Atg8: an autophagy-related ubiquitin-like protein family.

“Although mammalian Atg8 proteins were originally implicated in intracellular trafficking processes, they are all found within autophagosomes [73-75].”

“The motif in p62 responsible for binding Atg8, the LC3interacting region (LIR, also known as Atg8-interacting motif, AIM) [104,105], is common to other proteins that interact with Atg8 proteins, including Nbr1 [106], Atg3 [107], Nix [96] and the yeast cytoplasm-to-vacuole targeting receptor Atg19 [108].”

“[113] show the conservation (overall height) and residue prevalence of multiple alignment positions.”

“These adaptors bind simultaneously to Atg8 proteins and to ubiquitinated proteins, organelles, and in some cases pathogens [18,94,103].”

“Sequences for the alignments were taken from the CDD [114] and PFAM [115] database entries cd01611 and PF02991, respectively, and sequences”

“Sequences for the alignments were taken from the CDD [114] and PFAM [115] database entries cd01611 and PF02991, respectively, and sequences”

“Unlike ubiquitin, which is conjugated to a protein, Atg8 is covalently conjugated to PE lipid through its carboxy-terminal exposed glycine [64].”

“Thus, transgenic mice and tissue-culture cells expressing GFP-LC3 have been used to investigate autophagy [84,85].”

“Atg4 regulates a critical step in the autophagic process and is therefore subjected to regulation by reactive oxygen species, for example, which prevent it from reversing the conjugation of Atg8 proteins to PE during autophagy [83].”

“Although the transcriptional regulation of Atg8 genes is largely unknown, several reports have shown that the transcription factors FoxO1, FoxO3 and E2F1 regulate Atg8s in this process in mammalian cells [31-34].”

“Several approaches aimed at disrupting the functioning of Atg8 proteins by preventing their conjugation to PE have therefore been used, including the generation of Atg3-null mice and expression of a dominant-negative mutant of the promiscuous protease Atg4B.”

“Although the transcriptional regulation of Atg8 genes is largely unknown, several reports have shown that the transcription factors FoxO1, FoxO3 and E2F1 regulate Atg8s in this process in mammalian cells [31-34].”

“Although mammalian Atg8 proteins were originally implicated in intracellular trafficking processes, they are all found within autophagosomes [73-75].”

“autophagy-linked FYVE protein (Alfy) [98-100], nuclear dot protein 52 kDa (NDP52) [101], and Optineurin [102] as autophagic receptors.”

“This role was further established using an in vitro liposome-based system, where it can promote membrane fusion [20,22].”

“Choanoflagellates, unicellular species that are the closest known relatives of animals [6], have ATG8 genes that do not belong to the three metazoan subfamilies (Figure 1a).”

“The Atg5-Atg12 conjugate acts as an E3-like ligase, mediating the formation of Atg8-PE conjugate in vitro, suggesting a role for this complex in determining the Atg8 conjugation site in cells [67].”

“It was suggested that Atg16L (the mammalian Atg16) determines the lipida tion site of LC3 and serves as its E3 ligase [77].”

“These have led to the conclusion that PE-conjugated Atg8 proteins are active during autophagosome formation and are essential for the elongation and closure of the initial autophagic membrane, the phagophore [86-88].”

“Considerable progress in establishing this idea was achieved by the characterization of the ubiquitin-binding proteins p62/SQSTM1 [94], neighbor of Brca1 (Nbr1) [95], the Bcl2-related protein Nix [96,97], Shpilka et al.”

“Similar to many other cytoplasm-tovacuole targeting and autophagic factors, Atg8 is localized to the pre-autophagosomal structure under basal and nitrogen depletion conditions [65], a process dependent on the phagophore resident complex Atg5-Atg12Atg16 [66].”

“mediated by the cysteine protease Atg4, which is specific to Atg8 proteins [60,61].”

“autophagy-linked FYVE protein (Alfy) [98-100], nuclear dot protein 52 kDa (NDP52) [101], and Optineurin [102] as autophagic receptors.”

“Upregulation of Atg8s is observed under various stress conditions [27-30].”

“The motif in p62 responsible for binding Atg8, the LC3interacting region (LIR, also known as Atg8-interacting motif, AIM) [104,105], is common to other proteins that interact with Atg8 proteins, including Nbr1 [106], Atg3 [107], Nix [96] and the yeast cytoplasm-to-vacuole targeting receptor Atg19 [108].”

“Apparently, both the LC3 and the GABARAP/GATE-16 subfamilies are essential for autophagy, as they act at different stages of autophagosome formation: members of the LC3 subfamily are responsible for the elongation of the autophagic membrane, whereas GABARAP/GATE-16 subfamily members act downstream in a step coupled to…”

“GABARAP, however, was identified as a cytosolic protein that is localized to trans port vesicles, the Golgi network and the endoplasmic reticulum (ER) and that interacts with the γ2 subunit of GABA(A) receptors [40].”

“Atg8 also participates in formation of the autophagosome and is essential for elongation of the autophagic membrane [68,69].”

“The motif in p62 responsible for binding Atg8, the LC3interacting region (LIR, also known as Atg8-interacting motif, AIM) [104,105], is common to other proteins that interact with Atg8 proteins, including Nbr1 [106], Atg3 [107], Nix [96] and the yeast cytoplasm-to-vacuole targeting receptor Atg19 [108].”

“autophagy-linked FYVE protein (Alfy) [98-100], nuclear dot protein 52 kDa (NDP52) [101], and Optineurin [102] as autophagic receptors.”

“Furthermore, the overexpression approach is not optimal as LC3 proteins tend to form aggregates when transiently expressed [110].”

“These have led to the conclusion that PE-conjugated Atg8 proteins are active during autophagosome formation and are essential for the elongation and closure of the initial autophagic membrane, the phagophore [86-88].”

“autophagic factors The first link between Atg8 proteins and autophagy was established in Saccharomyces cerevisiae, where the single Atg8 protein was identified as an autophagy and cytoplasm-to-vacuole targeting essential factor [54-56].”

“These have led to the conclusion that PE-conjugated Atg8 proteins are active during autophagosome formation and are essential for the elongation and closure of the initial autophagic membrane, the phagophore [86-88].”

“Arrival of Atg8 proteins at the autophagic membrane depends on the hierarchical recruitment of numerous auto phagic factors, including phosphatidylinositol-3phosphate-binding proteins [18].”

“autophagy-linked FYVE protein (Alfy) [98-100], nuclear dot protein 52 kDa (NDP52) [101], and Optineurin [102] as autophagic receptors.”

“Although the transcriptional regulation of Atg8 genes is largely unknown, several reports have shown that the transcription factors FoxO1, FoxO3 and E2F1 regulate Atg8s in this process in mammalian cells [31-34].”

“The motif in p62 responsible for binding Atg8, the LC3interacting region (LIR, also known as Atg8-interacting motif, AIM) [104,105], is common to other proteins that interact with Atg8 proteins, including Nbr1 [106], Atg3 [107], Nix [96] and the yeast cytoplasm-to-vacuole targeting receptor Atg19 [108].”

“The cleaved Atg8 is than processed by a ubiquitin-like conjugation machinery composed of Atg7, an E1-like enzyme [62], and Atg3, a specific E2-like ligase [63].”

“Upregulation of Atg8s is observed under various stress conditions [27-30].”

“This study [22] revealed that the first amino-terminal α helix of the Atg8 proteins was both essential and sufficient for this activity.”

“Cytoplasm-to-vacuole targeting is responsible for transporting vacuolar protein residents, such as aminopeptidase I (Ape1) and α-mannosidase (Ams1), to the vacuole [57,58].”

“autophagy-linked FYVE protein (Alfy) [98-100], nuclear dot protein 52 kDa (NDP52) [101], and Optineurin [102] as autophagic receptors.”

“LC3 was first characterized as a protein that co-purified with microtubule-associated proteins 1A and 1B [38,39], but no direct involvement of this protein in membrane trafficking processes has been reported.”

“Atg8 also participates in formation of the autophagosome and is essential for elongation of the autophagic membrane [68,69].”

“Acetyla tion of the autophagic factors Atg5, Atg7, Atg8 and Atg12 is mediated by the acetyltransferase p300 and negatively regulates their activity [92].”

“Of the LC3 subfamily, LC3C, which is transcribed at lower levels than other members of this subfamily, is expressed predominantly in the lung [23,24].”

“brevicollis (Mbe) [6], the ichthyosporean Sphaeroforma arctica (Sar) and the amoeba Capsaspora owczarzaki (Cow) [111].”

“These homologs show different specificities toward Atg8 proteins: Atg4B shows a broad spectrum for all family members [78,79], whereas Atg4A is specific to the GABARAP and GATE-16 subfamilies [80,81].”

“autophagic factors The first link between Atg8 proteins and autophagy was established in Saccharomyces cerevisiae, where the single Atg8 protein was identified as an autophagy and cytoplasm-to-vacuole targeting essential factor [54-56].”

“Atg4 might function in pathways other than autophagy, as cleavage of Atg4D by caspase increases its activity and consequently promotes GABARAP-L1 processing [82].”

“Although the transcriptional regulation of Atg8 genes is largely unknown, several reports have shown that the transcription factors FoxO1, FoxO3 and E2F1 regulate Atg8s in this process in mammalian cells [31-34].”

“autophagic factors The first link between Atg8 proteins and autophagy was established in Saccharomyces cerevisiae, where the single Atg8 protein was identified as an autophagy and cytoplasm-to-vacuole targeting essential factor [54-56].”

“These homologs show different specificities toward Atg8 proteins: Atg4B shows a broad spectrum for all family members [78,79], whereas Atg4A is specific to the GABARAP and GATE-16 subfamilies [80,81].”

“In yeast, Atg8 expression is upregu lated during nitrogen starvation and is degraded in the vacuole following recruitment into autophagosomes [59].”

“Expression analysis of the different GABARAP proteins reveals a distinct pattern: GABARAP-L1 is expressed predominantly in the central nervous system, similar to GATE-16, which is strongly expressed in the brain, whereas the expression of GABARAP is more marked in the endocrine glands [25,26].”

“In addition to its interaction with the conjugation proteins, Atg8 also binds to other autophagic factors, such as Atg1, which might regulate its recruitment and activity on the autophagic membrane [60].”

“The role of GABARAP in intracellular transport is also supported by its interaction with NSF, a component essential for SNARE-mediated fusion [41,45].”

“The motif in p62 responsible for binding Atg8, the LC3interacting region (LIR, also known as Atg8-interacting motif, AIM) [104,105], is common to other proteins that interact with Atg8 proteins, including Nbr1 [106], Atg3 [107], Nix [96] and the yeast cytoplasm-to-vacuole targeting receptor Atg19 [108].”

“These homologs show different specificities toward Atg8 proteins: Atg4B shows a broad spectrum for all family members [78,79], whereas Atg4A is specific to the GABARAP and GATE-16 subfamilies [80,81].”

“Similar to LC3, GABARAP binds to tubulin through its amino-terminal region, an interaction that regulates clustering of GABA(A) receptors [8,46,47].”

“A conformational change was also reported in an in vitro reconstitution assay of yeast Atg8, in which accessibility of the amino terminus to antibody increased on conjugation of Atg8 to phosphatidylethanolamine (PE)-containing liposomes [19].”

“This interaction is thought to be essential for trafficking of the GABA(A) receptor to the plasma membrane [41].”

“Similar to many other cytoplasm-tovacuole targeting and autophagic factors, Atg8 is localized to the pre-autophagosomal structure under basal and nitrogen depletion conditions [65], a process dependent on the phagophore resident complex Atg5-Atg12Atg16 [66].”

“thaliana ATG8 genes showed distinct spatial and temporal expression patterns in different tissues [7].”

“These adaptors bind simultaneously to Atg8 proteins and to ubiquitinated proteins, organelles, and in some cases pathogens [18,94,103].”

“Thus, transgenic mice and tissue-culture cells expressing GFP-LC3 have been used to investigate autophagy [84,85].”

“Subsequent studies confirmed that these features are a unique characteristic of all Atg8 proteins [8-15] (Figure 2).”

“This interaction results in the arrival of glycogen at the lysosome and is therefore suggested to regulate cellular glucose levels [109].”

“GATE-16, the first Atg8 to be crystallized, contains two amino-terminal α-helices in addition to its carboxy-terminal ubiquitin core [13].”

“This interaction was suggested to control the forma tion of autophagosomes [72], indicating that Atg8 might provide a link between classical autophagic machinery and general trafficking factors that are crucial for autophagy.”

“In addition to their direct role in fusion events during autophagosome biogenesis, Atg8 proteins have an indirect role in which their binding to diabetes- and obesity-regulated protein (DOR) and to tumor protein 53-induced nuclear protein 1 (TP53INP2), two proteins that shuttle between the nucleus and the cytoplasm, r…”

“Although mammalian Atg8 proteins were originally implicated in intracellular trafficking processes, they are all found within autophagosomes [73-75].”

“Of the LC3 subfamily, LC3C, which is transcribed at lower levels than other members of this subfamily, is expressed predominantly in the lung [23,24].”

“Cytoplasm-to-vacuole targeting is responsible for transporting vacuolar protein residents, such as aminopeptidase I (Ape1) and α-mannosidase (Ams1), to the vacuole [57, 58 ].”

“Several approaches aimed at disrupting the functioning of Atg8 proteins by preventing their conjugation to PE have therefore been used, including the generation of Atg3-null mice and expression of a dominant-negative mutant of the promiscuous protease Atg4B.”

“Subsequent studies confirmed that these features are a unique characteristic of all Atg8 proteins [8-15] (Figure 2).”

“The cleaved Atg8 is than processed by a ubiquitin-like conjugation machinery composed of Atg7, an E1-like enzyme [62], and Atg3, a specific E2-like ligase [63].”

“GABARAP-L1 also binds directly to the G-protein-coupled κ-opioid receptor and mediates its transport in a manner dependent on modification of the carboxyl terminus [ 48 ,50,51].”

“In addition to their direct role in fusion events during autophagosome biogenesis, Atg8 proteins have an indirect role in which their binding to diabetes- and obesity-regulated protein (DOR) and to tumor protein 53-induced nuclear protein 1 (TP53INP2), two proteins that shuttle between the nucleus and the cytoplasm, r…”

“GATE-16 has been implicated in intra-Golgi transport through its interaction with Golgi SNARE protein 28 (GOS-28).”

“These homologs show different specificities toward Atg8 proteins: Atg4B shows a broad spectrum for all family members [78,79], whereas Atg4A is specific to the GABARAP and GATE-16 subfamilies [80,81].”

“Between these strands lie two α helices: α3 between β2 and β3, and α4 between β3 and β4 [16].”

“The molecular surface lying on the opposite side of the three-dimensional structure is poorly conserved and might mediate interactions specific to different Atg8 proteins [17], whereas the conserved domain is probably responsible for characteristic interactions of Atg8 proteins, such as binding to the conjugation mach…”

“This activity might involve the previously reported interaction of Atg8 with SNAREs in the yeast system [53].”

“Consistent with the involvement of mammalian Atg8s in membrane fusion events, the yeast Pichia pastoris Atg8 was recently found to mediate vacuolar fusion during adaptation from glucose- to methanol-containing medium [52].”

“GATE-16 has been implicated in intra-Golgi transport through its interaction with Golgi SNARE protein 28 (GOS-28).”

“Taken together, these findings suggest that the Atg8 amino-terminal region has a crucial role in the functions of these proteins [20-22].”

“GATE-16 GOS28 GABA(A) receptor Transferrin receptor κ Opioid receptor PXRICS ER Golgi G B GB G B 1 G B / G B 1 G B 1 Golgi PM G B NSF NSF G B / G B 1 GB/ GB1 β-catβ-cat N-cad herin (a) (b) mediated by the cysteine protease Atg4, which is specific to Atg8 proteins [60,61].”

“on processing of the GABARAP carboxyl terminus [42].”

“GABARAP’s involvement in vesicle transport is not limited to the GABA(A) receptor; other plasma membrane proteins, such as the transferrin receptor, interact with GABARAP, although the role of GABARAP in their transport is not well established [43].”

“Subsequent studies confirmed that these features are a unique characteristic of all Atg8 proteins [8-15] (Figure 2).”

“Expression analysis of the different GABARAP proteins reveals a distinct pattern: GABARAP-L1 is expressed predominantly in the central nervous system, similar to GATE-16, which is strongly expressed in the brain, whereas the expression of GABARAP is more marked in the endocrine glands [25,26].”

“binding to Atg19 receptor also disrupt autophagosome biogenesis [70,71].”

“It has been suggested that these factors mediate the transport of the transmembrane protein N-cadherin and its receptor β-catenin from the ER, thus controlling cell-cell adhesion [44].”

“Upregulation of Atg8s is observed under various stress conditions [27-30].”

“Subsequent studies confirmed that these features are a unique characteristic of all Atg8 proteins [8-15] (Figure 2).”

“GABARAP-L1 also binds directly to the G-protein-coupled κ-opioid receptor and mediates its transport in a manner dependent on modification of the carboxyl terminus [48,50,51].”

“Subsequent studies confirmed that these features are a unique characteristic of all Atg8 proteins [8-15] (Figure 2).”

“The role of Atg8 in intracellular trafficking processes Atg8s were originally implicated in membrane trafficking processes [35].”

“Subsequent studies confirmed that these features are a unique characteristic of all Atg8 proteins [8-15] (Figure 2).”

“binding to Atg19 receptor also disrupt autophagosome biogenesis [70,71].”

“and invariant proportions as previously described [112].”

“Upregulation of Atg8s is observed under various stress conditions [27-30].”

“For instance, the human genome codes for at least six distinct Atg8-like proteins, namely microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A; best known as LC3A), LC3B, microtubule-associated protein 1 light chain 3 gamma (MAP1LC3C; best known as LC3C), GABA type A receptor-associated protein (GABARAP), GA…”

“Yeast has only one Atg8 homolog, Caenorhabditis elegans and Drosophila melanogaster have two, whereas Arabidopsis thaliana has nine ATG8 homologs (reviewed by Shpilka et al., 2011).”