Atg8ylation as a general membrane stress and remodeling response

The yeast Atg8 protein and its paralogs in mammals, mammalian Atg8s (mAtg8s), have been primarily appreciated for their participation in autophagy. However, lipidated mAtg8s, including the most frequently used autophagosomal membrane marker LC3B, are found on cellular membranes other than autophagosomes. Here we put forward a hypothesis that the lipidation of mAtg8s, termed ‘Atg8ylation’, is a general membrane stress and remodeling response analogous to the role that ubiquitylation plays in tagging proteins. Ubiquitin and mAtg8s are related in sequence and structure, and the lipidation of mAtg8s occurs on its C-terminal glycine, akin to the C-terminal glycine of ubiquitin. Conceptually, we propose that mAtg8s and Atg8ylation are to membranes what ubiquitin and ubiquitylation are to proteins, and that, like ubiquitylation, Atg8ylation has a multitude of downstream effector outputs, one of which is autophagy.

Structural basis for membrane recruitment of ATG16L1 by WIPI2 in Autophagy

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed â-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 â-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

Structure-function relationship for a divergent Atg8 protein required for a non-autophagic function in malaria parasites

Atg8 family proteins are highly-conserved eukaryotic proteins with diverse autophagy and non-autophagic functions in eukaryotes. While the structural features required for conserved autophagy functions of Atg8 are well-established, little is known about the molecular changes that facilitated acquisition of divergent, non-autophagic functions of Atg8. The malaria parasite Plasmodium falciparum offers a unique opportunity to study non-autophagic functions of Atg8 family proteins because it encodes a single Atg8 homolog whose only essential function is in the inheritance of an unusual secondary plastid called the apicoplast. Here we used functional complementation to investigate the structure-function relationship for this divergent Atg8 protein. We showed that the LC3-interacting region (LIR) docking site (LDS), the major interaction interface of Atg8 protein family, is not sufficient for PfAtg8 apicoplast function. Other regions previously implicated in canonical Atg8 interactions, the ubiquitin-interacting motif (UIM) docking site (UDS) and the N-terminal helix are not required for PfAtg8 function. Finally, the unique Apicomplexan-specific loop previously implicated in interaction with membrane conjugation machinery in vitro, is not required in vivo neither for membrane conjugation nor for the effector function of PfAtg8. These results suggest that the effector function of PfAtg8 is mediated by structural features distinct from those previously identified for macroautophagy and selective autophagy functions.

Structural Basis for Membrane Recruitment of ATG16L1 by WIPI2 in Autophagy

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven bladed beta-propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Angstrom resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 beta-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand, and ATG8 lipidation on the other.

Critical role of mitochondrial ubiquitination and the OPTN–ATG9A axis in mitophagy

Damaged mitochondria are selectively eliminated in a process called mitophagy. Parkin and PINK1, proteins mutated in Parkinson’s disease, amplify ubiquitin signals on damaged mitochondria with the subsequent activation of autophagic machinery. Autophagy adaptors are thought to link ubiquitinated mitochondria and autophagy through ATG8 protein binding. Here, we establish methods for inducing mitophagy by mitochondria-targeted ubiquitin chains and chemical-induced mitochondrial ubiquitination. Using these tools, we reveal that the ubiquitin signal is sufficient for mitophagy and that PINK1 and Parkin are unnecessary for autophagy activation per se. Furthermore, using phase-separated fluorescent foci, we show that the critical autophagy adaptor OPTN forms a complex with ATG9A vesicles. Disruption of OPTN–ATG9A interactions does not induce mitophagy. Therefore, in addition to binding ATG8 proteins, the critical autophagy adaptors also bind the autophagy core units that contribute to the formation of multivalent interactions in the de novo synthesis of autophagosomal membranes near ubiquitinated mitochondria.

Leishmania donovani parasite requires Atg8 protein for infectivity and survival under stress

The importance of autophagy in parasites with a digenetic life cycle like Leishmania spp. is significant. The parasite survives as promastigotes in the insect gut and as immotile amastigotes in mammals. This study demonstrates increased autophagy in Leishmania parasite during progression of in vitro life cycle and upon exposure to stress stimuli like starvation, oxidative stress, and drugs. Autophagy inhibition during stress exposure increased cell death, indicating the importance of autophagy in cellular defense against adverse conditions. Atg8 protein, a homolog of mammalian autophagy protein LC3 is expressed in Leishmania parasite but its function remains unknown. Overexpression of Atg8 (Atg8-OE) rendered the parasites resistant to stress and capable of infecting macrophages in substantial numbers; however, disruption of the Atg8 gene (ΔAtg8) resulting in suppression of Atg8 protein expression, increased susceptibility to stress and reduced the capability to cause infection. A critical event in the Leishmania parasite lifecycle is the differentiation of promastigote forms to the disease causing amastigote forms. The failure of ΔAtg8 parasites lacking Atg8 protein to differentiate into amastigotes, unlike the Atg8-OE and vector-transfected parasites, clearly indicated Atg8 involvement in a crucial event. The inability of ΔAtg8 parasites to infect macrophages in vitro was verified in an in vivo mouse model of leishmaniases where infection could not be induced by the ΔAtg8 parasites. Autophagy is known to be involved in the remodeling of damaged organelles. The accumulation of Atg8 around damaged mitochondria suggested increase of autophagy in the vicinity of the organelle. This buildup was prevented when mitochondria generated reactive oxygen species that were quenched, suggesting them as possible signaling molecules for sensing mitochondrial instability. In summary, our study provides new evidences for a crucial role of Atg8 protein in sustaining Leishmania parasite survival during life cycle and stress exposure, differentiation to amastigotes, and their infective abilities.

Expression profiling of kinesins, involved in the development of autophagy in Arabidopsis thaliana, and the role of tubulin acetylation in the interaction of Atg8 protein with microtubules

Aim. To investigate the interrelation between changes in the expression levels of kinesin genes that are potentially involved in the development of stress-induced autophagy in Arabidopsis thaliana by means of microtubules, and the structural biology analysis of the role of α-tubulin acetylation in the regulation of interaction of α-tubulin with Atg8. Methods. The simulation of the influence of abiotic stresses. PCR analysis of changes in expression levels of kinesin genes. The molecular dynamics simulations of α-tubulin and Atg8 complexes were performed using the GROMACS 4.5.5 program. Results. It was shown that the changes in expression levels were caused by the influence of stressful stimuli. A significant increase in the transcriptional activity of the KIN5B, KIN12B, KIN12F genes after UV-B irradiation, the KIN6, KIN7O, KIN7D, KIN12B genes under osmotic-, and KIN6, KIN12B under salt stress was detected. By means of bioinformatics it was demonstrated that α-tubulin acetylation provides an enhanced interaction of α-tubulin and Atg8 protein. Conclusions. Obtained data point out the important role of kinesins and α-tubulin acetylation in realization of microtubules’ partaking in the development of stress-induced autophagy in plants. Keywords: microtubules, α-tubulin, kinesins, Atg8 protein, stress-induced autophagy.

GABARAP Like-1 enrichment on membranes: Direct observation of trans-homo-oligomerization between membranes and curvature-dependent partitioning into membrane tubules

The Atg8/LC3/GABARAP protein family has been implicated in membrane remodeling events on the growing autophagosome. In particular, each of these proteins can form a protein-lipid conjugate that has been shown in vitro to drive liposome aggregation and in some cases membrane fusion. Furthermore, yeast Atg8 has been described as a curvature sensing protein, through its natural capacity to concentrate on highly curved membranes. A key advance with yeast Atg8, was the introduction of Giant Unilamellar Vesicles (GUVs) as an in vitro support that could allow membrane deformation and tethering to be observed by simple microscopy. Further, micromanipulation of an individual GUV could be used to create local areas of curvature to follow Atg8 partitioning. Here, we use a recently developed method to decorate GUVs with the mammalian Atg8 protein GABARAPL1 and establish the generality of the observations made on yeast Atg8. Then we apply double micromanipulation, the capture and positioning of two independently prepared GUVs, to test elements of the mechanism, speed and reversibility of mammalian Atg8 protein-mediated tethering. We find that the membranes adhere through GABARAPL1/GABARAPL1 homotypic trans-interactions. On a single membrane with two regions with significantly different curvatures we observed that the regions of higher curvature can be enriched up to 10 times in GABARAPL1 compared to the planar regions. This approach has the potential to allow the formation and study of specific topographically-controlled interfaces involving Atg8-proteins and their targets on apposing membranes.