC3, hereafter referred to as LC3), γ-aminobutyric acid receptor-associated protein (GABARAP) and Golgi-associated ATPase enhancer of 16 kDa (GATE-16) .Humans have a single GATE-16 gene, two GABARAP genes and four LC3 genes.In addition, LC3A encodes two isoforms resulting from alternative splicing. All three subfamilies are also present in diverse other bilateral species and in the earlier diverging animals - cnidarians (such as sea anemones, corals and hydras) and sponges. Atg8 genes have been both duplicated and lost during evolution, leading to the extinction and expansion of some subfamilies in specific lineages .One example is in arthropods: the blacklegged tick Ixodes scapularis (an arachnid)  has GATE-16, GABARAP and LC3 genes, but in insects, the honey bee Apis mellifera has only a GABARAP and an LC3 gene  and the fruit fly Drosophila has two GABARAP genes . One of the fruit fly GABARAP genes has no coding region introns and could be the result of a retrotransposition of the other gene's transcript during the emergence of the fruit flies. The human LC3 gene MAP1LC3B2 also lacks coding region introns and its coding region has 376 out of 378 bases identical with the human gene MAP1LC3B, which has three coding region introns and is more than 10 kb long . The human genome also includes intron-less copies of all three ATG8 gene subfamilies, which are apparently inactive as a result of frameshifts and nonsense mutations.(3,4,5)

Autophagy
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. (7)

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).(7)

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.(8,9)

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. (9)

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), 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. (10,11)

Footnotes

1. Kirisako T; Baba M; Ishihara N; Miyazawa K; Ohsumi M; Yoshimori T; Noda T; Ohsumi Y. (1999). "Formation Process of Autophagosome Is Traced with Apg8/Aut7p in Yeast". J Cell Biol. 147 (2): 435–46. doi:10.1083/jcb.147.2.435. PMC 2174223. PMID 10525546. Cite error: Invalid <ref> tag; name "Kirisako" defined multiple times with different content (see the help page). 
2. 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. 
3. 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. 
4. 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. 
5. 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. 
6. 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. 
7. 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. 
8. 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. 
9. 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. 
10. 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. 
11. 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.