Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

Human GABARAP can restore autophagosome biogenesis in a C. elegans lgg-1 mutant

We recently described in C. elegans embryos, the acquisition of specialized functions for orthologs of yeast Atg8 (e.g., mammalian MAP1LC3/LC3) in allophagy, a selective and developmentally regulated autophagic process. During the formation of double-membrane autophagosomes, the ubiquitin-like Atg8/LC3 proteins are recruited to the membrane through a lipidation process. While at least 6 orthologs and paralogs are present in mammals, C. elegans only possesses 2 orthologs, LGG-1 and LGG-2, corresponding to the GABARAP-GABARAPL2/GATE-16 and the MAP1LC3 families, respectively. During allophagy, LGG-1 acts upstream of LGG-2 and is essential for autophagosome biogenesis, whereas LGG-2 facilitates their maturation. We demonstrated that LGG-2 directly interacts with the HOPS complex subunit VPS-39, and mediates the tethering between autophagosomes and lysosomes, which also requires RAB-7. In the present addendum, we compared the localization of autophagosomes, endosomes, amphisomes, and lysosomes in vps-39, rab-7, and lgg-2 depleted embryos. Our results suggest that lysosomes interact with autophagosomes or endosomes through a similar mechanism. We also performed a functional complementation of an lgg-1 null mutant with human GABARAP, its closer homolog, and showed that it localizes to autophagosomes and can rescue LGG-1 functions in the early embryo.     

Guidelines for monitoring autophagy in Caenorhabditis elegans

The cellular recycling process of autophagy has been extensively characterized with standard assays in yeast and mammalian cell lines. In multicellular organisms, numerous external and internal factors differentially affect autophagy activity in specific cell types throughout the stages of organismal ontogeny, adding complexity to the analysis of autophagy in these metazoans. Here we summarize currently available assays for monitoring the autophagic process in the nematode C. elegans. A combination of measuring levels of the lipidated Atg8 ortholog LGG-1, degradation of well-characterized autophagic substrates such as germline P granule components and the SQSTM1/p62 ortholog SQST-1, expression of autophagic genes and electron microscopy analysis of autophagic structures are presently the most informative, yet steady-state, approaches available to assess autophagy levels in C. elegans. We also review how altered autophagy activity affects a variety of biological processes in C. elegans such as L1 survival under starvation conditions, dauer formation, aging, and cell death, as well as neuronal cell specification. Taken together, C. elegans is emerging as a powerful model organism to monitor autophagy while evaluating important physiological roles for autophagy in key developmental events as well as during adulthood.     

Small differences make a big impact: Structural insights into the differential function of the 2 Atg8 homologs in C. elegans

The 2 C. elegans homologs of Atg8, LGG-1 and LGG-2, show differential function in the degradation of protein aggregates during embryogenesis. LGG-1 is essential for the degradation of various protein aggregates, while LGG-2 has cargo-specific and developmental stage-specific roles. LGG-1 and LGG-2 differentially interact with autophagy substrates and ATG proteins. LGG-1 and LGG-2 possess 2 hydrophobic pockets, the W-site and the L-site, which recognize the LIR motif in Atg8-binding proteins. The plasticity of the W-site and the size and shape of the L-site differ between LGG-1 and LGG-2, thus determining their preferences for distinct LIR motifs. The N-terminal tails of LGG-1 and LGG-2 adopt unique closed and open conformations, respectively, which may result in distinct membrane tethering and fusion activities. LGG-1 and LGG-2 have different affinities for ATG-7 and ATG-3, and lipidation of LGG-2 is regulated by levels of lipidated LGG-1. Taken together, the structural differences between LGG-1 and LGG-2 provide insights into their differential functions in the aggrephagy pathway.     

The two C. elegans ATG-16 homologs have partially redundant functions in the basal autophagy pathway

The presence of multiple homologs of the same yeast ATG genes endows an extra layer of complexity on the autophagic machinery in higher eukaryotes. The physiological function of individual homologs in the autophagy pathway remains poorly understood. Here we characterized the function of the two atg16 homologs, atg-16.1 and atg-16.2, in the autophagy pathway in C. elegans. We showed that atg-16.2 mutants exhibit a stronger autophagic defect than atg-16.1 mutants. atg-16.2; atg-16.1 double mutants display a much more severe defect than either single mutant. ATG-16.1 and ATG-16.2 interact with themselves and each other and also directly associate with ATG-5. atg-16.1 mutant embryos exhibit a wild-type expression and distribution pattern of LGG-1/Atg8, while LGG-1 puncta are markedly fewer in number and weaker in intensity in atg-16.2 mutants. In atg-16.2; atg-16.1 double mutants, the lipidated form of LGG-1 accumulates, but LGG-1 puncta are completely absent. ATG-16.2 ectopically expressed on the plasma membrane provides novel sites of LGG-1 puncta formation. We also demonstrated that the C-terminal WD repeats are dispensable for the role of atg-16.2 in aggrephagy (the degradation of protein aggregates by autophagy). Genetic epistasis analysis placed atg-16.2 upstream of atg-2, epg-6, and atg-18. Our study indicated that C. elegans ATG-16s are involved in specifying LGG-1 puncta formation and the two ATG-16 homologs have partially redundant yet distinct functions in the aggrephagy pathway.     

The scaffold protein EPG-7 links cargo–receptor complexes with the autophagic assembly machinery

The mechanism by which protein aggregates are selectively degraded by autophagy is poorly understood. Previous studies show that a family of Atg8-interacting proteins function as receptors linking specific cargoes to the autophagic machinery. Here we demonstrate that during Caenorhabditis elegans embryogenesis, epg-7 functions as a scaffold protein mediating autophagic degradation of several protein aggregates, including aggregates of the p62 homologue SQST-1, but has little effect on other autophagy-regulated processes. EPG-7 self-oligomerizes and is degraded by autophagy independently of SQST-1. SQST-1 directly interacts with EPG-7 and colocalizes with EPG-7 aggregates in autophagy mutants. Mutations in epg-7 impair association of SQST-1 aggregates with LGG-1/Atg8 puncta. EPG-7 interacts with multiple ATG proteins and colocalizes with ATG-9 puncta in various autophagy mutants. Unlike core autophagy genes, epg-7 is dispensable for starvation-induced autophagic degradation of substrate aggregates. Our results indicate that under physiological conditions a scaffold protein endows cargo specificity and also elevates degradation efficiency by linking the cargo–receptor complex with the autophagic machinery.     

The WD40 repeat PtdIns(3)P-binding protein EPG-6 regulates progression of omegasomes to autophagosomes

PtdIns(3)P plays critical roles in the autophagy pathway. However, little is known about how PtdIns(3)P effectors act with autophagy proteins in autophagosome formation. Here we identified an essential autophagy gene in C.?elegans, epg-6, which encodes a WD40 repeat-containing protein with PtdIns(3)P-binding activity. EPG-6 directly interacts with ATG-2. epg-6 and atg-2 regulate progression of omegasomes to autophagosomes, and their loss of function?causes accumulation of enlarged early autophagic structures. Another WD40 repeat PtdIns(3)P effector, ATG-18, plays a distinct role in autophagosome formation. We also established the hierarchical relationship of autophagy genes in degradation of?protein aggregates and revealed that the UNC-51/Atg1 complex, EPG-8/Atg14, and binding of lipidated LGG-1 to protein aggregates are required for?omegasome formation. Our study demonstrates that autophagic PtdIns(3)P effectors play distinct roles in autophagosome formation and also provides?a framework for understanding the concerted action of autophagy genes in protein aggregate degradation.     

Highlights from this issue

Germline P granules are specialized protein/RNA aggregates that are found exclusively in germ cells in C. elegans. During the early embryonic divisions that generate germ blastomeres, aggregate-prone P granule components PGL-1 and PGL-3 that remain in the cytoplasm destined for somatic daughters are selectively removed by autophagy. Loss-of-function of components of the autophagy pathway, including the VPS-34/BEC-1 complex, causes accumulation of PGL-1 and PGL-3 into aggregates in somatic cells (termed PGL granules). Formation of PGL granules depends on SEPA-1, which is an integral component of these granules. SEPA-1 is preferentially degraded by autophagy and is also required for the autophagic degradation of PGL-1 and PGL-3. SEPA-1 functions as a bridging molecule in mediating degradation of P granule components by directly interacting with PGL-3 and also with the autophagy protein LGG-1/Atg8. The defect in embryonic development in autophagy mutants is suppressed by mutation of sepa-1, suggesting that autophagic degradation of PGL granule components may provide nutrients for embryogenesis and/or also prevent the formation of aggregates that could be toxic for animal development. Our study reveals a specific physiological function of selective autophagic degradation during C. elegans development.     

The recycling endosome protein RAB-10 promotes autophagic flux and localization of the transmembrane protein ATG-9

Macroautophagy/autophagy involves the formation of an autophagosome, a double-membrane vesicle that delivers sequestered cytoplasmic cargo to lysosomes for degradation and recycling. Closely related, endocytosis mediates the sorting and transport of cargo throughout the cell, and both processes are important for cellular homeostasis. However, how endocytic proteins functionally intersect with autophagy is not clear. Mutations in the DAF-2/insulin-like IGF-1 (INSR) receptor at the permissive temperature result in a small increase in GFP::LGG-1 foci, i.e. autophagosomes, but a large increase at the nonpermissive temperature, allowing us to control the level of autophagy. In a RNAi screen for endocytic genes that alter the expression of GFP::LGG-1 in daf-2 mutants, we identified RAB-10, a small GTPase that regulates basolateral endocytosis. Loss of rab-10 in daf-2 mutants results in more GFP::LGG-1-positive foci at the permissive, but less GFP::LGG-1 or SQST-1::GFP foci at the nonpermissive temperature. As previously reported, loss of rab-10 alone resulted in an increase of GFP:LGG-1 foci. Exposure of rab-10 mutant animals to chloroquine, a known inhibitor of autophagic flux, failed to increase the number of GFP::LGG-1 foci. Moreover, colocalization between LMP-1::tagRFP and GFP::LGG-1 (the lysosome and autophagosome reporters) was decreased in daf-2; rab-10 dauers at the nonpermissive temperature. Intriguingly, RAB-10 was required to maintain the normal size of GFP::ATG-9-positive structures in daf-2 mutants at both the permissive and nonpermissive temperature. Finally, we found that RAB-10 GTPase cycling was required to control the size of GFP::ATG-9 foci. Collectively, our data support a model where rab-10 controls autophagic flux by regulating autophagosome formation and maturation.     

Induction of autophagic flux by amino acid deprivation is distinct from nitrogen starvation-induced macroautophagy

A number of signaling mechanisms have been implicated in the regulation of autophagic trafficking. Tor kinase activity, cAMP levels, and the GAAC pathway have all been suggested to be involved. Here, we closely analyzed the stimuli that underlie induction of autophagic trafficking in Saccharomyces cerevisiae. We find evidence for the existence of a novel aspect of the autophagic pathway that is regulated by intracellular amino acids, uncoupled from extracellular nutrient levels, and is absolutely dependent on Gcn2 and Gcn4. This requirement for Gcn2 and Gcn4 distinguishes amino-acid starvation induced autophagy from classic macroautophagy: Macroautophagic flux in response to nitrogen starvation is only partly diminished in gcn2Δ and gcn4Δ cells. However this maintenance of autophagic flux in gcn mutants during nitrogen starvation reflects the formation of larger numbers of smaller autophagosomes. We report that gcn2Δ and gcn4Δ cells are defective in the induction of Atg8 and Atg4 upon starvation, and this defect results, during total nitrogen starvation, in the formation of abnormally small autophagosomes, although overall autophagic flux remains close to normal due to a compensatory increase in the overall number of autophagosomes.     

Drosophila Gyf/GRB10 interacting GYF protein is an autophagy regulator that controls neuron and muscle homeostasis

Autophagy is an essential process for eliminating ubiquitinated protein aggregates and dysfunctional organelles. Defective autophagy is associated with various degenerative diseases such as Parkinson disease. Through a genetic screening in Drosophila, we identified CG11148, whose product is orthologous to GIGYF1 (GRB10-interacting GYF protein 1) and GIGYF2 in mammals, as a new autophagy regulator; we hereafter refer to this gene as Gyf. Silencing of Gyf completely suppressed the effect of Atg1-Atg13 activation in stimulating autophagic flux and inducing autophagic eye degeneration. Although Gyf silencing did not affect Atg1-induced Atg13 phosphorylation or Atg6-Pi3K59F (class III PtdIns3K)-dependent Fyve puncta formation, it inhibited formation of Atg13 puncta, suggesting that Gyf controls autophagy through regulating subcellular localization of the Atg1-Atg13 complex. Gyf silencing also inhibited Atg1-Atg13-induced formation of Atg9 puncta, which is accumulated upon active membrane trafficking into autophagosomes. Gyf-null mutants also exhibited substantial defects in developmental or starvation-induced accumulation of autophagosomes and autolysosomes in the larval fat body. Furthermore, heads and thoraxes from Gyf-null adults exhibited strongly reduced expression of autophagosome-associated Atg8a-II compared to wild-type (WT) tissues. The decrease in Atg8a-II was directly correlated with an increased accumulation of ubiquitinated proteins and dysfunctional mitochondria in neuron and muscle, which together led to severe locomotor defects and early mortality. These results suggest that Gyf-mediated autophagy regulation is important for maintaining neuromuscular homeostasis and preventing degenerative pathologies of the tissues. Since human mutations in the GIGYF2 locus were reported to be associated with a type of familial Parkinson disease, the homeostatic role of Gyf-family proteins is likely to be evolutionarily conserved.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Nutrient-driven O-GlcNAc cycling influences autophagic flux and neurodegenerative proteotoxicity

O-GlcNAcylation is an abundant post-translational modification implicated in human neurodegenerative diseases. We showed that loss-of-function of OGT (O-linked GlcNAc transferase) alleviated, while loss of OGA (O-GlcNAc selective β-N-acetyl-D-glucosaminidase) enhanced, the proteotoxicity of C. elegans neurodegenerative disease models including tauopathy, β-amyloid peptide and polyglutamine expansion. The O-GlcNAc cycling mutants act, in part, by altering insulin signaling, proteasome activity and autophagy. In mutants lacking either of these enzymes of O-GlcNAc cycling, there is a striking accumulation of GFP::LGG-1 (C. elegans homolog of Atg8 and LC3) and increased phosphatidylethanolamine (PE)-modified GFP::LGG-1 upon starvation. We speculate that O-GlcNAc cycling is a key nutrient-responsive regulator of autophagic flux acting at multiple levels including direct modification of BECN1 and BCL2.     

Resveratrol-mediated autophagy requires WIPI-1-regulated LC3 lipidation in the absence of induced phagophore formation

Canonical autophagy is positively regulated by the Beclin 1/phosphatidylinositol 3-kinase class III (PtdIns3KC3) complex that generates an essential phospholipid, phosphatidylinositol 3-phosphate (PtdIns(3)P), for the formation of autophagosomes. Previously, we identified the human WIPI protein family and found that WIPI-1 specifically binds PtdIns(3)P, accumulates at the phagophore and becomes a membrane protein of generated autophagosomes. Combining siRNA-mediated protein downregulation with automated high through-put analysis of PtdIns(3)P-dependent autophagosomal membrane localization of WIPI-1, we found that WIPI-1 functions upstream of both Atg7 and Atg5, and stimulates an increase of LC3-II upon nutrient starvation. Resveratrol-mediated autophagy was shown to enter autophagic degradation in a noncanonical manner, independent of Beclin 1 but dependent on Atg7 and Atg5. By using electron microscopy, LC3 lipidation and GFP-LC3 puncta-formation assays we confirmed these results and found that this effect is partially wortmannin-insensitive. In line with this, resveratrol did not promote phagophore localization of WIPI-1, WIPI-2 or the Atg16L complex above basal level. In fact, the presence of resveratrol in nutrient-free conditions inhibited phagophore localization of WIPI-1. Nevertheless, we found that resveratrol-mediated autophagy functionally depends on canonical-driven LC3-II production, as shown by siRNA-mediated downregulation of WIPI-1 or WIPI-2. From this it is tempting to speculate that resveratrol promotes noncanonical autophagic degradation downstream of the PtdIns(3)P-WIPI-Atg7-Atg5 pathway, by engaging a distinct subset of LC3-II that might be generated at membrane origins apart from canonical phagophore structures.     

Autophagosome formation and cargo sequestration in the absence of LC3/GABARAPs

It has been widely assumed that Atg8 family LC3/GABARAP proteins are essential for the formation of autophagosomes during macroautophagy/autophagy, and the sequestration of cargo during selective autophagy. However, there is little direct evidence on the functional contribution of these proteins to autophagosome biogenesis in mammalian cells. To dissect the functions of LC3/GABARAPs during starvation-induced autophagy and PINK1-PARK2/Parkin-dependent mitophagy, we used CRISPR/Cas9 gene editing to generate knockouts of the LC3 and GABARAP subfamilies, and all 6 Atg8 family proteins in HeLa cells. Unexpectedly, the absence of all LC3/GABARAPs did not prevent the formation of sealed autophagosomes, or selective engulfment of mitochondria during PINK1-PARK2-dependent mitophagy. Despite not being essential for autophagosome formation, the loss of LC3/GABARAPs affected both autophagosome size, and the efficiency at which they are formed. However, the critical autophagy defect in cells lacking LC3/GABARAPs was failure to drive autophagosome-lysosome fusion. Relative to the LC3 subfamily, GABARAPs were found to play a prominent role in autophagosome-lysosome fusion and recruitment of the adaptor protein PLEKHM1. Our work clarifies the essential contribution of Atg8 family proteins to autophagy in promoting autolysosome formation, and reveals the GABARAP subfamily as a key driver of starvation-induced autophagy and PINK1-PARK2-dependent mitophagy. Since LC3/GABARAPs are not essential for mitochondrial cargo sequestration, we propose an additional mechanism of selective autophagy. The model highlights the importance of ubiquitin signals and autophagy receptors for PINK-PARK2-mediated selectivity rather than Atg8 family-LIR-mediated interactions.     

Curvature‐sensitive trans‐assembly of human Atg8‐family proteins in autophagy‐related membrane tethering

In macroautophagy, de novo formation of the double membrane‐bound organelles, termed autophagosomes, is essential for engulfing and sequestering the cytoplasmic contents to be degraded in the lytic compartments such as vacuoles and lysosomes. Atg8‐family proteins have been known to be responsible for autophagosome formation via membrane tethering and fusion events of precursor membrane structures. Nevertheless, how Atg8 proteins act directly upon autophagosome formation still remains enigmatic. Here, to further gain molecular insights into Atg8‐mediated autophagic membrane dynamics, we study the two representative human Atg8 orthologs, LC3B and GATE‐16, by quantitatively evaluating their intrinsic potency to physically tether lipid membranes in a chemically defined reconstitution system using purified Atg8 proteins and synthetic liposomes. Both LC3B and GATE‐16 retained the capacities to trigger efficient membrane tethering at the protein‐to‐lipid molar ratios ranging from 1:100 to 1:5,000. These human Atg8‐mediated membrane‐tethering reactions require trans‐assembly between the membrane‐anchored forms of LC3B and GATE‐16 and can be reversibly and strictly controlled by the membrane attachment and detachment cycles. Strikingly, we further uncovered distinct membrane curvature dependences of LC3B‐ and GATE‐16‐mediated membrane tethering reactions: LC3B can drive tethering more efficiently than GATE‐16 for highly curved small vesicles (e.g., 50 nm in diameter), although GATE‐16 turns out to be a more potent tether than LC3B for flatter large vesicles (e.g., 200 and 400 nm in diameter). Our findings establish curvature‐sensitive trans‐assembly of human Atg8‐family proteins in reconstituted membrane tethering, which recapitulates an essential subreaction of the biogenesis of autophagosomes in vivo.     

The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice

Autophagy is an evolutionarily conserved bulk-protein degradation pathway in which isolation membranes engulf the cytoplasmic constituents, and the resulting autophagosomes transport them to lysosomes. Two ubiquitin-like conjugation systems, termed Atg12 and Atg8 systems, are essential for autophagosomal formation. In addition to the pathophysiological roles of autophagy in mammals, recent mouse genetic studies have shown that the Atg8 system is predominantly under the control of the Atg12 system. To clarify the roles of the Atg8 system in mammalian autophagosome formation, we generated mice deficient in Atg3 gene encoding specific E2 enzyme for Atg8. Atg3-deficient mice were born but died within 1 d after birth. Conjugate formation of mammalian Atg8 homologues was completely defective in the mutant mice. Intriguingly, Atg12–Atg5 conjugation was markedly decreased in Atg3-deficient mice, and its dissociation from isolation membranes was significantly delayed. Furthermore, loss of Atg3 was associated with defective process of autophagosome formation, including the elongation and complete closure of the isolation membranes, resulting in malformation of the autophagosomes. The results indicate the essential role of the Atg8 system in the proper development of autophagic isolation membranes in mice.     

Atg35, a micropexophagy-specific protein that regulates micropexophagic apparatus formation in Pichia pastoris

Autophagy-related (Atg) pathways deliver cytosol and organelles to the vacuole in double-membrane vesicles called autophagosomes, which are formed at the phagophore assembly site (PAS), where most of the core Atg proteins assemble. Atg28 is a component of the core autophagic machinery partially required for all Atg pathways in Pichia pastoris. This coiled-coil protein interacts with Atg17 and is essential for micropexophagy. However, the role of Atg28 in micropexophagy was unknown. We used the yeast two-hybrid system to search for Atg28 interaction partners from P. pastoris and identified a new Atg protein, named Atg35. The atg35? mutant was not affected in macropexophagy, cytoplasm-to-vacuole targeting or general autophagy. However, both Atg28 and Atg35 were required for micropexophagy and for the formation of the micropexophagic apparatus (MIPA). This requirement correlated with a stronger expression of both proteins on methanol and glucose. Atg28 mediated the interaction of Atg35 with Atg17. Trafficking of overexpressed Atg17 from the peripheral ER to the nuclear envelope was required to organize a peri-nuclear structure (PNS), the site of Atg35 colocalization during micropexophagy. In summary, Atg35 is a new Atg protein that relocates to the PNS and specifically regulates MIPA formation during micropexophagy.     

Scaffolding the expansion of autophagosomes

The conjugation of the small ubiquitin (Ub)-like protein Atg8 to autophagic membranes is a key step during the expansion of phagophores. This reaction is driven by 2 interconnected Ub-like conjugation systems. The second system conjugates the Ub-like protein Atg12 to Atg5. The resulting conjugate catalyzes the covalent attachment of Atg8 to membranes. Atg12–Atg5, however, constitutively associates with the functionally less well-characterized coiled-coil protein Atg16. By reconstituting the conjugation of Atg8 to membranes in vitro, we showed that after Atg8 has been attached to phosphatidylethanolamine (PE), it recruits Atg12–Atg5 to membranes by recognizing a noncanonical Atg8-interacting motif (AIM) within Atg12. Atg16 crosslinks Atg8–PE-Atg12–Atg5 complexes to form a continuous 2-dimensional membrane scaffold with meshwork-like architecture. Apparently, scaffold formation is required to generate productive autophagosomes and to deliver autophagic cargo to the vacuole in vivo.     

Mammalian Atg8s: One is simply not enough

Yeast Atg8, a key factor in the autophagic process, is a ubiquitin-like protein that undergoes a unique conjugation to phosphatidylethanolamine (PE). Atg8 plays a dual role in early stages of autophagosome formation: It was implicated in recruitment of cargo proteins such as Atg19 and Atg32 for Cvt and mitophagy, respectively, and in autophagosome biogenesis, serving as an elongation factor by mediating membrane hemi-fusion. Similarly, the mammalian Atg8 proteins, LC3s and GABARAPs, recruit cargo into autophagosomes by binding to adaptor proteins such as p62, NBR1 and Nix. These functions, however, are not essential for bulk autophagic flux. Other studies in which the activity of the mammalian Atg8s was blocked either by knockout of the E2-like enzyme Atg3 or by using a dominant negative mutant of the promiscuous protease Atg4B revealed, in agreement with the yeast Atg8 data, that the mammalian factors are crucial for the formation of normal and mature autophagosomes. While it seems that the single yeast Atg8 and the mammalian Atg8s share similar roles, it is still unclear why the mammalian system employs several homologs. Recent publications demonstrated that the mammalian Atg8s differ in their cargo specificity, as Nix, for example, binds exclusively to GABARAP-L1. This may suggest that these proteins exhibit distinct activity also in autophagosome biogenesis. In our study we divided the mammalian Atg8s into two subfamilies of homologs based on amino acid similarity, the LC3 and GABARAP/GATE-16 subfamilies, and tested their essentiality and role in autophagy. In agreement with previous studies we found that the mammalian Atg8s are essential for autophagy but, more importantly, that each of these subfamilies has a distinct role in the process of autophagosome biogenesis.